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Biomedical subjects

R B Freedman

Publications and source records attributed to R B Freedman.

At least 109 records · Page 6Linked to original sources
PubMed1990

Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase.

The role of protein disulphide isomerase (PDI) and other resident proteins of the endoplasmic reticulum (ER) lumen in co- and post-translational modification of secretory proteins has been studied in experiments on translation in vitro. We have devised procedures for extracting the lumenal content proteins of dog pancreas microsomal vesicles by alkaline buffer, or detergent washing, and for reconstitution of the depleted membrane fraction. When microsomal membranes are depleted of content by washing at pH 9.1, they are able to co-translationally glycosylate human interferon-gamma (IFN-gamma) and yeast pro-alpha-factor and the products appear to be identical to those produced by control microsomes. However, when microsomal membranes are depleted of content by washing with saponin they are still able to co-translationally translocate and glycosylate human IFN-gamma, but the products were of higher apparent Mr than those generated by control microsomes. When saponin-washed microsomal membranes were reconstituted with homogeneous protein disulphide isomerase (PDI), the generated vesicles gave the same pattern of co-translationally glycosylated IFN-gamma as saponin-washed microsomal membranes lacking PDI. These results are discussed in relation to the roles of resident ER proteins in co-translational modification; they suggest that PDI is not an essential component of the machinery of co-translational N-glycosylation, but that detergent washing may inactivate or remove some ER glycosidases.

Animals↗
PubMed1989

Conformational differences between two wheat (Triticum aestivum) 'high-molecular-weight' glutenin subunits are due to a short region containing six amino acid differences.

'High-molecular-weight' (HMW, high-Mr) glutenin subunits are protein constituents of wheat (Triticum aestivum) seeds and are responsible in part for the viscoelasticity of the dough used to make bread. Two subunits, numbered 10 and 12, are the products of allelic genes. Their amino acid sequences have been derived from the nucleic acid sequences of the respective genes. Subunit 10 has fewer amino acids than subunit 12, but migrates more slowly on SDS/PAGE (polyacrylamide-gel electrophoresis). This anomaly is due to between one and six of the amino acid differences between the subunits, localized towards the C-terminal end of the proteins. This has been established by making chimaeric genes between the genes for subunits 10 and 12, transcribing and translating them in vitro and analysing the products by SDS/PAGE. The postulated conformational differences between subunits 10 and 12 are discussed in relation to current hypotheses for the structure of HMW glutenin subunits.

Amino Acid Sequence↗
PubMed1989

Kinetics of bilirubin oxidation catalysed by bilirubin oxidase in a water-in-oil microemulsion system.

1. Bilirubin oxidase can catalyse the oxidation of its primary substrate, bilirubin, in a water-in-oil microemulsion, which consists of discrete nanometer-diameter water droplets dispersed in a continuous water-immiscible oil medium. The droplets are stabilized by a monolayer of the surfactant, cetyltrimethylammonium bromide present at the oil/water interface. 2. Spectroscopic evidence is presented to show that bilirubin solubilized in this system is located mainly in the surfactant layer, in a form accessible to the enzyme molecule. 3. Studies are presented on the enzyme-catalysed rate of bilirubin oxidation in this system, as a function of temperature, pH, water content, and substrate and enzyme concentrations. 4. The main conclusions are that the enzyme can efficiently oxidise bilirubin in microemulsions of low water content. The reaction obeys Michaelis-Menten kinetics. The optimal pH for the catalysis is 8.0. The efficiency of catalysis decreases sharply as the water content increases.

Bilirubin↗
PubMed1989

Analysis of the inactivation of liver alcohol dehydrogenase during storage in Aerosol-OT/isooctane microemulsions.

Changes in the enzymatic properties of horse liver alcohol dehydrogenase (HLADH; EC 1.1.1.1) were studied as a function of incubation time in Aerosol-OT/isooctane microemulsions. The enzyme was characterized by fluorimetric binding studies of the inhibitor isobutyramide to the binary complex, HLADH-NADH and by determination of Km,app and Vmax,app values for cyclohexanone. The Km,app values for cyclohexanone and the Kd,app for isobutyramide stay constant throughout a 48-h incubation, whereas the Vmax,app and the total number of inhibitor binding sites decrease. Thus the inactivation process previously described corresponds to progressive loss of functional sites, while the properties of the remaining functional sites are unchanged. If no co-enzyme is added to the system, the enzyme loses catalytic activity within less than an hour, but if co-enzyme is added, a fraction of the HLADH enzyme population retains enzyme activity over a long period of time. Hence the presence of bound co-enzyme significantly inhibits the process(es) leading to inactivation of the enzyme in the microemulsions.

