PHOSPHORYLATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

Glyceraldehyde-3-phosphate dehydrogenase (GPDH)* in rabbit skeletal muscle and brain extracts was found to be effectively phosphorylated by Ca2+/phospholipid- and Ca2+/calmodulin-dependent protein kinases. These protein kinases are tightly bound to GPDH which has been immobilized on Sepharose. They can be eluted from this affinity absorbent by washing the column with solutions of dehydrogenase or anti-GPDH antibodies. The elution of the protein kinases was most complete under conditions causing dissociation of the immobilized dehydrogenase tetramer to dimers. On this basis a method for isolating Ca2+/calmodulin-bound protein kinase has been devised which includes a stage of GPDH admixture removal by chromatography an phenyl-Sepharose. The capacity of GPDH to be phosphorylated by tissue protein kinases is also confirmed by analysis of GPDH preparations isolated from muscles. These preparations were found to contain sizable amounts of covalently linked endogenous phosphate, ie., 0.7 to 1.5 moles per mole of dehydrogenase tetramer.