PubMed Health⌕ Search

Biomedical subjects

A J Rivett

Publications and source records attributed to A J Rivett.

76 records · Page 5Linked to original sources
PubMed1981

Purification of the high-Km aldehyde reductase from rat brain and liver and from ox brain.

A procedure is described that yields an apparently homogeneous preparation of the high-Km aldehyde reductase from rat brain. This procedure is also applicable to the purification of this enzyme from rat liver and ox brain. In the latter case, however, the purified preparation could be resolved into two protein bands, both of which had enzyme activity, by polyacrylamide-gel electrophoresis. Since a sample of the ox brain enzyme from an earlier step in the purification procedure only showed the presence of a single band of activity after electrophoresis, this apparent multiplicity probably results from modification of the enzyme, possibly by oxidation, during the final step of the purification. A number of properties of the rat brain enzyme were determined and these were compared with those of the enzyme from rat liver. The two preparations were similar in their stabilities, behaviour during purification, kinetic properties, electrophoretic mobilities and amino acid compositions. Antibodies to the rat liver enzyme cross-reacted with that from brain and the inhibition of both these preparations by the antiserum was similar, further supporting the view that the enzymes from these two sources were closely similar if not identical.

Alcohol Oxidoreductases↗
PubMed1981

Kinetic studies with rat-brain succinic-semialdehyde dehydrogenase.

A simple procedure is described that gives an approximately 100-fold purification of rat brain succinic-semialdehyde dehydrogenase with a high yield. The enzyme exhibits a relatively low Km value for succinic semialdehyde (2.5 microM) and is inhibited by high concentrations of that substrate in an uncompetitive manner with respect to NAD+ (Ki = 150 microM). p-Hydroxybenzaldehyde was shown to give competitive inhibition with respect to succinic semialdehyde and uncompetitive inhibition with respect to NAD+. Initial rate studies in the presence of a fixed concentration of this inhibitor allowed a more accurate estimation of the kinetic parameters for the uninhibited reaction. The results of these studies, together with analysis of the dead-end inhibition by AMP and the effects of NAD+ and 3-acetylpyridine--adenine dinucleotide as alternative acceptors in the reaction, were consistent with the enzyme-catalysed reaction obeying a compulsory-order mechanism in which NAD+ was the first substrate to bind to the enzyme and NADH was the last product to dissociate from it.

Aldehyde Oxidoreductases↗
PubMedDate not supplied

Regulation of proteasome complexes by gamma-interferon and phosphorylation.

Proteasomes play a major role in non-lysosomal proteolysis and also in the processing of proteins for presentation by the MHC class I pathway. In animal cells they exist in several distinct molecular forms which contribute to the different functions. 26S proteasomes contain the core 20S proteasome together with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the ends of the 20S proteasome to form PA28-proteasome complexes and PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We have found differences in the subcellular distribution of the different forms of proteasomes. The gamma-interferon inducible PA28 alpha and beta subunits are predominantly located in the cytoplasm, while 19S regulatory complexes (present at significant levels only in 26S complexes) are present in the nucleus as well as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic reticulum (ER) where they may facilitate the generation of peptides for transport into the lumen of the ER. We have also investigated the effects of gamma-interferon on the levels and subcellular distribution of inducible subunits and regulator subunits. In each case gamma-interferon was found to increase the level but not to alter the distribution. Several subunits of proteasomes are phosphorylated including alpha subunits C8 (alpha7) and C9 (alpha3), and ATPase subunit S4 (rpt2). Our studies have shown that gamma-interferon treatment decreases the level of phosphorylation of proteasomes. We have investigated the role of phosphorylation of C8 by casein kinase II by site directed mutagenesis. The results demonstrate that phosphorylation at either one of the two sites is essential for the association of 19S regulatory complexes and that the ability to undergo phosphorylation at both sites gives the most efficient incorporation of C8 into the 26S proteasome.

Animals↗