IDENTIFICATION OF AN ECTOKINASE ACTIVITY IN CEREBELLAR GRANULE PRIMARY NEURONAL CULTURES

Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 nM [gamma-P-32]ATP. Phosphory lation of a 45-kDa endogenous protein was detected within 1 min and in creased linearly for similar to 20 min. Unlike what was seen with [gam ma-P-32]ATP, in the presence of [P-32]orthophosphate no visible phosph orylation of protein was detected after 10 min, but a different patter n of phosphorylation was obtained in 30 min. The phosphorylation of th e 45-kDa protein was reduced by 80-90% in the presence of 1 mu M unlab eled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 mM PO43-. Phos phorylation was inversely proportional to cell density and was unaffec ted by addition to the cells of 56 mM KCl or 100 mu M glutamate for 3 min. The presence of exogenously added cellular protein extracts or pr etreatment of the cells for up to 20 min in phosphorylation buffer als o did not affect the observed phosphorylation of the 45-kDa protein. T he phosphorylation was found to be insensitive to MgCl2 but inhibited in the presence of MnCl2 or NaF and in the absence of CaCl2. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80-90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylat ion of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore d istinguishable from a series of other presently characterized ecto-pro tein enzymes and intracellular kinases.