HUMAN LYMPHOCYTE-T CAMP-DEPENDENT PROTEIN-KINASE - SUBCELLULAR DISTRIBUTIONS AND ACTIVITY RANGES OF TYPE-I AND TYPE-II ISOZYMES

The role of the type I and type II protein kinase A isozymes in the regulation of human T lymphocyte immune effector functions has not been ascertained. To approach this question, we first characterized the distribution and enzyme activities of the type I and type II protein kinase A (PKA) isozymes in normal, human T lymphocytes. T cells possess both type I and type II isozymes with an activity ratio of 5.0:1 +/- 0.71 (mean +/- SD). The type I isozyme associates predominately with the plasma membrane whereas the type II isozyme localizes primarily to the cytosol. Analyses of isozyme activities demonstrated that T cells from approximately one-third of 16 healthy donors exhibited significantly higher type II isozyme activities (higher type II, type II(H)) than the remaining donors (lower type II, type II(L)) (mean = 605 +/- 75 pmol.min-1.mg protein-1, P < 0.001). Scatchard analyses of [H-3]cAMP binding in the cytosolic fraction demonstrated similar K(d) values (type II(H), 1.1 x 10(-7)M; type II(L), 9.0 x 10(-8) M); however, the B(max) (maximal binding) of the type II(H) was 400 fmol/mg protein compared to the B(max) of the type II(L) of 126 fmol/mg protein. Scatchard analysis of [H-3]cAMP binding to the type I isozyme associated with membrane fragments bad a K(d) of 5.6 x 10(-8) M and a B(max) of 283 fmol/mg protein. Eadie-Hofstee plots of type II(H) and type II(L) gave a K(m) and V(max) of 2.3 mg/ml and 1.5 nmol.mg.1.min-1, and 2.1 mg/ml and 1.6 nmol.mg-1.min-1, respectively. The 3.2-fold higher maximal binding of the type II isozyme in one-third of healthy donors may reflect a greater amount of isozyme protein. The compartmentalization of type I PKA isozyme to the plasma membrane and type II PKA isozyme to the cytosol may serve to localize the isozymes to their respective substrates in T lymphocytes.