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Soluble and Particulate Forms of Rat Catechol‐ O ‐Methyltransferase Distinguished by Gel Electrophoresis and Immune Fixation
Author(s) -
Grossman Mark H.,
Creveling C. R.,
Rybczynski Robert,
Braverman Muriel,
Isersky Chaviva,
Breakefield Xandra O.
Publication year - 1985
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1985.tb05432.x
Subject(s) - isoelectric focusing , gel electrophoresis , polyacrylamide gel electrophoresis , biochemistry , molecular mass , catechol o methyl transferase , chemistry , antiserum , electrophoresis , blot , biology , chromatography , microbiology and biotechnology , enzyme , antibody , gene , allele , immunology
Catechol‐ O ‐methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDSPAGE) of all tissue homogenates examined revealed three major immune‐specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 × g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an “aggregate” of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two‐dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pl of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1–5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is “soluble” with an apparent M r of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.