Article: Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing.
1990 Volume 270, Issue 2, Page(s) 483–489
Abstract: Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l ... class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c ... before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms ...
| Abstract | Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l.c.; five major proteins were isolated and identified as subunits 1, 2, 3, 4 and 8. F.p.l.c. chromatofocusing resolved the affinity-purified GST pool into nine different isoenzymes. The five basic (Alpha class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c. analysis revealed that subunit 8 was also present in the protein peaks designated 1-1, 1-2a and 1-2b. The four neutral (Mu class) isoenzymes were 3-3, 3-4, 3-6 and 4-4. The GST pool was methylated in vitro before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms (3-3, 3-4 and 4-4) were the primary GSTs methylated, and h.p.l.c. analysis confirmed that subunits 3 and 4 were the major methyl-accepting GST subunits. The addition of calmodulin stimulated the methylation in vitro of GST isoenzymes 3-3, 3-4 and 4-4 by 3.0-, 7.5- and 9.9-fold respectively. Reverse-phase h.p.l.c. also indicated that only the methylation of GST subunits 3 and 4 was stimulated by calmodulin. Basic GST isoenzymes were minimally methylated and the methylation was not enhanced by calmodulin. Investigation of the time course of methylation of GST subunits 3 and 4 indicated that at incubation times less than 4 h the methylation of both Mu class subunits was stimulated by calmodulin, and that under such conditions subunit 4 was the preferred substrate. In contrast, there was essentially no calmodulin-stimulated methylation at incubation times of 4 or 6 h, and the methylation of subunit 3 was predominant. Kinetic parameters at 2 h of incubation were determined in the presence and in the absence of calmodulin. The addition of calmodulin doubled the Vmax. for methylation of both subunits 3 and 4 and decreased the Km of subunit 4 for S-adenosyl-L-methionine 3.6-fold. Finally, methylation was substoichiometric and after 6 h of incubation ranged from 2.8 to 7.6% on a mole-to-mole basis for subunits 4 and 3 respectively. |
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| MeSH term(s) | Animals ; Calmodulin/pharmacology ; Chromatography, High Pressure Liquid ; Cytosol/enzymology ; Glutathione Transferase/isolation & purification ; Glutathione Transferase/metabolism ; Isoenzymes/isolation & purification ; Isoenzymes/metabolism ; Kinetics ; Liver/enzymology ; Male ; Methylation ; Rats ; Rats, Inbred Strains |
| Chemical Substances | Calmodulin ; Isoenzymes ; Glutathione Transferase (EC 2.5.1.18) |
| Language | English |
| Publishing date | 1990-09-01 |
| Publishing country | England |
| Document type | Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S. |
| ZDB-ID | 2969-5 |
| ISSN | 1470-8728 ; 0264-6021 ; 0006-2936 ; 0306-3275 |
| ISSN (online) | 1470-8728 |
| ISSN | 0264-6021 ; 0006-2936 ; 0306-3275 |
| DOI | 10.1042/bj2700483 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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