Ca2+/calmodulin-dependent protein phosphatase in bovine parotid gland: purification and characterization.
Calmodulin-dependent protein phosphatase (CaM-PPase) was isolated from bovine parotid gland by sequential application of DEAE-52, Affi-gel blue and calmodulin-affinity chromatography followed by gel filtration and high performance liquid chromatography. The enzyme was activated in the simultaneous presence of Ni2+ or Mn2+ and Ca2+ plus calmodulin. Ca2+/calmodulin-dependent activation of CaM-PPase was antagonized by inhibitors of calmodulin action, such as W-7 and trifluoperazine. Tryptophan fluorescence was quenched in the presence of Ni2+. CaM-PPase was a heterodimer. The molecular weights of large subunits which bound calmodulin (CaM) were 68 kD and 58 kD - the 68 kD subunit was predominant. Polyclonal antibodies against bovine calcineurin cross-reacted with both types of larger subunits. Using polyclonal antibodies against bovine calcineurin or the monoclonal antibody against subunit B of bovine calcineurin, the smaller molecular weight subunit (19 kD) was found to be immunologically identical to subunit B of bovine calcineurin. In bovine parotid gland, CaM-PPase was found both in acinar and duct cells.
Yokoyama
N
N
Department of Physiology, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Ozaki
I
I
Yamamoto
H
H
Furuyama
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D019703:Calcineurin; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007124:Immunoenzyme Techniques; D009532:Nickel / Q000494:pharmacology; D010306:Parotid Gland / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002413:Cations, Divalent; D009532:Nickel; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases