Protein kinases in brown adipose tissue of developing rats. II. two soluble kinase activities and their affinities for nucleotide and protein substrates.

A heat-stable, soluble component of brown adipose tissue from newborn rats was found to be readily phosphorylated by protein kinase of the same subcellular fraction. The concentration of this component in brown fat decreased with the age of the animals. A boiled crude microsomal preparation from rat liver was also phosphorylated by brown fat protein kinase. The GTP-linked phosphorylation of the endogenous heat-stable protein was not stimulated by ATP (in contrast to phosphorylation of histone). The maximum velocity of phosphorylation achieved with GTP was about 2.5 times higher than that with ATP as nucleotide substrate. This difference was not due to ATPase activity in the assay. With histone as the protein acceptor both activities were the same. The affinity of protein kinase(s) for ATP was lower with the endogenous heat-stable brown-fat protein and with boiled microsomes (Km of 0.21 mM and 0.17 mM, respectively) than with histone (Km of 0.05 M). No detectable ATPase activity was present in either acceptor protein. It is concluded that the 100 000 times g supernatant fraction from brown fat of infant rats contains two protein kinase activities. One preferentially uses ATP and histone as substrates and the other uses endogenous heat-stable soluble proteins and either ATP or GTP.