title: 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.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912, USA.
authors.forename: X L
authors.initials: XL
authors.lastname: Wang
authors.forename: R A
authors.initials: RA
authors.lastname: Akhtar
authors.forename: A A
authors.initials: AA
authors.lastname: Abdel-Latif
mesh_terms: 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
chemical_list: 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