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C N Oliver

Publications and source records attributed to C N Oliver.

27 records · Page 2Linked to original sources
PubMed1985

Covalent modification of proteins by mixed-function oxidation: recognition by intracellular proteases.

Mixed-function oxidation of E. coli glutamine synthetase is a site-specific reaction involving covalent modification of specific amino acid residues. It causes loss of a specific histidine residue which is thought to be at one of the metal binding sites. The modified enzyme is catalytically inactive. Oxidative modification causes enhanced susceptibility to proteolytic attack and several different types of proteases recognize the oxidatively modified enzyme. This specific covalent modification increases the rate of degradation of glutamine synthetase to about the same extent as major structural modifications such as relaxation, subunit dissociation and denaturation. Moreover, the oxidative modification is one that is likely to occur in vivo. Adenylylation, which causes reversible inactivation of glutamine synthetase, has no effect on rate of proteolysis. We propose that the degradation of E. coli glutamine synthetase occurs by a two-step process. Control of the degradation is likely to be at the first step, which is inactivation by mixed-function oxidation of the enzyme. Metabolic control of the degradation process and the link with the nutritional state of the cell could be achieved by substrate protection against oxidative modification. At present, the apparent energy requirement of the degradation process is unexplained. Although most of our studies have involved E. coli glutamine synthetase, there is evidence that oxidative modification may be a general mechanism by which proteins are marked for degradation. Many enzymes are inactivated by oxidative modification which can be catalyzed by a variety of mixed-function oxidase systems. Several different types of intracellular proteases in E. coli and mammalian cells preferentially degrade the oxidized form of glutamine synthetase. Oxidative modification of proteins can occur in vivo and may be involved in intracellular protein turnover. It has also been implicated in host defense mechanisms and in aging.

Amino Acid Sequence↗
PubMed1984

Silica-stimulated monocytes release fibroblast proliferation factors identical to interleukin 1. A potential role for interleukin 1 in the pathogenesis of silicosis.

Previous study strongly suggests that silicotic fibrosis is mediated by macrophages and their soluble mediators. The biochemical properties of the mediators involved in silicotic fibrosis, however, are as yet ill defined. The current study, therefore, determined whether human monocyte-macrophages treated with fibrogenic silica dust released factors capable of activating fibroblasts as measured by an increase in fibroblast proliferation. Silica, but not nonfibrogenic diamond dust, stimulated the release of fibroblast proliferation factors. Moreover, the level of fibroblast proliferation activity was comparable with the level of thymocyte proliferation (interleukin-1) activity in the same culture supernatants. The factors responsible for these seemingly diverse activities were found to behave identically when analyzed by gel filtration chromatography, size exclusion chromatography, isoelectrofocusing, ion exchange chromatography, and hydrophobic chromatography. Moreover, the response of these factors to four different proteases and heat (56 degrees C) was also identical, which shows that their comigration on various separation media could not be explained by noncovalent interaction between otherwise unrelated species. The data demonstrate that a monocyte-derived thymocyte proliferation factor having the molecular properties of interleukin 1 is capable of regulating fibroblast proliferation. In silicosis and other fibrotic diseases, the local release of interleukin 1 may contribute to abnormal connective tissue deposition by stimulating fibroblast proliferation, and thereby, amplifying other signals stimulating the synthesis of connective tissue components.

Cells, Cultured↗
PubMed1983

Inactivation of key metabolic enzymes by mixed-function oxidation reactions: possible implication in protein turnover and ageing.

