chunk_id int64 1M 11.9M | document stringlengths 3 4.98k | embedding listlengths 384 384 |
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10,003,800 | At pH 7.0 and 25 degrees C, NBF-Cl (4-chloro-7-nitrobenzofurazan) reacts rapidly with rabbit muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) to yield a product with an absorption maximum at 402 nm, which is shifted to 422 nm upon acid denaturation. The reaction involves arylation of a single cysteine residue per subunit of tetrameric aldolase, as shown by the molar absorptivity of NBF-aldolase and by titration of sulfhydryl groups of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid), DTNB. The site of arylation appears to be Cys-237, which is the cysteine residue that reacts most rapidly with DTNB. The site of arylation appears to be Cys-237, which is the cysteine residue that reacts most rapidly with DTNB, and which is not essential for aldolase activity. Arylation of the enzyme is 13-20-times more rapid than that of model compounds. The relatively high rate of arylation is not due to medium effects, to an anomalously low pKa of Cys-237, or to the presence of a binding site for NBF-Cl, and is tentatively assigned to acid-base catalysis by other functional groups in the vicinity of the reactive sulfhydryl group. The NBF-Cl reaction provides the most efficient means of titrating Cys-237 residues in rabbit muscle aldolase. | [
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10,003,801 | Modification of the essential amino acids of human pancreatic carboxylic-ester hydrolase. | [
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10,003,802 | Chemical modification of human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) were performed using organophosphorus compounds, ethoxyformic anhydride and Woodward's K reagent. It has been shown that: (1) the inhibition of the enzyme activity by organophosphorus compounds is due to the phosphorylation of only one alcohol residue, probably a serine residue which may represent the acylable group of the enzyme: (2) 35-36 free carboxyl groups are modified by Woodward's K reagent but only one is responsible for the loss of enzyme activity. This carboxyl group has a pKa of 5.2.: (3) carbethoxylation of histidine residues leads to the inhibition of the enzyme activity. All nine histidine residues are reactive but only one is essential for activity. Taking into account the probable formation of an acyl-enzyme intermediate (Lombardo, D. and Guy, O. (1981) Biochim. Biophys. Acta 657, 425-437) during substrate hydrolysis and the present data we discuss a possible mechanism for carboxylic-ester hydrolase catalysis, a mechanism similar to that described for chymotrypsin. | [
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10,003,803 | Catalytic properties of modified human pancreatic carboxylic-ester hydrolase. | [
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10,003,804 | Human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) modified by specific reagents (diisopropyl phosphorofluoridate, diethyl p-nitrophenyl phosphate, Woodward's K reagent and ethoxyformic anhydride) was studied for its activity. The three residues probably implicated in the active site acting on soluble substrate are shown to be essential for the activity on emulsified substrates such as tributyrin or cholesterol esters solubilized by bile salts (hydrolyzing and synthetizing activities). The modified enzyme is still able to bind to interfaces. Nevertheless, one supplementary carboxylic acid group is responsible for the stability of the enzyme bound to interfaces. | [
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10,003,805 | Stability and inhibition of Aspergillus and Rhizopus lipases. | [
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] |
10,003,806 | A lipase (triacylglyceral aclhydrolase, EC 3.1.1.3) secreted by Aspergillus contains two chains each of molecular weight 25000, with 12 mannose, two galactose and two N-acetylglucosamine residues per chain. The average hydrophobicity is close to the mean fov globular proteins. Thermal denaturation was biphasic when followed by circular dichroism but the stability is not unusual for globular proteins, nor does partial removal of the carbohydrate affect it. (delta G=46kJ). Refolding did not occur. The inhibitory effect of 2H2O on the rate of reaction implicates a histidine in the active centre. The inhibitory effect of concanavalin A was complex but the active centre was blocked. A lipase from Rhizopus arrhizus showed immunological identity with Aspergillus lipase, but unlike that one, was not inhibited by the antiserum. Similarly it was not inhibited by concanavalin -A although Con A-Sepharose was used in its isolation. It was 4-fold as active as the Aspergillus enzyme but had low thermal stability (delta K= 16kJ). The interfacial location of lipases does not impose any special requirement on the overall structure of the enzyme. | [
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10,003,807 | Tryptophan fluorescence of human hemoglobin. II. Effect of inositol hexaphosphate on the T-R transition. | [
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10,003,808 | Fluorescence spectra of tryptophan residues of human hemoglobin in the absence and presence of inositol hexaphosphate were measured at room temperature. The tryptophan fluorescence intensity of deoxy HbA was observed to decrease in accordance with the binding with inositol hexaphosphate. The fluorescence intensity of HbA, Hb Kempsey (beta 99 Asp-Asn), Hb Chesapeake (alpha 92 Arg-Leu) and NES-des-Arg Hb (des-141 alpha Arg and beta 93 Cys-N-ethylsuccinimide derivative) in the presence of inositol hexaphosphate exhibits a considerable decrease in the deoxy to oxy transition, while no or slight fluorescence intensity change was observed in the deoxy to oxy transition of Hb Kempsey and NES-des-Arg Hb in the absence of inositol hexaphosphate. The tryptophan fluorescence behavior suggest that the inositol hexaphosphate-induced structural change in these hemoglobins is attributable to the formation of a different T type of structure from that of the normal T-R transition. | [
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10,003,809 | Assembly of formyltetrahydrofolate synthetase monomers. Kinetic evidence for a temperature-induced conformational change. | [
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10,003,810 | The assembly of Clostridium acidi-urici formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) monomers induced by low concentrations of CsCl to form the catalytically active tetramer shows a pronounced lag phase in the time-course progress curve. Preincubation of the monomer for 20 min at 20 degree C in a medium of either high ionic strength, 10% dimethylsulfoxide or 1 M glycerol removes the lag period. Shorter preincubation periods produce the same effect if the medium contains both high ionic strength and dimethylsulfoxide or glycerol. Preincubation at 0 degree C in the presence of these agents has little effect. The reassociation reaction is first-order with respect to monomer concentration, before preincubation at 20 degree C, but second-order after. It is proposed that two conformers of the monomer exist in equilibrium in a temperature-dependent reaction and that the conformer able to reassociate is more favored at 20 degree C than at 0 degree C. High ionic strength or the presence of the organic solvents shift the equilibrium far in the direction of the conformer capable of reassociation, changing the rate-limiting step from a first-order conformational step to a second-order monomer association step. | [
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10,003,811 | Sulfhydryl groups of native myosin and of the myosin heavy chains from Physarum polycephalum compared to vertebrate skeletal, smooth, and non-muscle myosins. | [
