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 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.
Wang
X L
XL
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912, USA.
Akhtar
R A
RA
Abdel-Latif
A A
AA
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D007498:Iris / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D013379:Substrate Specificity
D002147:Calmodulin; D007295:Inositol Phosphates; C081655:inositol-1,3,4,5-tetrakisphosphate; D015240:Phorbol 12,13-Dibutyrate; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D007545:Isoproterenol; D002118:Calcium