PURIFICATION AND PROPERTIES OF D-MYO-INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE FROM BOVINE IRIS SPHINCTER SMOOTH-MUSCLE - EFFECTS OF PROTEIN-PHOSPHORYLATION IN-VITRO AND IN INTACT MUSCLE

Stimulation of bovine iris sphincter muscle with carbachol (10 mu M) i ncreased accumulation of Ins(1,4,5)P-3 (InsP(3)) and Ins(1,3,4,5)P-4 ( InsP(4)) by 86 and 32% respectively. Addition of isoproterenol (5 mu M ) to muscle pretreated with carbachol reduced the H-3-radioactivity in InsP(3) by 30 % and increased that of InsP(4) by 41%. InsP(3) 3-kinas e was predominantly localized in the soluble fraction (110 000 g super natant) of the iris sphincter. The enzyme was purified from this fract ion by sequential chromatography on DEAE-cellulose, calmodulin (CAM)-a garose affinity, and Mono-Q anion-exchange columns. The specific activ ity of the purified enzyme was 1.94 mu mol/min per mg protein with a p urification of 114-fold, compared with the cytosolic fraction of the m uscle. SDS/PAGE showed the enzyme to be associated with a protein band corresponding to 50 kDa. In the presence of 10 mu M Ca2+, CaM dose-de pendently stimulated the enzyme. InsP(3) 3-kinase specifically phospho rylated InsP(3) with an apparent K-m of 0.56 mu M and a V-max. of 2.5 mu mol/min per mg protein. The stimulatory effect of CaM was due to a change in V-max. and not in its K-m. The enzyme was maximally active a t pH 7.0-7.5. Phosphorylation of the purified InsP(3) 3-kinase with pr otein kinase A increased its activity; in contrast, phosphorylation wi th protein kinase C inhibited the enzyme activity. Treatment of the in tact iris sphincter with isoproterenol or phorbol 12,13-dibutyrate res ulted in stimulation of InsP(3) 3-kinase activity in the soluble fract ion and this activation was preserved on SDS/PAGE and renaturation. Th ese results indicate that the bovine iris sphincter contains a Ca-CaM- dependent InsP(3) 3-kinase which can be differentially regulated, both in vitro and in intact muscle, by protein kinases A and C.