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10,003,812 | Although a previously reported analysis of Physarum myosin detected no cysteine residues in the molecule, the myosin ATPase activity was inhibited by p-chloromercuribenzoate. We have re-examined this apparently contradictory finding. We found highly purified plasmodial myosin to be very sensitive to N-ethylmaleimide inhibition of the K+, Ca2+ -activated ATPase. An estimate of the number of reactive sulfhydryls of the native myosin using Ellman's reagent showed only 1.5 mol 11 min-reactive sulfhydryl/mol as compared to 4.5 for chicken breast myosin in 5 min. 3H- and 14C-labelled N-ethylmaleimide was used to estimate the total sulfhydryls of the SDS-denatured heavy chains. Plasmodial myosin heavy chains bound 10-13% of the N-ethylmaleimide bound by chicken breast myosin heavy chains. Smooth muscle myosin heavy chains as well as heavy chains of embryonic chicken presumptive myoblasts had 65-70% of the reactive groups of chicken myotube myosin heavy chains. Amino acid analyses of purified Physarum myosin showed that some preparations contained cysteic acid residues even before performic oxidation. After the performic oxidation a mean value of 3 mol cysteic acid per 10(5) g Physarum myosin was found, or less than half that reported for striated muscle myosin. Our results show that in the sulfhydryl-poor plasmodial myosin each heavy chain contains at least two sulfhydryls, and probably more, but that there is variable oxidation of the total sulfhydryls. It has been reported that plasmodial myosin lacks rapidly reacting sulfhydryls groups when tested with an ATP analogue which reacts with light chains of vertebrate muscle myosins. Therefore, the 1-2 sulfhydryls of plasmodial myosin which react rapidly with Ellman's reagent appear to be on the heavy chain. Our results also suggest that during development of myotubes changes occur in the myosin heavy chains. | [
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10,003,813 | An affinity label for the anion binding site in ovotransferrin. Implications for iron release to reticulocytes. | [
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10,003,814 | Bromopyruvate has been found to be an affinity label for the anion binding site in ovotransferrin. Spectral and radioactive labeling studies indicate that the bromopyruvate first forms a reversible ternary complex with ovotransferrin and iron and then reacts to yield the covalently bound product. This derivative is partially resistant to iron loss by protonation and suggests that protonation and loss of anion is a requisite first step for iron release to reticulocytes. | [
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10,003,816 | Glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) from human erythrocytes as well as from other sources was inactivated irreversibly by the arginine modifying reagents 2,3-butanedione, 1,2-cyclohexanedione and phenylglyoxal. The inactivation caused by these reagents increased with time and was biphasic in nature. Inactivation by 2,3-butanedione in borate buffer could be partially reversed by dialysis against phosphate buffer or by incubation with hydroxylamine. The dicarbonyl reagents simultaneously inactivated the transhydrogenase activity of glutathione reductase that is expressed by the pyridine nucleotide sites. Amino acid analysis of enzyme modified with 2,3-butanedione showed a decrease by about 5 in the number of arginine residues. Analysis of enzyme inactivated with phenylglyoxal indicated modification of two arginine residues. NADPH, NADP/ and other adenosine 2'-phosphate nucleotides partially protected the enzyme against inactivation. GSSG did not protect the enzyme significantly. Modified glutathione reductase displayed a diminished affinity for the nucleotides as shown by affinity chromatography on 2',5'-ADP-Sepharose and by stopped-flow kinetics of the reduction of the enzyme. Modification did not cause any gross structural changes of enzyme molecule. It is concluded that some of the arginine residues modified are located at the pyridine-nucleotide-binding sites of the enzyme. | [
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10,003,817 | The isolation, purification, and properties of a ribonuclease (Mr 18 000) from human uremic serum, and its relation to the human urinary ribonuclease (Mr 33 000). I. Isolation of the low molecular weight ribonuclease. | [
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10,003,818 | The isolation, purification, and properties of a ribonuclease (Mr 18 000) from human uremic serum, and its relation to the human urinary ribonuclease (Mr 33 000). II. Properties of the enzymes. | [
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] |
10,003,819 | A low molecular weight ribonuclease (Mr 18 000) isolated and purified from human uremic serum was found to have similar properties to the high molecular weight ribonuclease (Mr 33 000) isolated from human urine. A detailed comparative study of both enzymes was undertaken to investigate the relationship between them. It is suggested that poly(C)-avid human ribonucleases have similar amino acid compositions but variable carbohydrate contents and that variations in the sugar content are responsible for variations in the molecular weight. | [
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10,003,820 | Evidence for cooperative effects in human liver arginase. | [
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10,003,821 | The reaction kinetics of human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) in terms of arginine concentration is strikingly altered by varying the pH. Lowering the pH from the optimum (9.5) toward a more physiological value (7.5) there is a transition from hyperbolic to sigmoidal kinetics. The cooperative effects are observed in the presence and absence of the product ornithine. Dimers of arginase exhibit typical Michaelis-Menten kinetics even in the presence of ornithine. Dimer-dimer interactions are suggested to explain the kinetic properties of arginase at pH 7.5. | [
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10,003,822 | Iron content and molecular weight of uteroferrin and a comparison of its iron and copper forms. | [
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10,003,823 | The molecular weight of the purple, iron-containing glycoprotein uteroferrin has been determined to be 35140 +/- 1610 by sedimentation equilibrium centrifugation. This is consistent with measurements carried out by other procedures. The dry weight of a solution of uteroferrin with an absorption of 1.00 cm -1 at 280 nm was 0.87 mg/ml. Highly purified uteroferrin has a ratio of absorbance at 280 nm to 545 nm of about 13.2 and a revised extinction coefficient at 545 nm of 3.1 . 10(3) M -1 is presented. The iron content of uteroferrin has been determined both colorimetrically and by atomic absorption spectroscopy. One iron atom is present per polypeptide chain. Reduction with dithionite leads to loss of iron, allowing the apoprotein to be prepared. Enzymatic activity can be restored to the apoprotein with Fe(III) or with Cu(II). The copper enzyme has no visible color and has a higher pH optimum than the Fe-uteroferrin. Whereas the phosphatase activity of the latter is increased several-fold by beta-mercaptoethanol, reduction inactivates Cu-uteroferrin. Both forms of uteroferrin have similar Km values for p--nitrophenyl phosphate and are inhibited by molybdate but not by tartrate. Excess Cu(II) and Fe(III) strongly inhibit uteroferrin phosphatase activity and these results may explain the failure of other to restore activity to the apoprotein using Cu(II) and Fe(III) salts. | [
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10,003,824 | Drug-induced oxidation and precipitation of hemoglobins A, S and C. | [
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10,003,825 | Analysis of optical absorption spectra of drug-hemoglobin reaction mixtures show that oxidation of oxyhemoglobins A, S and C by menadione and acetylphenylhydrazine and the consequent formation of methemoglobin, hemichromes and 'choleglobin' proceed at identical rates for all three hemoglobins. Nevertheless, precipitate formation, which is linked to hemichrome production, occurs in a definite order: S greater than C greater than A. Differences in precipitability among these three hemoglobins are linked to an as yet unidentified property of the aggregate hemichromes, which is not directly related to the rate of total hemichrome production. | [
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10,003,826 | A new electrophoretic variant of hemoglobin (Ogi) in which a leucine residue is replaced by an arginine residue at position 34 of the alpha-chain. | [
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] |
10,003,827 | Hemoglobin Ogi, in which an arginine is substituted for a leucine residue at position 34 of the alpha-chain, was detected in a Japanese family. Although slightly increased oxygen affinity is associated with this amino acid substitution in the alpha 1 beta 1 contact, it is without obvious deleterious effect on the hematological parameters of the individuals heterozygous for this variant. | [
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10,003,829 | The anti-B specific lectin produced by Streptomyces sp. was shown to have two carbohydrate-binding sites with binding constants of 8.3 . 10(3) M-1 (15 degrees C) and 2.2 . 10(3) M-1 (4 degrees C) for L-rhamnose and D-galactose, respectively, calculated according to Scatchard plots. The binding of specific sugars to the lectin not only induced a peculiar ultraviolet difference spectrum showing a blue shift of tryptophan absorption, but also caused crystallization of the lectin at a concentration of 1 mg per ml or more. The solvent-perturbation studies on the lectin showed that the number of solvent-exposed tryptophan (or average extent of exposure) was two in the absence of L-rhamnose, and three in the presence of the sugar. This suggests that one tryptophan residue appears outside as the result of sugar-binding to the lectin, which is reflected by the difference spectra. Oxidation of two tryptophan residues with N-bromosuccinimide led to complete loss of carbohydrate-binding activity of the lectin, indicating that these residues are important for retaining the activity. | [
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10,003,830 | Comparative study of the conformational features of rat immunoglobulin G subclasses by circular dichroism. | [
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10,003,831 | Four subclasses of rat immunoglobulin G and some of their fragments were studied by circular dichroism. These studies were carried out in different conditions in order to determine whether conformational differences exist between each subclass. The results showed that in their native state the subclasses cannot be distinguished by characteristic differences. Moreover, differences between monoclonal immunoglobulins G of the same subclass were found, particularly in the 200-250 nm region, and their relationship to aromatic side chain contributions (especially those of tyrosine) has been investigated. However, the conformations of the immunoglobulin G were found to be differently affected by denaturing treatments and a relation to subclass has been demonstrated. | [
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10,003,832 | Lipid composition of tracheobronchial secretions from normal individuals and patients with cystic fibrosis. | [
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10,003,833 | The lipid composition of tracheobronchial secretions from normal individuals and patients with cystic fibrosis was investigated. Lipids were extracted from he dialyzed and lyophilized samples, and fractionated on silicic acid columns into neutral lipids, glycolipids and phospholipids. The lipids contained each fraction were separated into individual components by thin-layer chromatography and quantified. The secretions of patients with cystic fibrosis and were found to contain about 30% more lipids than that of normal individuals and exhibited elevated levels of cholesterol, phospholipids and glycosphingolipids. The level of free fatty acids and glyceroglucolipids was higher in the normal secretions. The phospholipids of cystic fibrosis secretions exhibited higher content of sphingomyelin and phosphatidylserine, while the normal samples contained more lysophosphatidylcholine. The glycosphingolipids of both types of samples consisted mainly of glucosyl- and lactosylceramides. The major glyceroglucolipid of the normal tracheobronchial secretions was tetraglucosyl glyceroglucolipid, whereas hexa-and octaglucosyl glyceroglucolipids were the predominant compounds of the cystic fibrosis secretions. | [
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10,003,834 | Studies on the in vivo synthesis of triacylglycerol in mouse liver. | [
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10,003,835 | The in vivo biosynthesis of hepatic glycerolipids was examined by studying the incorporation of [2-3H] glycerol into triacylglycerol in the mouse. The isotope was administered by rapid injection into the portal vein. The incorporation of glycerol was linear for about 1 min and maximal rates were seen in the presence of additional oleic acid. At 20 mumol of oleic acid bound to 1 mumol of bovine serum albumin, the highest level tested, glycerol incorporation was still increasing linearly, whereas a plateau was reached at 4 mumol of glycerol. The liver incorporated 200-300 nmol of [2-3H] glycerol into triacyglycerol per min when 20 mumol of albumin-bound oleic acid plus 4 mumol of glycerol were injected in a 0.2 ml volume. Studies on the uptake by the liver after intraportal injection revealed that at 0.5 min the liver retained 2 mumol of glycerol and 5 mumol of oleic acid, indicating that the uptake of substrate was not rate-limiting. The results suggest that the availability of substrate is a major factor in the regulation of triacyglycerol biosynthesis by the liver. | [
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10,003,836 | Biosynthesis of platelet-activating factor. I. Evidence for an acetyl-transferase activity in murine macrophages. | [
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10,003,837 | Platelet-activating factor (PAF-acether; 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is released from murine peritoneal adherent cells by inflammatory and non-inflammatory stimuli. We have found, in extracts from these cells, an enzyme activity that synthesizes. PAF-acether from synthetic lyso-PAF-acether by transferring the acetyl moiety of acetyl-coenzyme A onto the lyso-PAF-acether molecule. The enzyme is stabilized by 1 mM dithiothreitol, is calcium-dependent, has an apparent Km of 172 microM for acetyl-CoA and is active in a 6-8 pH range. When the acetyl-CoA substrate is replaced by propionyl-CoA, an ether lipid is produced which turns out to be as potent an aggregating agent as PAF-acether. In all cases, the products of the reaction were characterized by their behaviour in platelet-aggregation tests and their high-pressure liquid chromatography (HPLC) elution profiles. The precise definition of this acetyl-transferase is of primary importance for the development of new pharmacological agents capable of moduling a potent platelet aggregating factor. | [
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10,003,838 | Biosynthesis of dolichol and cholesterol during embryonic development of the chicken. | [
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10,003,839 | The polyisoprenol dolichol, in its phosphorylated form, serves as the lipid intermediate in N-linked glycoprotein synthesis, and shares the portion of its biosynthetic pathway with cholesterol. The synthesis of dolichol and cholesterol was followed over the course of development by incubating chicken embryos with [3H] acetate for 24-h intervals. Dolichol and cholesterol were isolated from the non-saponifiable lipids and identified by gel filtration and thin-layer chromatography. It was found that the rate of acetate incorporation into these two compounds per g of embryo decreased from days 8 to 14 of development. In the whole embryo, over the course of development the ratio of the rates of acetate incorporation into dolichol to that into cholesterol decreased markedly. These results suggest that mechanisms exist for controlling the differential rates of dolichol and cholesterol synthesis during development. When labeled organs from day 10 embryos were analysed, it was found that the ratio of the rates of synthesis of dolichol to cholesterol in brain was about 14-fold lower than that in liver and heart. Similar values for the rates of synthesis of these two compounds were obtained when isolated organs were labeled in vitro. These data suggest that these three organs are capable of synthesizing dolichol and cholesterol de novo, and that each organ independently regulates the rates of synthesis of dolichol and cholesterol. | [
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10,003,840 | The turnover of molecular species of phosphatidylinositol in Ehrlich ascites tumor cells. | [
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10,003,841 | It has been shown previously the 32Pi is incorporated into phosphatidylinositol 30 times faster than into the other phospholipids classes in Ehrlich ascites tumor cells, whereas [1-14C] glycerol is incorporated at almost the same rate (Waku, K., Nakazawa, Y. and MOri, W. (1976) J. Biochem. 79, 407-411). It was therefore suggested that there is a recirculating system (phosphatidylinositol leads to diacylglycerol leads to phosphatidic acid leads to CDP diacylglycerol leads to phosphatidylinositol) of phosphatidylinositol in Ehrlich ascites tumor cells. In this work, 32Pi or [1-3H] glycerol was injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells from which the lipids were extracted after selected periods. Phosphatidylinositol was prepared and fractionated in the form of dimethylphosphatidic acid into six molecular species by AgNO3-impregnated TLC. The specific radioactivities of the fractionated species were determined. 32Pi was incorporated into diene molecular species and [1-3H] glycerol into monoene species with a higher rate than the other species and both precursors were incorporated into tetraene species rather slowly. 32P/3H values appeared to be at almost the same for each molecular species, although monoene species showed slightly lower values. These results suggest that there could be a recirculating of the phosphorylinositol moiety in each of the molecular species of phosphatidylinositol. | [
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10,003,842 | Relative acylglycerol acyltransferase activities in homogenates of enzymically dispersed rat jejunal villus and crypt cells. | [
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10,003,843 | The relative acyltransferase activities were compared in homogenates of rat jejunal villus and crypt cells isolated by differential scraping and hyaluronidase dispersion. The contributions of the monoacylglycerol and phosphatidic acid pathways to the higher acylglycerol and phospholipid biosynthesis were assessed using 2-oleoyl-sn-[3H] glycerol and [I-14C] palmitic acid as tracers. The stereochemical course of the diacylglycerol biosynthesis was determined by stereospecific analysis. Using 2-oleoyl-sn-glycerol as a tracer, the villus cells exhibited for times higher diacylglycerol and 19 times higher triacylglycerol biosynthesis than crypt cells on an equivalent protein basis. Furthermore, while the villus cell homogenates yielded a preponderance (75%) of the 1, 2-diacyl-sn-glycerols, the crypt cell homogenates formed essentially racemic proportions of 1, 2- and 2,3-diacyl-sn-glycerols. Both villus and crypt cell homogenates exhibited comparable acyl acceptor and acyl donor concentration dependence and the same cofactor requirements. It is unlikely that these acyltransferase activities in the crypt cell preparation are due to contamination with the villus cells, because then more comparable proportions of the enantiomeric diacylglycerols and triacylglycerols would have been anticipated. It is concluded that the crypt cells possess intrinsic monoacylglycerol and to a much lesser extent diacylglycerol acyltransferase activities, which are acquired prior to the development of a distinct brush border and which probably do not require dietary stimulus for induction. | [
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10,003,845 | We have determined the sites of degradation of low density lipoprotein in rats using covalently linked [14C] sucrose as tracer. On degradation, 14C is trapped intracellularly as a cumulative measure of the amount of protein catabolized by each tissue. [14C] Sucrose-labeled rat low density lipoprotein (d 1.02-1.05 g/ml) was cleared from the plasma at a rate (0.092 +/- 0.003 h-1) similar to that for 125I-labeled LDL (0.096 +/- 0.22 h-1). Tissues were examined for total 14C content 24 h after injection of 14C-labeled lipoprotein. At death, animals were perfused thoroughly to remove trapped plasma. Recovery of 14C in tissue was 100 +/- 23% of catabolized 14C-labeled lipoprotein (calculated from plasma decay kinetics). In three test tissues, leakage of 14C over 5 days was less than 10%/day; leakage from liver was 10%/day, predominantly into bile; 14C content of kidney increased slightly. Thus, 14C trapping was adequate. The 14C-labeled lipoprotein was catabolized 66.8 +/- 2.5% by liver. No other organ catabolized more than 8%. Liver, adrenal and ovary were the most active per unit wet weight, followed by spleen. Urinary excretion, in 24h, was 3% and biliary excretion was 7% of catabolized. Human low density lipoproteins were similarly examined with similar results; this similarity may be due to exchange of rat apolipoproteins onto human lipoprotein in the circulation. | [
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10,003,846 | Increased hepatitic cholesterol production due to liver hypertrophy in rat experimental nephrosis. | [
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10,003,847 | Control and nephrotic rats were compared as to the liver contents of cholesterol, phospholipid and the activity of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Whole liver homogenates as well as endoplasmic reticulum membrane samples showed increased free cholesterol and phospholipid mass in the nephrotics. Correction of the values by the protein content indicated membrane expansion, i.e. liver hypertrophy. However, total hepatic cholesterol synthesis as measured by the reductase activity was increased in the nephrotic rat. These results are in accordance with previous studies showing enhanced cholesterol production in experimental nephrosis. In short, enhanced cholesterol mass in the liver coexists with increased hepatic synthesis in the experimental model used. | [
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10,003,848 | Lysolecithin acyltransferase and alveolar phosphatidylcholine palmitate in experimental acute alveolar injury in the dog lung. | [
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10,003,849 | Lysolecithin acyltransferase (EC 2.3.1.23) activities in lung homogenates and in subcellular fractions, and fatty acid composition of phosphatidylcholine (PC) in lung lavage were studied in dogs with acute alveolar injury induced by N-nitroso-N-methylurethane. The specific activity in the microsomal fraction was 10 and 3 times higher than those of homogenate and mitochondrial fractions, respectively. Both the lysolecithin acyltransferase activities and the proportions of palmitate in alveolar lavage PC increased during the early phase of injury (days 2-4), and decreased during peak injury (days 6-8). Such correlation was not found during the recovery period (day 15). During recovery, specific and total activities of the enzyme were nearly 2- and 3-fold, respectively, those of controls. Nevertheless, the palmitate proportions in PC were normal, indicating that the increased enzyme activity in vitro was not reflected in increased PC palmitate during recovery. This finding indicates that the enzyme activity per cell was normal during recovery and suggests that the increase in specific and total activities is due to massive regeneration of type II cells and that the enzyme is localized mainly in these cells. The decrease in the proportion of palmitate in lavage PC during peak injury may lead to abnormality of surfactant function. | [
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10,003,850 | Effects of lipid peroxidation on prostaglandin synthesis in rabbit kidney medulla slices. | [
