CALCIUM-DEPENDENT PHOSPHORYLATION OF SYMBIOSOME MEMBRANE-PROTEINS FROM NITROGEN-FIXING SOYBEAN NODULES - EVIDENCE FOR PHOSPHORYLATION OF NODULIN-26

By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca2+-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca2+ (K0.5 = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca2+-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with P-32-phosphate. Overall, the results show the existence of a Ca2+-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca2+-dependent phosphorylation.