PubMed Health⌕ Search

Biomedical subjects

J W Porter

Publications and source records attributed to J W Porter.

At least 37 records · Page 2Linked to original sources
PubMed1982

Liberation, purification, and properties of thioesterase component of pigeon liver fatty acid synthetase complex.

Proteolysis of pigeon liver fatty acid synthetase with elastase results in the quantitative cleavage of the thioesterase component from the enzyme complex. This thioesterase component is two or three times more active catalytically in the isolated state than in the native fatty acid synthetase, and its activity is not affected by the presence or absence of reducing thiols. The proteolytically cleaved thioesterase is separated from the core enzyme in one step by size-exclusion chromatography on a Sephadex G-75 column. The peptide obtained by gel permeation is homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. Size-exclusion chromatography on Bio-Gel A 0.5 m and Sephadex G-75 columns, sucrose density gradient ultracentrifugation, and N-terminal amino acid analysis also indicate that the proteolytically cleaved thioesterase is homogeneous. The sedimentation coefficient of the thioesterase is approximately 2.9 S. Proteolytic cleavage with elastase also quantitatively releases the [1,3-14C]- or [1,3-3H]diisopropylphosphofluoridate-labeled thioesterase component from the correspondingly labeled fatty acid synthetase. Binding studies with 14C- or 3H-labelled diisopropylphosphofluoridate and fatty acid synthetase show that 2 mol of the label are bound per mol of the enzyme when complete loss of fatty acid-synthesizing activity occurs. The molecular weight of the thioesterase component is estimated to be 36000 by size-exclusion chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis.

Amino Acids↗
PubMed1982

Isolation and characterization of an acyl carrier protein from pigeon liver fatty acid synthetase by controlled proteolysis with elastase.

Controlled proteolytic cleavage of 4'-phospho[14C]pantetheine-labeled pigeon liver fatty acid synthetase generates two 4'-phospho[14C]pantetheine-labeled peptides, Ec1 and Ec2. These are separated from each other and the core enzyme by gel permeation chromatography on a Sephadex G-75 column. The two radioactively labeled peptides constitute 50% of the radioactivity initially present in the 4'-phospho[14C]pantetheine-labeled fatty acid synthetase. The remaining label in the core enzyme is released quantitatively by proteolytic cleavage with trypsin. The molecular weights of Ec1 and Ec2 peptides, as determined by size exclusion chromatography and SDS-polyacrylamide gel electrophoresis, are 12000 and 6000, respectively. Both the higher and lower molecular weight peptides are homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. The higher molecular weight peptide, Ec1, is characterized as an acyl carrier protein by the transacylation reaction between the unlabeled Ec1 peptide and radioactively labeled acetyl- and malonyl-CoA. Since Ec2 peptide also contains the prosthetic group present in the Ec1 peptide, the Ec2 peptide appears to result from the proteolytic cleavage of the higher molecular weight peptide, Ec1. Amino acid composition of the acyl carrier protein shows the presence of 1 mol of 4'-phosphopantetheine per mol of protein. 2 mol of acyl carrier protein are present per mol of the fatty acid synthetase. The amino acid analysis is in good agreement with the molecular weight of the Ec1 peptide, as determined by gel filtration and SDS-polyacrylamide gel electrophoresis. N-Terminal amino acid analysis of this peptide shows the presence of an arginine residue.

Acyl Carrier Protein↗
PubMed1982

An improved purification and further characterization of the messenger ribonucleic acid for the fatty acid synthetase from rat liver.

High purity fatty acid synthetase mRNA has been prepared from rat liver. The translational purity of the mRNA preparation was at least 27% as judged by the percentage of the radioactivity incorporated into acid-insoluble material that was precipitated by anti-fatty acid synthetase antibody. The specific activity of the mRNA was 220-times greater than that reported previously from this laboratory [1]. The large increase in the specific activity was achieved by the repeated use of high resolution linear-log sucrose density gradient centrifugation and the removal of 28 S rRNA by Sepharose 4B chromatography, as well as by the optimization of the K+ concentration (160 mM) in the reticulocyte lysate translation system. The mRNA preparation showed a single major band on agarose gel electrophoresis under denaturing conditions, and the translational activity of the fatty acid synthetase mRNA on the gel was found to coincide with this band. The molecular weight of the fatty acid synthetase mRNA is 2.5.10(6) Da. The mRNA directed the synthesis of fatty acid synthetase with a molecular weight indistinguishable from that of the authentic enzyme subunit (Mr = 240 000). The copurification of the translation product and authentic enzyme revealed that the fatty acid synthetase polypeptides synthesized in the reticulocyte lysate system are assembled in vitro into dimers, the native form of the enzyme.

Animals↗
PubMed1982

Limited elastase digestion of pigeon liver fatty acid synthetase with retention of all partial enzyme activities.

Pigeon liver fatty acid synthetase which contains two subunits of 240,000 daltons each has been treated with elastase. This treatment yields four protein fragments which can be separated on sodium dodecyl sulfate (SDS)-gel electrophoresis. After the subunit protein has been treated with elastase, all of the partial enzyme activities catalyzed by the complex are present, but enzyme activity for fatty acid synthesis is lost. The formation of protein fragments during proteolysis has been followed by densitometric scanning of the SDS gels. The results of these scans have suggested that (a) there are two peptide components present in the highest molecular weight band, (b) both are rapidly digested to yield the second and third largest peptides, and (c) a further cleavage of the third largest peptide gives rise to the smallest of the four major peptides. Crossed-rocket immunoelectrophoretic analysis of the four protein fragments has confirmed these conclusions and established also that the three smallest peptides are homogeneous. Each of the four peptides has been isolated by preparative SDS-gel electrophoresis, and antibody to one has been prepared. This antibody fraction immunotitrates overall fatty acid synthetase activity and immunoprecipitates the native enzyme. Immunoelectrophoresis of the four elastase-digested synthetase products against this antibody showed some cross-reactivity with a peptide that was neither the precursor nor the product of the immunogen. This cross-reacting antibody was removed by reaction with the nonrelated protein to yield antibody specific for one region of the fatty acid synthetase complex.

Animals↗
PubMed1982

Perturbation and change in coral reef communities.

Ninety-six percent of surveyed shallow-water Dry Tortugas reef corals died during the severe winter of 1976-1977. Data from skeletal stains indicate that death occurred during the mid-January intrusion of 14 degrees C water onto the reef. In deeper water, community parameters such as percent cover, species number, and relative abundance showed no significant change. However, an analysis of competitive interactions at the growing edges of adjacent colonies reveals a 70% reduction in space competition during this environmental disturbance. These results can explain high variability in the growth rate of Floridian reefs and demonstrate the importance of obtaining long-term spatial information to interpret successional dynamics of complex communities.

Journal Article↗
PubMed1982

Regulation of short-term changes in hepatic beta-hydroxy-beta-methylglutaryl-CoA reductase activity.

Immunotitrations of rat liver hydroxymethylglutaryl-CoA (HOMeGlt-CoA) reductase activity were performed before and after short-term changes in the nutritional or hormonal state of the animals. Changes in enzyme activity (increase or decrease) within 1 h following cholesterol feeding or glucagon or mevalonolactone administration to normal rats, or insulin administration to diabetic rats were accompanied by no change in the specific activity of the enzyme, as determined from the quantity of enzyme activity inactivated by a fixed quantity of antibody. These results support the conclusion that the loss in enzyme activity was due to conversion of the enzyme to immuno-unreactive products. In agreement with this conclusion the enzyme activity lost after these short-term physiological changes was not restorable by phosphoprotein phosphatase action. On the other hand, incubation of rat liver microsomes with ATP and Mg2+ decreased the specific activity of HOMeGlt-CoA reductase about tenfold, as determined by immunotitration. The low specific activity produced under these conditions was increased by phosphatase action to nearly the original level. The above evidence suggests that the changes in HOMeGlt-CoA reductase activity that resulted from short-term physiological changes in hormonal or nutritional states of an animal were brought about by a change in the quantity of enzyme, and not by reversible phosphorylation of pre-existing enzyme.

Animals↗
PubMed1981

Demonstration of the occurrence of inactive fatty acid synthetase in rat liver by immunotitration and its in vitro partial activation.