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10,003,851 | Ascorbic acid and Fe2+ had a more powerful stimulatory effect on the lipid peroxidation of rabbit kidney medullar slices in 0.15 M KCl/0.02 M Tris-HCl buffer than in Krebs-Henseleit buffer. The lipid peroxidation induced by ascorbic acid and Fe2+ enhanced the release of unsaturated fatty acids from tissue lipids, but inhibited medullary generation of prostaglandin E. These results suggest that the lipid peroxidation induced by ascorbic acid and Fe2+ may inhibit the formation of prostaglandin E and that this inhibitory effect may be mediated by lipid peroxides via the inhibition of prostaglandin synthetase. | [
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10,003,852 | Increased cholesterol esterification in rat liver microsomes in purified non-specific phospholipid transfer protein. | [
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10,003,853 | The effect of the non-specific phospholipid transfer protein purified from rat liver on the activity of acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26) in rat liver microsomes was studied. The activity of cholesterol acyltransferase was measured from the rate of incorporation of [1-14C] oleoyl-CoA into cholesteryl oleate. Activity was stimulated by preincubation by the microsomes with the non-specific phospholipid transfer protein alone, but most effectively when vesicles consisting of phosphatidylcholine/cholesterol (molar ratio 1:1) also were present in the preincubation mixture. Preincubation with vesicles consisting of only phosphatidylcholine or phosphatidylcholine/phosphatidylethanolamine (molar ratio 1:1) had no effect. The stimulating effect is dependent on transfer protein and vesicle concentration and on the length of preincubation. Treatment of the transfer protein with N-ethylmaleimide abolished its effect on cholesterol ester formation. Preincubation of the microsomes with transfer protein and phosphatidylcholine/cholesterol vesicles containing radioactively labeled cholesterol shows that exogenous cholesterol is converted readily to cholesterol ester. The data are explained by the ability of non-specific phospholipid transfer protein to effect net transfer of cholesterol to those microsomes that contain cholesterol acyltransferase. Enlargement of the cholesterol substrate pool would then give rise to stimulation of the cholesterol acyltransferase activity. This study suggests a role for the transfer protein in modulating cholesterol metabolism by its ability to transport cholesterol between membranes. | [
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10,003,854 | Regulation of mouse hepatic ATP-citrate lyase activity by dietary fat. Evidence for the presence of inactive enzyme. | [
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10,003,855 | The induction of ATP-citrate lyase activity in mouse liver dietary carbohydrate (glucose) in markedly reduced by including in the diet a source of polyunsaturated fatty acids. Within 72 h after changing from a standard mouse chow diet to a high carbohydrate diet containing 15% (W/W) of hydrogenated cottonseed oil (as a source of saturated fatty acids), the activity of mouse liver ATP-citrate lyase per milligram cytosolic protein was approx. 3-fold higher than that from mice fed a similar diet containing 15% (W/W) of corn oil. The rate of synthesis of ATP-citrate lyase relative to that for total protein and the rate of degradation of the enzyme were similar for both dietary groups. Elevated levels of enzyme activity in the hydrogenated cottonseed oil-fed livers were not accompanied by a similar increase in the amount of enzyme protein., To explain such findings, we propose that the activity of hepatic ATP-citrate lyase is regulated by dietary polyunsaturated fatty acids through a mechanism involving the conversion of a catalytically active form of the enzyme to a catalytically inactive form. A reversal of this conversion (inactive-active) is evident within 72 h of removing the mice from the corn oil diet and placing them on the hydrogenated cottonseed oil diet. Furthermore, the conversion appears to be independent of the in vivo rate of synthesis of the enzyme. | [
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10,003,856 | Effects of local anesthetics and related compounds on the endocytosis and catabolism of asialo-glycoproteins in isolated hepatocytes. | [
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10,003,857 | The effects of local anesthetics, including procaine and dibucaine, and some related amine compounds, such as dansyl-cadaverine, were studied with respect to their effects on the uptake and degradation of asialo-glycoproteins in isolated hepatocytes. 0.5 mM of either dibucaine or dansyl-cadaverine reduced the rates of uptake to 18-19% of control value; other amines were less effective. Dibucaine and dansyl-cadaverine both acted by reducing the surface binding capacity of the cells as well as by reducing the rate of internalization of surface-bound asialo-glycoprotein. All of the compounds that affected the uptake, including dansyl-cadaverine, also reduced the rate of degradation. This effect could be studied separately from their effect on uptake. The concentrations that were required in order to reduce degradation were, in general 0.5-0.25 of those which caused a reduction in the uptake. Even though dibucaine, lidocaine and dansyl-cadaverine were found to accumulate in the lysosomes, it was concluded from studies with isopycnic centrifugation in sucrose gradients that all three compounds inhibited the rate of transfer of endocytosed protein from endocytic vesicles to lysosomes. This effect could be due to a reduced rate of fusion between endocytic vesicles and lysosomes. | [
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10,003,858 | Role of glucose metabolism in acid formation by isolated gastric glands. | [
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10,003,859 | The role placed by glucose in providing energy for acid formation was studied in isolated gastric glands from rabbit. The widely-used inhibitors of glycolysis, iodoacetic acid and iodoacetamide were found to inhibit glucose oxidation as well as the indicators of acid formation, respiration and accumulation of aminopyrine. However, the potent inhibitor of acid formation was found to involve a nonspecific mechanism other than the simple inhibition of glycolysis. An alternative approach involved use of the glucose transport inhibitor, phloretin. Phloretin blocked glucose oxidation and also inhibited functional responses. Acid formation was restored easily by the addition of pyruvate or various other oxidizable substrates. Measurement of lactate formation in the absence of exogenous glucose showed that the gastric glands contain very little glycogen. Addition of external glucose resulted in a 10-fold increase in lactate formation and this rate was stimulated further by histamine and rotenone. Rotenone also inhibited both respiration and aminopyrine accumulation; however, the inhibition was not complete. Phloretin treatment resulted in total inhibition of the residual aminopyrine accumulation after rotenone treatment. The results are interpreted to indicate that gastric glands are dependent almost totally on external substrate supply to support acid formation; and, that while anaerobic glucose metabolism can sustain a very low level of acid formation, the major role of glucose is to yield pyruvate equivalents for subsequent oxidation. | [
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10,003,861 | Psoriatic epidermis has a rapid rate of turnover and produces a stratum corneum with an abnormal tonofilament composition. One polypeptide chain, (Mr 70,000) is absent or greatly decreased in relative amount and two other chains, (Mr 63,000 and 55,000), which are normally modified in the living cells, persists into the stratum corneum. Increasing the turnover of normal epidermis has been shown to cause the persistence of 63 and 55 kilodalton chains in the stratum corneum but does not affect the relative amount of 70 kilodalton chain. It has, therefore, been suggested that, in psoriasis, the deficiency of the 70 kilodalton chain may occur prior to or simultaneously with the induction of increased tissue turnover. In the present study, the polypeptide chain composition of living psoriatic epidermis has been examined. It is shown that the relative amounts of 70 kilodalton chain in psoriatic stratum corneum and involved living epidermis from the same site are not significantly different. The abnormality is, therefore, already present in the living cells and it appears that, in psoriasis, the synthesis of the 70 kilodalton chain is defective. The uninvolved epidermis of psoriatics is intermediate between normal and involved psoriatic epidermis both in the ability to synthesise the 70 kilodalton chain and to modify the 63 and 55 kilodalton chains. Comparisons of amino acid compositions of proteins containing different proportions of 70 kilodalton chain suggest that it has a considerably higher content of glycine and serine than the other tonofilament chains. These studies suggest that the 70 kilodalton chain may be functionally different from the other tonofilament chains. The defect in its synthesis in psoriasis is a relatively early event and may be involved with the induction of increased tissue turnover or induced by the same abnormal conditions as the increased tissue turnover. | [
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10,003,862 | Photoaffinity labelling of the serotonin carrier protein in platelets and brain synaptosomes. | [
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10,003,863 | Azidoimipramine, a photoaffinity labelling reagent for the serotonin transport protein, was synthesized. This reagent, upon irradiation, binds covalently to brain synaptosomes preparation and to gel-filtered platelets. Two-dimensional SDS-polyacrylamide gel electrophoresis-isoelectric focusing and tritium fluorography analysis indicate that two synaptosomal proteins and four platelets proteins were labelled by [3H]azidoimipramine. | [
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] |
10,003,864 | Structural organization of the carbohydrate moiety of human transcortin as determined by methylation analysis of the whole glycoprotein. | [
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10,003,865 | Methylation analysis of human transcortin showed that this glycoprotein contains N-glycosidically linked oligosaccharide chains of N-acetyllactosamine type, most of the chains being biantennary and others tri- and/or tetra-antennary. The carbohydrate chains of transcortin are also heterogeneous with respect to the content of fucose and the position of the glycosidic linkages. | [
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10,003,866 | De novo purine synthesis in skeletal muscle. | [
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10,003,867 | Myoblast and primary muscle cultures from rat were found to contain the complete pathway of de novo purine nucleotide synthesis. Quantitative assessment of the pathway in skeletal muscle in mice in vivo, revealed a more intensive purine production in muscle than in liver. Skeletal muscle is thus a major site of de novo purine production in the mammalian body. | [
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10,003,868 | Extracts of bone contain a potent regulator of bone formation. | [
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10,003,869 | We prepared aqueous extracts of whole femorae and tibiae of embryonic chicks. An amount of extract containing 25 microgram of protein resulted in a 500% increase in DNA synthesis in calvarial cell culture, and significant effects were detected with 5 microgram (55%). The time course for stimulation of DNA synthesis showed a peak occurring 16-20 h after addition of the extract. This matrix factor is nondialyzable, and fractionation on a column of Sephadex G-100 indicated a molecular weight of 60-80,000. At the maximum dose used, [3H]proline incorporation into total protein of calvarial cells was increased by 55%, and thus far, all fractions active in promoting DNA synthesis have been found to increase collagen synthesis in cultured chick tibiae. These data are consistent with an effect on osteoblasts as well as bone precursor cells. Extracts prepared from tibiae of 2-day-old chicks, from which the marrow had been removed, also stimulated DNA synthesis (280% increase), thus ruling out the possibility that the factor is a relatively nonspecific nitrogen from the hematopoietic cell line. We conclude that bone matrix contains a substance which could regulate bone formation in vitro by control of mitosis in osteogenic precursors and/or stimulation of osteoblast activity. | [
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10,003,870 | Protein degradation in rat liver. Evidence for populations of protein degradation rates in cellular organelles. | [
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10,003,871 | Protein degradation in liver subcellular and submitochondrial fractions from adult rats has been measured by a double isotope technique. Protein subunits have been resolved by two-dimensional polyacrylamide gel electrophoresis. No consistent relationship between subunit size or isoelectric point and degradation rate has been found in any fraction. Frequency analysis of isotope ratios for protein subunits in subcellular and submitochondrial fractions has been carried out. The analyses demonstrate that degradation rates of resolved protein subunits in organelles or organelle subcompartments can be grouped into populations. Resolved protein subunits in subcellular and submitochondrial fractions can be grouped into one, two or three populations. The results suggest limited heterogeneity of protein degradation rates within each subcellular organelle and support the notion that morphological subcompartment rather than molecular property may be the prime determinant of rates of degradation of individual proteins. | [
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10,003,872 | Protein degradation in mammary gland explants. Evidence for limited heterogeneity of protein degradation rates. | [
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10,003,873 | Mammary gland explants in organ culture were subjected to hormonal manipulation, and rates of protein degradation during 1 and 2 day periods were measured by a double-isotope method. Isotope ratios for protein subunits in subcellular fractions were measured after resolution by two-dimensional polyacrylamide gel electrophoresis. Frequency distribution analysis shows that isotope ratios for each fraction are grouped predominantly in exponential distributions corresponding to populations of protein subunits with different mean degradation rates. The results also suggest that heterogeneity of protein degradation rates within each population is limited. There is no consistent correlation of degradation rate with protein isoelectric point or subunit molecular weight either overall or within any population of degradation rates. Therefore, the similarity of protein degradation rates within each population is clearly related to these molecular properties of the proteins. | [
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10,003,874 | Enzyme and protein turnover in adipose tissue in pregnancy and lactation. | [
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10,003,875 | 1. The relative rates of synthesis of fatty acid synthetase and the pyruvate dehydrogenase complex were measured in adipose tissue in virgin, late pregnant and early lactating rats after injection of L-[2,3-3H]alanine. The relative rate of synthesis of fatty acid synthetase decreased approximately 4-fold between 2 days prepartum and 2 days postpartum. The relative rate of synthesis of the pyruvate dehydrogenase complex did not change. 2. The fractional rate of total adipose tissue protein synthesis was measured by constant infusion with L-[U-14C]tyrosine. Total protein synthesis did not differ in virgin and 2-day lactating rats. The half-life of adipose tissue protein in virgin rats determined by decay of 14C label from protein after injection of NaH14CO3 was 86.9 +/- 6.7 h. This is in close agreement with the half-life (82.5 +/- 20 h) calculated from the fractional rate of protein synthesis determined by the constant infusion method. | [
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10,003,876 | Dowex-1-induced reactions of reduced nicotinamide adenine dinucleotide (NADH). | [
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] |
