Demonstration of calmodulin-stimulated phosphatase isozymes by monoclonal antibodies.
A procedure combining immunoprecipitation and immunotransblot employing subunit-specific monoclonal antibodies of the brain phosphatase, VJ6 and VA1, was used on tissues including heart, muscle, lung, spleen, pancreas, uterus, and liver. The various tissue extracts were subjected to immunoprecipitation by the beta subunit-specific VA1-immunoabsorbant, the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunotransblot, using both the alpha and beta subunit-specific antibodies VJ6 and VA1, respectively. Protein bands corresponding to alpha and beta subunits and the immunostain of beta subunit were detected in all samples, whereas alpha subunit was strongly stained only in the brain extract, weakly in heart and muscle extracts, and essentially negatively in all the other samples. In contrast, a polyclonal antiserum of bovine brain calmodulin-stimulated phosphatase could immunostain both alpha and beta subunits from all tissues. Calmodulin-binding protein fractions from a number of bovine tissues were all shown to contain the immunoprecipitable alpha subunit, as well as calmodulin-stimulated p-nitrophenylphosphatase activity. Micropeptide mapping showed that alpha subunits of bovine brain and bovine lung calmodulin-stimulated phosphatase isozymes were distinct molecular species. These results provide direct evidences for the existence of calmodulin-stimulated phosphatase isozymes in mammalian tissues.
Yokoyama
N
N
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Furuyama
S
S
Wang
J H
JH
D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000032:analysis*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000032:analysis*; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D010179:Pancreas / Q000201:enzymology; D010749:Phosphoprotein Phosphatases / Q000032:analysis*; D051381:Rats; D013154:Spleen / Q000201:enzymology; D014018:Tissue Distribution; D014599:Uterus / Q000201:enzymology
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D007527:Isoenzymes; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases