Properties of inositol polyphosphate 1-phosphatase.
We recently described inositol polyphosphate 1-phosphatase, an enzyme which cleaves the 1-phosphate from inositol 1,4-bisphosphate (Ins(1,4)P2) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) (Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 15946-15952). We have now purified the enzyme to homogeneity from calf brain. The enzyme hydrolyzes 50.3 mumol of Ins(1,4)P2/min/mg protein. The enzyme has an apparent mass of 44,000 daltons as determined both by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that it is monomeric. Lithium ions inhibit Ins(1,3,4)P3 hydrolysis uncompetitively with an apparent Ki of approximately 0.3 mM LiCl. Calcium inhibits hydrolysis of Ins(1,4)P2 and Ins(1,3,4)P3 equally, with approximately 40% inhibition occurring at 1 microM free Ca2+. Rabbit polyclonal antiserum against purified inositol polyphosphate 1-phosphatase was prepared which immunoprecipitates approximately 0.3 milliunits of activity/microliter serum (1 unit = 1 mumol of Ins(1,4)P2 hydrolyzed per min). This antiserum was used to determine the enzyme content in several bovine tissues, all of which had a similar intrinsic specific activity (i.e. approximately 0.3 milliunits/microliter antiserum). Tissues studied included brain, heart, kidney, liver, lung, parotid, spleen, testis, and thymus. Approximately 10-15% of the total inositol polyphosphate 1-phosphatase activity in calf brain homogenates remains in a particulate fraction; antiserum also binds 0.3 milliunits of membrane-associated activity/microliter antiserum. Thus, a single enzyme can account for Ins(1,4)P2 hydrolytic activity in the bovine tissues. Ins(1,3,4)P3 metabolism was also investigated in bovine tissue homogenates. Inositol polyphosphate 1-phosphatase accounts for greater than 80% of the hydrolytic activity in all tissues studied except brain, where inositol polyphosphate 4-phosphatase is the major enzyme that hydrolyzes Ins(1,3,4)P3. The apparent Km of inositol polyphosphate 1-phosphatase for Ins(1,3,4)P3 varies approximately 3-4-fold among the bovine tissues.
Inhorn
R C
RC
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Majerus
P W
PW
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D006882:Hydroxyapatites; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D018021:Lithium Chloride; D009928:Organ Specificity; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D013950:Thymus Gland / Q000201:enzymology
D002712:Chlorides; D006882:Hydroxyapatites; D007295:Inositol Phosphates; D015544:Inositol 1,4,5-Trisphosphate; D017886:Durapatite; D008094:Lithium; D010744:Phosphoric Monoester Hydrolases; C053087:inositol-1,4-bisphosphate 1-phosphatase; D018021:Lithium Chloride