10,003,877 | Dowex 1-formate has been found to cause both anomerization and oxidation of NADH, and when NADH is chromatographed on a column of this resin, the major products observed are NAD+ and alpha-NAD+. Competing with the oxidation reaction is the conversion of NADH and alpha-NADH to unstable acid-modification products that subsequently break down during chromatography to give ADP-ribose and a variety of neutral and cationic degradation products. The effects of Dowex 1-formate on NADH differ from those of acid as oxidation is minimal when NADH is incubated in acid solution, although anomerization, acid-modification, and degradation to ADP-ribose and other products readily occur. The neural and cationic acid-degradation products that are formed from acid-modified NADH have been resolved by chromatography into 12 compounds, 6 of which react with 3-methyl-2-benzothiazolinone hydrazone and thus are identified as carbonyls. These substances gradually disappear from acid solution over a period of days and are replaced by polymeric pigments. | [
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10,003,878 | Regulation of putrescine metabolism in Ehrlich ascites carcinoma cells exposed to hypotonic medium. | [
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10,003,879 | When exposed to hypotonic growth medium, Ehrlich ascites carcinoma cells showed a rapid stimulation of ornithine decarboxylase (EC 4.1.1.17) activity in 4 h, followed by a rise in their putrescine content. This effect was totally abolished by addition of a slightly hypertonic concentration of sodium chloride or sucrose to the medium. The general protein synthesis was unaffected by the hypotonic treatment. The uptake of putrescine and, to a lesser extent, spermidine was enhanced, the conversion of the radioactive putrescine into spermidine appeared partially inhibited during later stages of the hypotonic treatment. As a result, the half-life of putrescine increased from 2.8 h under isoosmotic conditions to 7.3 h in hypoosmotic medium. Both exogenous ([14C]-putrescine-derived) and endogenous ([14C]ornithine-derived) putrescine degraded at similar rates in control and hypotonic cells, yet the putrescine taken from the medium degraded preferably to nonpolyamine products, while the putrescine synthetized in the cell was converted evenly to spermidine and to other metabolites. Adenosyl-methionine decarboxylase activity (EC 4.1.1.50), which provides the second precursor for spermidine and spermine synthesis, was distinctly inhibited in the hypotonic medium. Inhibition was likewise observed in spermidine synthase activity, while spermine synthase was marginally stimulated. It appears that the hypotonic treatment serves a special condition under which not only the formation of putrescine is enhanced dramatically but the cells also attempt to converse the diamine by preventing its further metabolism to higher polyamines. | [
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10,003,880 | The steroid ligands of estrogen binding proteins in Xenopus laevis liver cytosol are primarily metabolites 17beta-estradiol. | [
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10,003,881 | The interactions in vitro between [3H]estradiol and liver proteins from Xenopus laevis have been examined to determine if the binding reaction meets criteria of steroid-receptors which may function in the induction of vitellogenesis. Estrogenic hormones associated with proteins is serum and liver cytosol from Xenopus laevis. However, the interactions between soluble liver proteins and estrogens apparently do not result from serum contamination of liver as specific binding was distinguishable by ligand affinity and by differential mobility on polyacrylamide gels. Steroid ligands bound by liver proteins during incubation in vitro were examined by solubility and by thin-layer chromatography. Only a small percentage (13%) of the bound radioactive ligand was recovered as the original tritium-labeled steroid, 17beta-estradiol. The major ligand was recovered as a water-soluble metabolite of estradiol which was identified tentatively as an estradiol-glucoside. To investigate whether the protein-bound estradiol metabolite(s) merely masks a small amount of authentic estradiol-receptor complexes or if the metabolite could be an intermediate in estrogen function, isolated liver nuclei were incubated with liver cytosol containing 3H-labeled steroid-protein complexes or with serum protein-steroid complexes relative to [3H]estradiol. Nuclei preferentially accumulated 3H-labeled steroids from liver cytosol protein-steroid complexes relative to [3H[estradiol from serum proteins. However, analysis of the steroids recovered in the nuclei after incubation with liver cytosol revealed that both 17beta-[3H]estradiol and the 3H-labeled water-soluble metabolite were retained in vitro by nuclei.U | [
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10,003,882 | Synthesis of sialoglycoconjugates during dimethylsulfoxide-induced erythrodifferentiation of friend leukemia cells. | [
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] |
10,003,883 | We have previously shown that erythroid differentiation of Friend murine leukemia cells by dimethylsulfoxide results in a decrease in sialic acid content and net negative surface charge. The mechanism responsible for the decrease in sialic acid content was examined by measuring the synthesis of sialic acid from N-acetylmannosamine and its catabolic removal from sialoconjugates during the maturation process. A decrease in the incorporation of N-[3H]acetylmannosamine into sialoglycoconjugates occurred as early as 12 h after exposure to dimethylsulfoxide. Radioactivity incorporated into sialoglycoconjugates was relatively stable in untreated and dimethyl-sulfoxide-treated cells, implying that catabolic removal of sialic acid residues was not a factor in the decreased surface sialic acid content of differentiated erythroleukemia cells. In addition, no difference existed between control and treated cells in sialyltransferase activity. Significant decreases occurred, however, in the incorporation of radioactivity from N-[3H]acetylmannosamine into N-acetylneuraminic acid, CMP-N-acetylneuraminic acid and a material tentatively identified as N-acetylmannosamine-6-phosphate, 48 h after the addition of dimethylsulfoxide. The decrease in sialic acid biosynthesis in differentiated erythroleukemia cells was reflected by an 83% decrease in the amount of radioactively-labeled sialic acid released by neuraminidase treatment of cells exposed to dimethylsulfoxide. These findings are consistent with a cellular aging phenomenon triggered by the polar solvent-induced differentiation of the leukemic cells into more mature forms. | [
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10,003,884 | Calmodulin and acidic compounds alter basic protein phosphorylation by the protein kinase from human platelets. | [
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] |
10,003,885 | A cyclic nucleotide-independent protein kinase of human platelets, which phosphorylated histones, myelin basic protein and protamine and did not catalyze the phosphorylation of acidic proteins such as casein, phosvitin and myosin light chain, has been purified approx. 1,500-fold from the crude extract by steps of DEAE-cellulose, Sephadex G-200, hydroxylapatite and phosphoryl cellulose column chromatography. The substrate phosphorylation by this kinase was markedly enhanced by calmodulin even in the absence of Ca2+, when mixed histone was used as a substrate. The interaction of the kinase with mixed histone resulted in an irreversible inactivation of the enzyme. Calmodulin prevented this inactivation, and this compound produced an apparent increase in histone phosphorylation by the kinase. It should be noted that acidic polypeptides such as troponin-C, phospholipids and nucleic acids have a similar ability. The addition of Ca2+ reduced the effect of calmodulin more than the effects of other acidic compounds. | [
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10,003,886 | Selective appearance of heparin in mammalian tissues during development. | [