Several mixed-function oxidation systems catalyze the inactivation of Escherichia coli glutamine synthetase. Inactivation involves modification of a single histidine residue in each enzyme subunit and makes the enzyme susceptible to proteolytic degradation. We show here that 10 key enzymes in metabolism are inactivated by a bacterial NADH oxidase and by an oxidase system comprised of NADPH, cytochrome P-450 reductase, and cytochrome P-450 isozyme 2 from rabbit liver microsomes. Most of the inactivatable enzymes require a divalent cation for activity and all but one (enolase) possess a nucleotide binding site. Glutamine synthetase, pyruvate kinase, and phosphoglycerate kinase are protected from inactivation by their substrates; substrate protection of other enzymes was not tested. We propose that inactivation involves mixed-function oxidization system-catalyzed synthesis of H(2)O(2) and reduction of Fe(III) to Fe(II) followed by oxidation of enzyme-bound Fe(II) by H(2)O(2) to generate oxygen radicals that attack a histidine (or other oxidizable amino acid) at the metal binding site of the enzyme. This is supported by the following: (i) most of the inactivation reactions are inhibited by EDTA and by catalase, (ii) both mixed-function oxidation systems reduce Fe(III), and (iii) H(2)O(2) together with Fe(II) catalyzes nonenzymic inactivation of glutamine synthetase. In view of the fact that inactivation of glutamine synthetase makes it susceptible to proteolytic degradation, it is possible that mixed-function oxidation system-catalyzed inactivation of enzymes is a regulatory step in enzyme turn-over. In addition, the implication of oxidative inactivation reactions in ageing is suggested by the fact that many of the enzymes inactivated by mixed-function oxidation systems are known to accumulate as inactive forms during ageing.

Aging↗
PubMed1983

A proteolytic artifact associated with the lysis of bacteria by egg white lysozyme.

Polyacrylamide gel electrophoresis of cell-free extracts of Escherichia coli that had been grown in a medium containing 32Pi disclosed the presence of several 32P-labeled proteins. Comparison of the electrophoretic patterns obtained in the presence of carrier unlabeled purified E. coli glutamine synthetase before and after treatment with trypsin, subtilisin, or snake venom phosphodiesterase showed that most of the 32P was present in the adenylyl moieties of adenylylated glutamine synthetase. Low molecular weight 32P-labeled degradation products of glutamine synthetase were also observed in extracts prepared by treatment of cells with lysozyme but not in extracts prepared by sonic oscillation. The degradation of glutamine synthetase in lysozyme-prepared extracts is likely due to an intrinsic proteolytic activity of egg white lysozyme. Proteolysis probably occurs at the esterase site of lysozyme described by Piszkiewicz and Bruice [Piszkiewicz, D. & Bruice, T.C. (1968) Biochemistry 7, 3037-3047]. Selective carboxymethylation of lysozyme histidine-15 leads to simultaneous loss of esterase and protease activities but only to partial loss of lytic activity. In view of these findings, caution is needed in the interpretation of results obtained with extracts of cells prepared by lysozyme treatment, especially when such extracts are used to investigate the properties of proteolytic enzymes.

Animals↗
PubMed1981

Turnover of bacterial glutamine synthetase: oxidative inactivation precedes proteolysis.

We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover.

Adenosine Triphosphate↗
PubMedDate not supplied

Optimization of poly(ethylene glycol) precipitation of hepatitis A virus used to prepare VAQTA, a highly purified inactivated vaccine.

Poly(ethylene glycol) precipitation has been successfully used to concentrate and purify hepatitis A virus from crude lysate preparations for production of VAQTA, a highly purified, formalin-inactivated hepatitis A vaccine. Initial results showed that nucleic acids present in the starting material were problematic for the performance of the poly(ethylene glycol) precipitation step. Extensive experiments were carried out to identify processing conditions suitable for vaccine manufacture which would enhance product yield and improve purity. Results of these studies indicated that the earlier practice of concentrating crude virus-containing lysate using semipermeable membranes led to aggregation of high molecular weight nucleic acids. This aggregated material coprecipitated with the virus during the subsequent poly(ethylene glycol) precipitation step; variable amounts of nucleic acids led to inconsistent virus recovery and product purity. Nuclease treatment of the crude lysate preparations decreased the molecular size of the nucleic acids and significantly reduced their coprecipitation with the virus. Further experiments demonstrated that optimal placement of the nuclease treatment was at the lysate stage followed by a capture step using anion exchange chromatography. These steps combined with optimization of the virus concentration, ionic strength, and pH of the poly(ethylene glycol) precipitation led to effective and selective concentration of the virus which significantly enhanced process reproducibility and control.

Chemical Precipitation↗