Aerosols↗
PubMed1989

Rat liver type I iodothyronine deiodinase is not identical to protein disulfide isomerase.

This study was done to test the recent hypothesis (Boado et al. (1988) Biochem. Biophys. Res. Commun. 155, 1297-1304) that type I iodothyronine deiodinase (ID-I) is identical to protein disulfide isomerase (PDI). Autoradiograms of rat liver microsomal proteins, labeled with N-bromoacetyl-[125I]triiodothyronine (BrAc[125I]T3) and separated by SDS-PAGE, show predominantly 2 radioactive bands of Mr 27 and 56 kDa. Substrates and inhibitors of ID-I inhibited labeling of the 27 kDa band but not that of the 56 kDa band. Treatment of microsomes with trypsin abolished labeling of the 27 kDa protein and destroyed the activity of ID-I but did not prevent labeling of the 56 kDa protein. Following treatment of microsomes at pH 8.0-9.5 or with 0.05% deoxycholate (DOC) PDI content and labeling of the 56 kDa protein were strongly diminished but ID-I activity and labeling of the 27 kDa protein were not affected. The latter decreased in parallel after treatment at pH greater than or equal to 10. Rat pancreas microsomes contain high amounts of PDI but show no ID-I activity. Reaction of these microsomes with BrAc[125I]T3 results in extensive labeling of a 56 kDa protein but no labeling of a 27 kDa protein. Pure PDI (Mr 56 kDa) was readily labeled by BrAc[125I]T3 but showed no deiodinase activity. These results strongly suggest that the 27 kDa band represents (a subunit of) ID-I while the 56 kDa band represents PDI. From these and other data it is concluded that PDI and ID-I are not identical proteins.

Affinity Labels↗
PubMed1989

Preparation and characterization of dog pancreas microsomal membranes specifically depleted of protein disulphide-isomerase.

1. The selective release of protein disulphide-isomerase from dog pancreas and rat liver microsomal membranes was studied to throw light on the mechanisms of retention of this enzyme within the endoplasmic reticulum, and in order to prepare microsomal membranes specifically depleted of the enzyme. 2. Protein disulphide-isomerase was quantitatively released from dog pancreas microsomal membranes by washing at pH 9 and above, as demonstrated both by enzyme assay and by immunoblotting analysis. 3. Integral membrane proteins implicated in the process of translocation and segregation of secretory proteins were retained in pH 9-washed dog pancreas microsomal membranes. 4. After pH 9 washing, dog pancreas microsomal membranes were fully active in the translocation, segregation and processing of nascent secretory proteins; these membranes therefore provide a useful experimental system for testing the action of protein disulphide-isomerase on nascent secretory proteins. 5. Protein disulphide-isomerase was not released from rat liver microsomal membranes by pH 9 washing, and was much less readily released from these membranes by sonication, washing etc. than from dog pancreas microsomal membranes. 6. The mechanism of retention of protein disulphide-isomerase, and of other resident proteins of the lumen of the endoplasmic reticulum, is discussed in the light of these findings.

Animals↗
PubMed1989

Induction of expression of protein disulphide-isomerase during lymphocyte maturation stimulated by bacterial lipopolysaccharide.

Protein disulphide-isomerase (PDI) activity, and the level of immunodetectable PDI protein, were monitored in splenic lymphocytes and in BCL1 cells during culture in the presence of various activating factors. Bacterial lipopolysaccharide stimulated induction of PDI in splenic B cells and BCL1 cells. The time-course and specificity of induction indicated that the increase in expression of PDI is closely coupled to the final stages of B cell differentiation into antibody-producing plasma cells. The system will prove valuable in studies on the control of expression of PDI.

Animals↗
PubMed1989

Structure and dynamics of staphylococcal nuclease mutants as studied by fluorescence quenching techniques.

Fluorescence techniques have been used to investigate the effect of mutations on the structure and dynamics of staphylococcal nuclease. An estimate of the accessibility to acrylamide of the enzyme's single tryptophan residue (Trp140) was obtained from the Stern-Volmer constant for fluorescence quenching. This was indicative of a partially buried tryptophan in the wild-type nuclease. Five single-site mutant nucleases (H124L, V66L, G88V, G79S and F76V) and one double mutant (V66L + G88V), with widely differing stabilities to denaturants, gave Stern-Volmer constants which were very similar to that of their parent enzyme. Studies of the temperature- and viscosity-dependence of quenching suggest that access by acrylamide to Trp140 is limited by diffusion rather than by protein structural fluctuations. Lifetime-resolved fluorescence anisotropy studies using steady-state instrumentation suggest that there is very little segmental motion of the Trp140; most of the anisotropy therefore decays due to protein rotation in the solution. Rotational correlation times for several nuclease mutants have been determined and these are very similar to that of the native nuclease. Thus it appears that these substitutions in the primary amino acid sequence, which have significant effects on the stability of the folded proteins, do not cause a significant change in the protein structure or dynamics around Trp140.

