Studies on the properties of myo-inositol-1,4,5-trisphosphate 5-phosphatase and myo-inositol monophosphatase in bovine iris sphincter smooth muscle: effects of okadaic acid and protein phosphorylation.
In bovine iris sphincter, myo-inositol 1,4,5-trisphosphate (IP3) 5-phosphatase and myo-inositol 1-phosphate (IP1) monophosphatase are mainly localized in the microsomal and soluble fractions, respectively. Studies on the properties of these enzymes can be summarized as follows. (1) The microsomal IP3 5-phosphatase hydrolyzed IP3 to myo-inositol 1,4-bisphosphate with an apparent Km of 28 microM and Vmax of 32 nmol/min per mg protein. The IP1 monophosphatase in the soluble fraction hydrolyzed IP1 into free inositol with an apparent Km of 89 microM and Vmax of 7 nmol/min per mg protein. (2) IP3 5-phosphatase and IP1 monophosphatase had optimal pH values at 8.0 and 7.0, respectively. (3) Both enzymes required Mg2+ and their highest specific activities were at a cation concentration of 2 mM. (4) Ca2+ (> 0.5 microM) exerted an inhibitory effect on IP3 5-phosphatase activity, and marked inhibition (47%) was observed at a concentration of 10 microM. Higher concentrations of the cation (> 100 microM) were required to inhibit IP1 monophosphatase. (5) IP1 monophosphatase, but not IP3 5-phosphatase, was inhibited by Li+. Li+ had no effect on the contractile response in this smooth muscle. (6) Both enzymes were inhibited by ATP and by the thiol-blocking agent, disulfiram. In addition, thimerosal, a thiol reagent, also inhibited the IP3 5-phosphatase activity. (7) Protein phosphorylation of the microsomal and soluble fractions with PKA or PKC had no effect on the activities of these enzymes. (8) Okadaic acid, a protein phosphatase inhibitor, had no effect on the activity of IP3 5-phosphatase. However, in the intact iris sphincter the toxin significantly reduced the carbachol-induced IP3 production, 1,2-diacylglycerol formation, measured as phosphatidic acid, and caused muscle relaxation.
Wang
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Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912.
Akhtar
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Abdel-Latif
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D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D004221:Disulfiram / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D000072181:Inositol Polyphosphate 5-Phosphatases; D007498:Iris / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000201:enzymology*; D019319:Okadaic Acid; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D004988:Ethers, Cyclic; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D013439:Sulfhydryl Reagents; D019319:Okadaic Acid; D002217:Carbachol; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D000072181:Inositol Polyphosphate 5-Phosphatases; D008274:Magnesium; D004221:Disulfiram