Isozyme-specific monoclonal antibodies of CaM-stimulated phosphatase: identification of an enzyme domain involved in metal ion activation.
Bovine brain and lung extracts each contain three forms of CaM-stimulated phosphatase, designated BPI, BPII, and BPIII, and LPI, LPII, and LPIII, respectively. The different forms show uniform immunoreactivity toward one alpha subunit-specific monoclonal antibody, VD3, but differential reactivity toward another antibody, VJ6. Using the procedure of limited clostripain digestion, the binding sites for mAb VD3 and mAb VJ6 have been localized to the carboxylterminal region of about 40 amino acid residues and to the carboxylterminal one-third of the alpha subunit, respectively. The result has substantiated our previous suggestion that the isolated multiple forms of the brain and lung phosphatases represent isozymes rather than in vitro proteolysis artifacts. The effects of the two monoclonal antibodies on p-nitrophenylphosphatase activities of the bovine brain phosphatase isozymes have been examined. BPI and BPIII, the two VJ6 reactive brain isozymes, can be inhibited by VJ6 antibody in a metal ion and CaM-dependent manner; none of the isozymes are inhibited by mAb VD3. The inhibition of BPI by mAb VJ6 is most pronounced when the assay is carried out in the presence of CaM and Ni2+, whereas little or no effect is seen if Mg2+ plus Ca2+ are used as metal ion activators. Thus, VJ6 appears to identify an alpha-subunit domain important for the Ni(2+)-induced activation of CaM-stimulated phosphatase.
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
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Yokoyama
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Z
Ali
J H
JH
Wang
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002118:Calcium / Q000494:pharmacology*; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000276:immunology*; D002413:Cations, Divalent; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015151:Immunoblotting; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000276:immunology*; D008168:Lung / Q000201:enzymology; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009532:Nickel / Q000494:pharmacology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000276:immunology*
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D002413:Cations, Divalent; D007527:Isoenzymes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D009532:Nickel; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D008274:Magnesium; D002118:Calcium