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10,003,887 | The distribution of heparin in tissues of bovine and porcine fetuses of different ages as well as in the adult animals is reported. Heparin is present almost exclusively in the hematopoietic and lymphoid tissues of fetuses (spleen, liver, thymus and lymph nodes), whereas ileum, skin and lung besides lymph nodes and bovine thymus are the heparin-rich tissues in adult animals. The small amount of heparin in fetal tissues, its decrease in porcine liver and spleen, its appearance in skin, ileum and lung, tissues directly exposed to the environment after birth, its preferential localization at the maternal side of the placenta together with other data led to the suggestion that heparin may be involved with defense mechanisms. | [
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10,003,888 | Plasma membrane-associated component(s) that confer(s) cholera toxin sensitivity to adenylate cyclase. | [
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10,003,889 | Incubation of crude normal rat kidney membranes with activated cholera toxin in the presence of DTP, ATP and NAD results in a 10--20 fold stimulation of adenylate cyclase activity. Optimal choleragen activation f the cyclase is shown to be dependent upon the presence of a plasma membrane-associated reconstituting activity, which can be dissociated from the membranes by washing with 10 mM potassium phosphate buffer, pH 7.5, containing 5 mM EDTA and 0.1 mM dithiothreitol. choleragen-catalyzed ADP ribosylation of plasma membrane substrate proteins also requires the presence of reconstituting activity factor. Sephadex G-150 filtration of solubilized reconstituting activity shows a peak activity eluting in the region corresponding to a protein with a molecular weight of approx. 13,000. Reconstituting activity is eluted from DEAE-cellulose at a salt concentration of 40--100 mM KCl. This active factor is not destroyed by trypsin treatment or by boiling for 30 min. These observations indicate that an endogenous membrane-associated factor, along with GTP, may be involved in modulating the ability of choleragen to activate adenylate cyclase. | [
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] |
10,003,890 | Ornithine oxoacid aminotransferase found in AH 130 ascites hepatoma cells. | [
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] |
10,003,891 | We found heterogenous ornithine oxoacid aminotransferase (L-ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.1.3) in rat ascites hepatoma AH 130 cells. Compared with enzymes from normal rat tissues, this heterogenous enzyme showed larger Km values for 2-oxoglutarate, a different elution-profile upon affinity chromatography with 2-oxoglutarate, more anionic mobility upon polyacrylamide gell electrophoresis, and a clearly different salting-out property upon ammonium sulfate fractionation. Similar heterogeneity of this aminotransferase was found in human cancer cells. | [
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10,003,892 | High intralysosomal levels of lipoprotein cholesterol influence the endocytic activity and acid hydrolase content of CH-23 fibroblasts. | [
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] |
10,003,893 | Lipoprotein extracts isolated from hyperlipemic rabbit serum were used to study the effects of abnormal lipid levels on the functions of the lysosomal system in Chinese hamster CH-23 fibroblasts. The ability of cells enriched with lipid to endocystose [3H]inulin was largely unimpaired and utilising density gradient fractionation procedures the fusion between incoming inulin and low density lysosomes ladened with esterified cholesterol could be demonstrated. Studies in which cells were exposed to short 'bursts' of [3H]inulin indicated, however, that the rate of fusion between inulin and secondary lysosomes was reduced. The incorporation of lipid material also produced a rapid though transient increase in several acid hydrolase activities. The stimulus for increased enzyme activity does not appear to be the deposition of ingested lipid within the lysosomes but rather at some stage prior to this, possibly the formation of endocytic vesicles. The current findings suggest that the intralysosomal incorporation of lipid material which occurs in several pathological conditions has marked effects on lysosomal function. | [
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10,003,894 | The interactions of tropolone with magnesium ions and tubulin. | [
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10,003,895 | Tropolone, a single analog of colchicine, interacts with Mg2 with the formation of a 1 : 1 complex and an apparent equilibrium binding constant Kb of 1.4 x 10(4) M(-1) in neutral aqueous solution at 25 degrees C. The tropolone-Mg2 complex, but not tropolone, is fluorescent. Since tubulin binds Mg2 (Frigon, R.P. and Timasheff, S.N. (1975) Biochemistry 14, 4567-4573), previous reports of tropolone interaction with tubulin in Mg2 -containing buffers must be critically re-examined. Fluorescence and difference absorption spectroscopy experiments performed at essentially constant Mg2 activity indicate that tubulin does bind tropolone, but the optical effects are too weak to use in quantitative studies. | [
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10,003,896 | 19F nuclear magnetic resonance studies of the coat protein of bacteriophage M13 in synthetic phospholipid vesicles and deoxycholate micelles. | [
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10,003,897 | The nonlytic, filamentous coliphage M13 offers an excellent model system for the study of membrane-protein interactions. We prepare derivatives of the protein containing fluorine-labeled amino acids and use 19F nuclear magnetic resonance (NMR) to study the protein in both deoxycholate micelles and phospholipid vesicles. We have previously described the in vivo preparation of an m-fluorotyrosyl derivative of M13 coat protein and also a method for incorporation of high levels of this protein into small, uniformly sized phospholipid vesicles of defined composition. Herein we describe the in vivo preparation and the characterization of an m-fluorophenylalanine derivative. We simultaneously compare the environment and mobility of the tyrosine and phenylalanine residues (the former in the hydrophobic region of the protein and the latter in the hydrophilic regions) as influenced by bile salt detergent or lipid interactions. | [
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] |
10,003,898 | Bilayer acyl chain dynamics and lipid-protein interaction: the effect of the M13 bacteriophage coat protein on the decay of the fluorescence anisotropy of parinaric acid. | [
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10,003,899 | Nanosecond fluorescence polarization anisotropy decay is used to determine the effect of the bacteriophage M13 coat protein on lipid bilayer acyl chain dynamics and order. The fluorescent acyl chain analogues cis- and trans-parinaric acid were used to determine the rate and extent of the angular motion of acyl chains in liquid crystalline (39 degrees C) dimyristoylphosphatidylcholine bilayers free of coat protein or containing the coat protein at a protein:lipid ratio of 1:30. Subnanosecond time resolution was obtained by using synchrotron radiation as the excitation source for single photon counting detection. Previous measurements of Förster energy transfer from coat protein tryptophan to cis- or trans-parinaric acid have shown that these probes are randomly distributed in the bilayer with respect to the protein. The anisotropy decay observed for pure bilayers has the form of a rapid drop, followed by a nonzero constant region extending from roughly 3 ns to at least 12 ns. The magnitude of the anisotropy in the plateau region is simply related to the acyl chain order parameter. The effect of the M13 coat protein is to increase the acyl chain order parameter significantly while having only a small effect on the rate of angular relaxation. This behavior is rationalized in terms of a simple microscopic model. The order parameters for pure lipid and coat protein containing bilayers are compared to 2H-NMR values. | [
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