PROTEIN-PHOSPHORYLATION IN GOLGI, ENDOSOMAL, AND ENDOPLASMIC-RETICULUM MEMBRANE-FRACTIONS OF LACRIMAL GLAND

Ca2+/calmodulin- and cAMP-dependent protein kinase activities were cha racterized in two subcellular membrane samples. Membranes from rat lac rimal gland were isolated by differential and density gradient centrif ugation into six density windows. The present study focused on membran es from density windows III and V which contain mixtures of apical, Go lgi, endosomal, and endoplasmic reticulum membranes in different propo rtions. Phosphorylation of membrane proteins was measured by incubatin g the samples in [g-P-32]ATP and separating the proteins by discontinu ous SDS-PAGE followed by autoradiography. The amount of phosphate inco rporated into specific peptide bands was quantified by densitometry. C a2+/calmodulin-dependent protein kinase phosphorylated a 52,000 MW pep tide in membranes from both density windows with a maximal increase fr om 0.3 to 66 mu M free Ca2+. Trifluoperazine and promethazine, two inh ibitors of Ca2+/calmodulin-dependent protein kinases, inhibited this p hosphorylation. cAMP-dependent protein kinase phosphorylated a 22,000 MW peptide and a 91,000 MW peptide which were present in membranes fro m density window III only. We conclude that a Ca2+/calmodulin-dependen t protein kinase activity is present in membranes from both density wi ndow III and V whereas a cAMP-dependent protein kinase activity is pre sent only in membranes from density window III.