320 Biochimica etBiophysicaActa 928 (1987) 320-331 Elsevier BBA12020 Purification and properties of a multifunctional calcium/calmodulin-dependent protein kinase from rat pancreas Jonathan A. Cohn, Barbara Kinder, James D. Jamieson, Nancy G. Delahunt and Fred S. Gorelick Departments of Medicine, Surgery and Cell Biology, Yale University School of Medicine, New Haven, CT (U.S.A.) (Received 25 November 1986) Key words: Protein kinase; Substrate specificity; (Rat pancreas) A ealcium/calmodulin-dependent protein kinase (Ca/calmodulin protein kinase) was purified from rat pancreas using hydrophobic chromatography followed by gel filtration and affinity chromatography. Ca/ calmodulin protein kinase from pancreas resembled previously described multifunctional Ca/calmodulin protein kinases from other tissues with respect to substrate specificity, autophosphorylation on serine and threonine residues, and catalytic and hydrodynamic properties. While Ca/calmodulin protein kinase from other tissues contains subunits of 53-60 kDa with variable proportions of a smaller 50-52 kDa subunit, pancreatic Ca/calmodulin protein kinase was found to contain a single component of 51 kDa. Experiments mixing brain Ca/calmodulin protein kinase with pancreatic homogenate suggest that the absence of a larger subunit in the pancreatic Ca/calmodulin protein kinase is not due to proteolytic degradation during enzyme preparation. Ca/calmodulin protein kinase binding to 1251-labeled caimodulin in solution was demonstrated using the photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azidobenzoate. 12Sl-labeled calmodulin bind- ing to Ca/calmodulin protein kinase was also demonstrated using filters containing Ca/calmodulin protein kinase transferred from polyacrylamide gels after two-dimensional gel electrophoresis. Finally, the ribosomal substrate for Ca/calmodulin protein kinase was identified as the ribosomal protein, $6. The purification procedure presented in this study promises to be useful in characterizing Ca/calmodulin protein kinase in other tissues and in clarifying the role of these enzymes in cellular function. Introduction The exocrine pancreas is an excellent model system for studying intracellular events leading to protein secretion in response to extracellular neu- rohumoral stimuli [1,2]. In this tissue, several types Abbreviations: HSAB, N-hydroxysuccinimidyl-4-azido- benzoate;' PMSF, phenylmethylsulfonyl fluoride; Hepes, 4-(2- hydroxyethyl)-I -piperazineethanesulfonic acid. Correspondence (present address): J. Cohn, Box 8124, Washington University Medical Center, 660 South Euclid Ave., St. Louis, MO 63110, U.S.A. of stimuli produce prompt increases in cyto- plasmic calcium [3-6] and associated changes in the phosphorylation state of cellular proteins, in- cluding the ribosomal protein, $6 [7-11]. Using this ribosomal protein as a model substrate, previ- ous studies from this laboratory have demon- strated that pancreatic tissue fractions contain calcium-dependent protein kinase activities that are activated by calmodulin and by phospholipid [12]. Both of these calcium-dependent kinase ac- tivities have subsequently been demonstrated in pancreas using other substrates [13,14]. These pro- tein kinase activities are of interest because each provides a mechanism by which calcium-depen- 0167-4889/87/$03.50 © 1987 Elsevier Science Pubfishers B.V. (Biomedical Division)