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Biomedical subjects

R B Freedman

Publications and source records attributed to R B Freedman.

At least 127 records · Page 7Linked to original sources
PubMed1985

The latency of rat liver microsomal protein disulphide-isomerase.

Protein disulphide-isomerase (PDI) activity was not detectable in freshly prepared rat liver microsomes (microsomal fraction), but became detectable after treatments that damage membrane integrity, e.g. sonication, detergent treatment or freezing and thawing. Maximum activity was detectable after sonication. Identical latency was observed in microsomes prepared by gel filtration and in those prepared by high-speed centrifugation. PDI activity was latent in all particulate subcellular fractions, but not latent in the high-speed supernatant. When all fractions were sonicated to expose total PDI activity, PDI was found at highest specific activity in the microsomal fraction and co-distributed with marker enzymes of the endoplasmic reticulum. Washing of microsomes under various conditions that removed peripheral proteins and, in some cases, bound ribosomes did not remove significant quantities of PDI, nor did it affect the latency of PDI activity. Treatment of microsomes with proteinases, under conditions where the permeability barrier of the microsomal vesicles was maintained intact, did not inactivate PDI significantly or affect its latency. PDI was very readily solubilized from microsomal vesicles by low concentrations of detergents, which removed only a fraction of the total microsomal protein. In all these respects, PDI resembled nucleoside diphosphatase, a marker peripheral protein of the luminal surface of the endoplasmic reticulum, and differed from NADPH: cytochrome c reductase, a marker integral protein exposed at the cytoplasmic surface of the membrane. The data are compatible with a model in which PDI is loosely associated with the luminal surface of the endoplasmic reticulum, a location consistent with the proposed physiological role of the enzyme as catalyst of formation of native disulphide bonds in nascent and newly synthesized secretory proteins.

Animals↗
PubMed1985

Dechlorination of halocarbons by microsomes and vesicular reconstituted cytochrome P-450 systems under reductive conditions.

A spectrophotometric assay of the reductive dechlorination of halocarbons was developed and used to determine the kinetic characteristics of dechlorination of a range of haloethanes catalysed by microsomes from rat and rabbit liver. Analysis of the typical reaction of hexachloroethane shows that the reaction is catalysed by cytochrome P-450 and results in the formation of olefinic products as well as less chlorinated alkanes: in other respects the reaction resembles that known to occur with carbon tetrachloride. The dechlorination of haloethanes catalysed by a vesicular reconstituted system of cytochrome P-450 enzymes from rabbit liver was also studied and found to be similar to that catalysed by microsomes: both reductase and a phenobarbital inducible form of cytochrome P-450 were essential. There is no substantial dependence of maximum dechlorination rate on compound structure, suggesting that the reduction of substrate is not the rate limiting step in the overall reaction. The main factor in determining the apparent binding constant to the enzyme is the partition coefficient into the lipid membrane, as assessed by calculated log P values.

Anaerobiosis↗
PubMed1984

Protein disulphide-isomerase of chick-embryo tendon.

Protein disulphide-isomerase can be partially purified from the high-speed-supernatant fraction of extensively disrupted chick-embryo tendon tissue. The catalytic properties of the preparation resemble those of the enzyme from mammalian liver. Gel electrophoresis and isoelectric focusing show the enzyme to be very acidic, with pI 4.4 +/- 0.3. Gel filtration indicates an Mr for the active enzyme of 140 000. The enzyme can be partially purified by preparative gel filtration or isoelectric focusing, but its limited stability has prevented purification to homogeneity; active fractions from both gel filtration and isoelectric focusing show two major polypeptide components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The major polypeptides present in partially purified preparations have Mr 45 000 and 55 000; the latter band co-distributes with the enzyme activity in fractionations by both gel filtration and isoelectric focusing. The subcellular location of the enzyme cannot be established from work on homogenates of whole tissue, which are extensively disrupted. In homogenates from isolated tendon cells, the enzyme is located in a vesicle fraction that is excluded from Sepharose 2B but is of low density and can only be sedimented at very high speeds. This fraction is identified as deriving from the endoplasmic reticulum on the grounds of marker-enzyme studies and electron microscopy.

Animals↗
PubMed1984

Catalysis by protein disulphide-isomerase of the assembly of trimeric procollagen from procollagen polypeptide chains.

Type-I procollagen, 14C-biosynthetically labelled, was reduced under denaturing and non-denaturing conditions. Reoxidation to disulphide-linked trimers occurred with non-denatured chains in the presence of an oxidant system containing oxidized and reduced glutathione. Dimeric intermediates were not detected. This reoxidation was accelerated by homogeneous beef liver protein disulphide-isomerase.

Animals↗
PubMed1983

Two-dimensional electrophoresis of membrane proteins. Factors affecting resolution of rat-liver microsomal proteins.

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI greater than 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112-120) gives good resolution of proteins of pI 5-8, including cytochromes P-450, but fails to resolve a major acidic protein of pI less than 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R . N+(CH3)2 . CH2CH2CH2SO3- are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C14 or C16) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.

Animals↗
PubMed1983

Immunological identity between bovine preparations of thiol:protein-disulphide oxidoreductase, glutathione-insulin transhydrogenase and protein-disulphide isomerase.

Five preparations of bovine thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase (EC 1.8.4.2) and one preparation of bovine liver protein-disulphide isomerase (EC 5.3.4.1) from four different laboratories showed immunological identity in double immunodiffusion and rocket-line immunoelectrophoresis. Consequently, thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase and protein-disulphide isomerase, formerly classified as two separate enzymes, should be considered as alternative activities of the same enzyme.

Animals↗
PubMed1983

Characterisation of hepatocyte sub-populations generated by centrifugal elutriation.

We describe protocols for the fractionation of isolated hepatocytes into eight sub-populations using centrifugal elutriation. The distribution of fluorescein isothiocyanate and acridine orange in hepatocytes prepared from livers pre-perfused with one of these dyes is described and used as an indicator of acinar zone derivation for each population. The cytochrome P-450 content and response to induction by 3-methylcholanthrene and phenobarbitone; the distribution of lactate dehydrogenase, glucose-6-phosphatase, pyruvate kinase and tyrosine aminotransferase activities in the sub-populations is also reported. A marked asymmetry of distribution in all these activities was observed. On the basis of putative zone derivations (based on data of fluorescent dye distribution) of eight factors studied, the distributions of six were consistent with the sub-populations being derived from different acinar zones. Two major discrepancies were noted however, the distribution of pyruvate kinase activity and the response of the sub-populations to phenobarbitone. We conclude from this study that while a metabolic heterogeneity was revealed in the sub-populations generated, further characterisation is required to determine whether acinar zone separation has occurred and if so to what extent.

Acridine Orange↗
PubMed1983

Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure.

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

Amino Acids↗
PubMed1983

Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction.

The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of 'scrambled' ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources, that have been referred to as protein disulphide-isomerase, disulphide-interchange enzyme, thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase are identical or homologous proteins. The assay, nomenclature and physiological role of this enzyme are discussed.

Animals↗
PubMed1983

Identification of protein disulphide-isomerase as a major acidic polypeptide in rat liver microsomal membranes.

Protein disulphide-isomerase was purified to homogeneity from rat liver by a rapid high-yielding procedure. Structural properties of the pure enzyme were very similar to those of the bovine liver enzyme purified by the same method. The purified rat liver enzyme was subjected to two-dimensional gel electrophoresis in the presence and in the absence of microsomal membranes, and shown to co-electrophorese with a major acidic polypeptide clearly identifiable in the two-dimensional electrophoretic profile of microsomal membranes. This identification was confirmed by peptide 'mapping' of the pure enzyme and of the defined spot from a two-dimensional electrophoresis gel.

Animals↗
PubMed1982

Alterations in the enzyme activity and polypeptide composition of rat hepatic endoplasmic reticulum during acute exposure to 2-acetylaminofluorene.

Studies have been made of the morphology, enzyme activity and protein composition of liver endoplasmic reticulum in rats exposed to acute doses of the carcinogen, 2-acetylaminofluorene (2-AAF). Electron microscopic examination revealed numerous ultrastructural changes in the hepatocyte; most consistent alterations were the disorganisation of endoplasmic reticulum system with apparent increase of smooth endoplasmic reticulum. Administration of 2-AAF to rats immediately depressed microsomal glucose-6-phosphatase activity and eventually induced epoxide hydratase activity 6--7-fold over control activity. The induction was time-dependent and maximal rates of induction were observed at dosages greater than 40 mg/kg body wt. The treatment also induced cytochrome b5 content, NADH and NADPH cytochrome c reductase activities (1.0--1.5-fold). Only very small changes in the total content of cytochrome P-45- were noted. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of microsomal proteins from 2-AAF pretreated animals showed time-dependent induction of two polypeptides which differed slightly in migration, in the region of Mr = 48000; the fast-migrating induced polypeptide has been identified as epoxide hydratase. Two-dimensional PAGE analysis of microsomal proteins from 2-AAF exposed rats showed a reproducible deletion of a protein with molecular weight in the region of 67000. The basis for the alterations in the protein composition of endoplasmic reticulum in response to 2-AAF treatment is discussed.

2-Acetylaminofluorene↗
PubMed1981

Specific labelling of microsomal proteins by reactive intermediates generated from 2-acetylaminofluorene in vitro.

Incubation of 2-[9-14C]acetylaminofluorene (2-[9-14C]AAF) in vitro with rat liver microsomes, leads to covalent binding of label to microsomal proteins. The binding is NADPH-dependent, increases linearly with time, and is inhibited by SKF-525A and 7,8-benzoflavone (7,8-BF). Binding is increased more than 8-fold in microsomes from 3-methylcholanthrene(MC)-pretreated rats, but only less than 2-fold in those from phenobarbital(PB)-pretreated rats. In the presence of cytosolic proteins, there is slight enhancement of the labelling of microsomes and some labelling of the cytosolic proteins. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis indicate that covalent labelling by 2-AAF derivatives is concentrated in specific proteins. The pattern of labelling varies between microsomes from animals pretreated with PB, MC and 2-AAF. Factors which may contribute to the specificity of labelling are discussed.

2-Acetylaminofluorene↗