Escherichia coli↗
PubMed1989

Role of protein disulphide-isomerase in the expression of native proteins.

Formation of native disulphide bonds is a post-translational modification associated with the folding and assembly of secretory proteins. The process is catalysed within the lumen of the endoplasmic reticulum by the enzyme protein disulphide-isomerase (PDI), which is abundant in secretory cells and catalyses thiol: protein-disulphide interchange in vitro with very broad protein substrate specificity. The presence of PDI within microsomal vesicles is essential for efficient and rapid cotranslational disulphide bond formation during protein synthesis in vitro. The sequence of PDI is now known from several species, and shows the presence of two domains closely homologous to thioredoxins. Chemical modification data confirm the role of the thioredoxin domains in thiol:disulphide interchange activity, and structural models of these domains can be built based on homology with thioredoxin. Thus PDI is both strongly implicated in the process of native protein folding in vivo and well characterized at the molecular level. In addition to its disulphide-isomerase activity, PDI participates as a component of the enzyme prolyl-4-hydroxylase, and further functions have also been proposed.

Amino Acid Sequence↗
PubMed1988

Defective co-translational formation of disulphide bonds in protein disulphide-isomerase-deficient microsomes.

The formation of disulphide bonds in mammalian secretory and cell-surface proteins occurs in the lumen of the endoplasmic reticulum and is believed to be catalysed by the enzyme protein disulphide-isomerase (PDI). The evidence for this physiological role for PDI is circumstantial and relates to the cell and tissue distribution of the enzyme, its developmental behaviour and its catalytic properties in vitro. A clear requirement for PDI in the correct folding or assembly of disulphide-bonded proteins during biosynthesis has not been demonstrated. We have prepared dog pancreas microsomes which are deficient in soluble lumenal proteins, including PDI, but which are still able to translocate and process proteins synthesized in vitro. Using the formation of intramolecular disulphide bonds during the in vitro synthesis of gamma-gliadin, a wheat storage protein, as a model, we have demonstrated that these microsomes are defective in co-translational formation of disulphide bonds. Reconstitution of these microsomes with purified PDI reverses this defect.

Animals↗
PubMed1988

The transcription and translation in vitro of individual cereal storage-protein genes from wheat (Triticum aestivum, cv. Chinese Spring). Evidence for translocation of the translation products and disulphide-bond formation.

Genes coding for the high-Mr ['high-molecular-weight' (HMW)] glutenin subunit 12 and for a gamma-gliadin from wheat (Triticum aestivum, cv. Chinese Spring) were subcloned into transcription-translation vectors. In each case transcription in vitro yielded a RNA transcript which when added to a rabbit reticulocyte cell-free translation system directed the synthesis of a polypeptide of appropriate Mr by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE). When dog pancreatic microsomal vesicles were added to the translation system, translocation of the newly synthesized polypeptides occurred, as judged by protection from proteolysis. When translation and translocation of the gamma-gliadin was carried out under conditions favouring the formation of disulphide bonds, a polypeptide was synthesized which had a faster mobility on SDS/PAGE carried out under non-reducing conditions than under reducing conditions. This suggests that the processed and translocated gamma-gliadin forms an intramolecular disulphide bond or bonds during synthesis in vitro.

Cell-Free System↗
PubMed1987

Biosynthesis and degradation of collagen in X-irradiated mouse lung.

Fibrosis, characterized by accumulation of collagen, is a delayed result of radiation injury in many tissues, including lung. To investigate its development, synthesis and degradation of collagen were measured in lungs of mice after X irradiation of the whole thorax. The ratio of type I (coarse fibered) to type III (meshwork) collagen was also determined. Synthesis of procollagen, measured as the activities of prolyl-4-hydroxylase and protein disulfide isomerase in lung tissue, was increased at 2 months after X-ray doses of 5, 7.5, and 9 Gy. Maximal increases were observed 6 to 7 months after doses of 9 Gy and persisted up to 15 months after exposure. Increases after 5 and 7.5 Gy were more gradual, but by 1 year after irradiation they had reached levels similar to those after 9 Gy. X irradiation had no effect on the degradation of collagen as assessed by collagenase activity in lung. The ratio of type I to type III collagen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of collagen-derived cyanogen bromide peptides, was the same in irradiated lungs as in age-matched controls. Therefore, increased synthesis of procollagen, rather than decreased degradation of collagen or changes in collagen type, is an important factor in the accumulation of collagen in irradiated lung.

Animals↗