Direct immunotitrations of rat liver fatty acid synthetase in crude tissue homogenates with monospecific rabbit anti-rat liver fatty acid synthetase antibody enabled us to make a comparison of fatty acid synthetase protein and activity (percentage of maximal activity) as a function of the nutritional state in normal, diabetic, and insulin- and glucagon-insulin treated animals. Previous results, in which large changes in fatty acid synthetase activity were related to protein synthesis and degradation rather than to enzyme activation, were confirmed. It was also shown that fatty acid synthetase activation does not occur immediately on synthesis but follows the synthesis of fatty acid synthetase protein. In order to characterize the enzymatically inactive protein found on immunotitration and to develop an in vitro system for fatty acid synthetase activation, conditions were sought to obtain large amounts of fatty acid synthetase protein free from, or low in, activity. It was found that treatment of hypophysectomized rats with triiodothyronine meets these requirements, yielding milligram quantities of inactive fatty acid synthetase protein with less than 2% of maximal activity. A part of the inactive fatty acid synthetase was found to be the apoenzyme as indicated by beta-ketoreductase and thioesterase activities, by its ability to incorporate label from [G3H]CoA, and by its partial in vitro activation, which led to an increase in overall synthetase activity in crude and partially purified cell-free systems. The components required for activation include magnesium ion and a transferase fraction prepared from livers of 48-h fasted, 12-h refed rats.

Animals↗
PubMed1981

pH properties and chemical mechanism of action of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The pH variation of the kinetic parameters V and V/K for the oxidation of mevaldate by NADP+ in the presence of CoA (reverse reaction) and for the reduction of mevaldate by NADPH in the presence or absence of CoA (forward reaction) for the reactions catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was examined. In the reverse reaction a group, X, on the enzyme with a pK of 7.9 must be unprotonated for NADP+ binding and catalysis. The presence of NADP+ shifts this pK to a value below 6. The V/K profile for mevaldate shows that deprotonation of a group, Y, with a pK of 6.7 decreased the reaction rate by a factor of 27. In the forward reaction, the pK of the X group was about 6.9 except when CoA and mevaldate were both present, in which case it was shifted to 7.8. CoA decreased the Kms for mevaldate about 10-fold without changing the Vmax at the optimum protonation state. The catalytic group, X, was identified as a cationic acid, probably histidine. A catalytic mechanism is proposed in which the protonated form of histidine induces hydride transfer from the A side of NADPH by donating a proton to the carbonyl of HMG-CoA or to the aldehyde form of mevaldate. The role of the Y group, which from its pK of 6.7 and the chemistry involved may be a carboxyl group, is presumably to catalyze conversion of mevaldate thiohemiacetal formed in the reduction of HMG-CoA to CoA and the free aldehyde form of mevaldate. Mevaldate was shown by 1H NMR to contain 44% hydrate in D2O and 39% in H2O. When an enzymatic method was used, it was also determined that only one stereoisomer of mevaldate is used by HMG-CoA reductase.

Buffers↗
PubMed1981

The influence of dietary unsaturated cis and trans and saturated fatty acids on tissue lipids of swine.

A feeding trial was conducted to evaluate the effects of dietary trans unsaturated fatty acids (trans fat) and of the interplay of dietary saturated fatty acids (saturated fat), cis unsaturated fatty acids, (cis fat) and trans fat on tissue lipids, particularly those effects suggestive of angiotoxicity. Swine were fed for 10 months a diet containing 17% added fat. Seven blends of varying proportions of the 3 fat components provided sufficient sample points to permit an examination of the interplay. Parameters under study included weight gain, serum cholesterol and triglyceride concentrations, lipoprotein lipid profile, total lipid and cholesterol concentrations of liver, heart and aorta, fatty acid composition of liver and aorta lipids and hepatic fatty acid synthesis and cholesterol synthesis and oxidation. Fat blends containing disproportionately high levels of saturated or cis fat generally elicited responses consistent with results reported by others. The notable exception was the serum cholesterol concentration. Throughout the study, the swine were hypercholesterolemic. Swine fed the high saturated fat blend had serum cholesterol levels equal to those swine fed the high cis fat blend. Serum cholesterol levels in the swine fed the other fat blends were more elevated. Another apparent anomaly was the lower concentration of lipid in the aortas of swine fed the high-saturated fat diet. The impact of the trans fat was modulated by the relative proportions of saturated and cis fat in the diet. The impact of trans fat was of greater magnitude for most parameters when the fat blend was low in saturated fat. The sole parameter suggestive of trans fat-mediated angiotoxicity was the distribution of lipids in lipoprotein fractions. Swine fed diets containing trans fat had lower relative proportions of the alpha-lipoprotein lipids. Although hypercholesterolemic, the high fat diets were not overtly angiotoxic except when fed to swine that carried a specific immunogenetically-defined low density lipoprotein.

Acetyl-CoA Carboxylase↗