Abstract
Stimulation of bovine iris sphincter muscle with carbachol (10 microM) increased accumulation of Ins(1,4,5)P3 (InsP3) and Ins(1,3,4,5)P4 (InsP4) by 86 and 32% respectively. Addition of isoproterenol (5 microM) to muscle pretreated with carbachol reduced the 3H-radioactivity in InsP3 by 30% and increased that of InsP4 by 41%. InsP3 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 mumol/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 microM Ca2+, CaM dose-dependently stimulated the enzyme. InsP3 3-kinase specifically phosphorylated InsP3 with an apparent K(m) of 0.56 microM and a Vmax. of 2.5 mumol/min per mg protein. The stimulatory effect of CaM was due to a change in Vmax. and not in its K(m). The enzyme was maximally active at pH 7.0-7.5. Phosphorylation of the purified InsP3 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 InsP3 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 InsP3 3-kinase which can be differentially regulated, both in vitro and in intact muscle, by protein kinases A and C.