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N J Bulleid

Publications and source records attributed to N J Bulleid.

48 records · Page 3Linked 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

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↗
PubMed1988

Induction of microsomal epoxide hydrolase by nitrosamines in rat liver. Effect on messenger ribonucleic acids.

Nitrosomethylethylamine and nitrosomethylpropylamine were found to be more potent inducers of rat liver microsomal epoxide hydrolase (styrene oxide hydrolase) than nitrosodiethylamine or nitrosodimethylamine. The time course of induction following a single administration of nitrosodimethylethylamine, nitrosomethylpropylamine or nitrosodiethylamine each showed a delay of 24 hr during which enzyme activity was unaltered. After that time activity increased and reached a maximum at between 72 and 120 hr. Increased enzyme activity following NDEA was paralleled by changes in the content of epoxide hydrolase in microsomes as measured by Western blots. Nitrosamines caused an increase of mRNA for epoxide hydrolase which was detected by probing Northern blots with a [32]-P labelled epoxide hydrolase cDNA and by in vitro translation of polyadenylated mRNA. Both methods showed a maximal increase at 72 hr after nitrosodiethylamine treatment but a significant increase was also observed at 24 hr although at this time no increase in enzyme activity was apparent.

Animals↗
PubMed1986

Microsomal epoxide hydrolase of rat liver. Purification and characterization of enzyme fractions with different chromatographic characteristics.

Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified enzyme, which varied in their contents of phospholipid, were obtained by ion-exchange chromatography. One fraction (A), which did not bind to CM-cellulose, had a high phospholipid content, and a second fraction (B), which was eluted from CM-cellulose at high ionic strength, had a low phospholipid content. Removal of most of the phospholipid from fraction A altered its chromatographic behaviour. When the delipidated material was re-applied to CM-cellulose, most of the enzyme bound to the cation-exchanger. The specific activities of all the fractions described (with styrene epoxide [(1,2-epoxyethyl)benzene] as substrate) were altered by adding the non-ionic detergent Lubrol PX or phospholipid. Lubrol PX inhibited enzyme activity, and phospholipid reversed this inhibition. The various enzyme fractions isolated appeared to be different forms of the same protein, as judged by their minimum Mr values and immunochemical properties. These results indicate that different fractions of epoxide hydrolase isolated by ion-exchange chromatography probably are not different isoenzyme forms.

Animals↗
PubMed1985

Alterations in the metabolism of 7,12-dimethylbenz[a]anthracene and various xenobiotics by rat hepatic microsomes following Sudan III treatment in vivo.

Sudan III treatment of Long-Evans rats results in increased hepatic monooxygenase activity using ethoxycoumarin and aniline as substrates. Monooxygenase activity towards amino-pyrine and nitrosodimethylamine is not affected. Sudan III treatment results in increased microsomal cytochrome P448 and increased amounts of a protein band which comigrates with purified cytochrome P448 during SDS polyacrylamide gel electrophoresis. The proportions of the different dihydrodiols formed during the incubation of 7,12-dimethylbenz[a]anthracene with microsomes vary between untreated and treated animals. Thus, extracts of microsomes from untreated rats were found to contain materials with chromatographic properties identical to those of the 3,4-dihydrodiol and the 5,6-dihydrodiol when examined on two different h.p.l.c. systems. Extracts of microsomes from Sudan III treated animals were found to contain materials with chromatographic properties identical to those of the 5,6-dihydrodiol and the 8,9-dihydrodiol when similarly examined. These findings suggest that the protective effect of Sudan III against DMBA induced leukaemia is mediated by an alteration in monooxygenase activity.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1984

Effects of metyrapone and norharmane on microsomal mono-oxygenase and epoxide hydrolase activities.

This study was undertaken to examine the possibility that metyrapone and norharmane stimulate epoxide hydrolase and inhibit mono-oxygenase activities by binding to a cytochrome P-450 component of a stable complex containing the two enzymes. The concentration of metyrapone and norharmane which inhibited mono-oxygenase activities of hepatic microsomes from untreated and diethylnitrosamine treated rats was lower than that required to stimulate epoxide hydrolase of the same microsomes. The ability of metyrapone and norharmane to stimulate epoxide hydrolase in these microsomes was not inhibited by the addition of carbon monoxide and reductant. Epoxide hydrolase activity was inhibited by detergents but the enzyme was still stimulated by metyrapone and norharmane under conditions of total membrane disaggregation. When microsomes were solubilized, epoxide hydrolase could be quantitatively recovered by immunoprecipitation. The immunoprecipitate contained no detectable cytochrome P-450 but was stimulated by metyrapone and norharmane. A purified epoxide hydrolase was stimulated by metyrapone but not by norharmane. The response of the enzyme to norharmane was not restored by the inclusion of cytochrome P-448. These findings suggest that metyrapone and norharmane act at separate sites on both cytochrome P-450 and epoxide hydrolase.

Alkaloids↗
PubMedDate not supplied

Protein-specific chaperones: the role of hsp47 begins to gel.

Collagen biosynthesis involves a complex series of post-translational modifications, controlled by a number of general and specific molecular chaperones. A recent study has shed new light on the role played in this process by the procollagen-specific chaperone Hsp47.

Animals↗
PubMedDate not supplied

Studies on the interaction and exchange of inhibitors and proteases on the surface of tumour cells in frozen sections.

We have used frozen sections of squamous cell carcinoma as a convenient source of a cell surface protease associated with tumour cells. This protease has been referred to as guanidinobenzoatase (GB) and is now known to be functionally identical to tissue plasminogen activator (t-PA). The use of a fluorescent competitive inhibitor of GB enabled the enzymic status of GB to be determined, i.e. was the enzyme active, latent or removed from our test system. The cell surface GB was then demonstrated to interact with extractable cytoplasmic inhibitors obtained from these sections. We then used a protected form of the GB in the absence of these internal inhibitors; such sections were used to transfer the GB to fibrin fibrils, thus exposing the presumptive receptor on the tumour cell surfaces. Texas red labelled t-PA was then shown to bind to the tumour cells in these pretreated sections from which the GB had previously been removed. We believe that the surface of tumour cells can be used to study the interaction of the naturally occurring inhibitors with GB and also that the cell surface receptors for GB can be used to study the binding of t-PA to cell surfaces.

Aminacrine↗