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) increased 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-kinase was predominantly localized in the soluble fraction (110 000 g supernatant) of the iris sphincter. The enzyme was purified from this fraction by sequential chromatography on DEAE-cellulose, calmodulin (CAM)-agarose affinity, and Mono-Q anion-exchange columns. The specific activity of the purified enzyme was 1.94 mu mol/min per mg protein with a purification of 114-fold, compared with the cytosolic fraction of the muscle. 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-dependently stimulated the enzyme. InsP(3) 3-kinase specifically phosphorylated 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 at pH 7.0-7.5. Phosphorylation of the purified InsP(3) 3-kinase with protein kinase A increased its activity; in contrast, phosphorylation with protein kinase C inhibited the enzyme activity. Treatment of the intact iris sphincter with isoproterenol or phorbol 12,13-dibutyrate resulted in stimulation of InsP(3) 3-kinase activity in the soluble fraction and this activation was preserved on SDS/PAGE and renaturation. These 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.