Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have characterized 11 porcine liver cytosolic glutathione S-transferase (GST) subunits from their precise molecular mass, immunoreactivity and partial amino acid sequence. Four Alpha-, six Mu- and one unexpected Pi-class GST subunits were found with average molecular masses of 24.984-25.228 kDa, 25.039-25.657 kDa and 23.510 kDa respectively. Molecular masses were established using electrospray-ionization mass spectrometry, with a precision of +/- 3-4 mass units. Glutathione (GSH) and S-hexylglutathione (ShGSH) were tested as affinity ligands in the purification procedure. The binding selectivity of GSH was better than that of ShGSH, although non-GST proteins were retained on both matrices. As already described in other studies, a number of non-GST proteins bound to the affinity resins. Two of them were tentatively identified as mevalonate kinase and carbonyl reductase. The characterization of pig liver cytosolic GST subunits pattern achieved in this work should constitute a useful tool for rapid evaluation of these enzymes' expression in modulation studies.
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PMID:Characterization of pig liver glutathione S-transferases using HPLC-electrospray-ionization mass spectrometry. 876 Mar 77

Sequencing of polymerase chain reaction-amplified cDNAs from cultured cells of three patients with mevalonate kinase deficiency revealed a G --> A transversion at nucleotide 1000 of the coding region, converting alanine to threonine at position 334 (A334T). To characterize this defect, we expressed wild-type and mutant cDNAs in Escherichia coli as the glutathione S-transferase fusion proteins, with purification by affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis for wild-type and mutant fusion proteins indicated an expected molecular mass of 42-43 kDa. Kinetic characterization of the wild-type fusion protein yielded Km values of 150 +/- 23 and 440 +/- 190 microM (mean +/- S.E.) for substrates (RS)-mevalonate and ATP, respectively. Expressed wild-type mevalonate kinase (MKase) had a maximum velocity of 13.6 +/- 1.4 units/mg of protein (n = 22, +/-S.E.), whereas the A334T mutation yielded an enzyme with average Vmax of 0.26 +/- 0.02 unit/mg of protein (n = 6, +/-S.E.), representing a decrease to 1.4% of control Vmax. Restriction digestion with HhaI, in conjunction with direct sequencing of cDNAs, revealed that two patients were homozygous and one heterozygous for the A334T allele, establishing autosomal recessive inheritance within families. Although the A334T enzyme had a normal Km for ATP of 680 +/- 226 microM (n = 3, +/-S.E.), the Michaelis constant for (RS)-mevalonate was increased >30-fold to 4623 +/- 1167 microM (n = 4, +/-S.E.) under standard assay conditions. Comparable kinetic results were obtained using extracts of lymphoblasts, which were homozygous for the A334T allele. Alanine 334 is invariant in MKase from bacteria to man and located in a glycine-rich region postulated to have homology with ATP-binding sequences. Our results indicate that the bacterial expression system for human MKase will provide a useful model system in which to analyze inherited mutations and identify the first active site residue in MKase associated with stabilization of mevalonate binding.
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PMID:Identification of an active site alanine in mevalonate kinase through characterization of a novel mutation in mevalonate kinase deficiency. 933 62