Abstract
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.
Highlights
We recently described inositol polyphosphate 1- glycerol, arachidonic acid, and thesoluble inositol phosphates phosphatase, an enzyme which cleaves the 1-phosphate
We have studied the properties of 15%of the totalinositol polyphosphate 1-phosphatase the enzyme in other bovine tissues. activity incalf brain homogenates remains ina particulate fraction; antiserum binds 0.3 milliunits of membrane-associated activity/plantiserum.a single enzyme can account for Ins(1,4)Pz hydrolytic activity in thebovine tissues
Purification of Inositol Polyphosphate I-Phosphatase-We previously reported a 3600-fold purification of inositol polyphosphate 1-phosphatase from calf brain (14)
Summary
M NHdCOOH (pH 3.45) gradient as described (13). Eighty-two per- polyacrylamide gel. Inositol polyphosphate 1-phosphatase hydrolysis of Ins(l,4)Pzwas assayed by one of two methods as described (14). DEAE-Sepharose-Ten calf brains were homogenized and centrifuged, and the supernatant was chromatographed on DEAE-Sepharose as described (14).Inositol polyphosphate 1-phosphatase activity was estimated inthis andall remaining steps using Assay 1.Enzymecontaining fractions were pooled and concentrated by precipitation in 75% ammonium sulfate. Hydroxylapatite-The concentrated, dialyzed enzyme from DEAESepharose chromatography was passed onto a Bio-Gel HTP hydroxylapatite column (7.7 X 10 cm) equilibrated in buffer A containing 2 mM sodium phosphate. Peak enzyme-containing fractions werepooled and concentrated by 75% ammonium sulfate precipitation as described ambMovNe.aTCh1e,3pmeMlletMwgaCs1r2e,1su0smpeMnd0e-dmienrc2a0ptmoMethHanEoPl E(bSuf(fpeHr c7).0.), 150 Gel Filtration Chromatography-The concentrated HPLC-DEAE enzyme pool was applied to three Bio-Si1TSK-250 columns (600 X 7.5 mm) attached in series which were equilibrated in buffer C. Peak enzyme-containing fractions (97 and 98) and side fractions (96 and 100; see “Results”) were pooled separately and concentrated to 200 PI in a Micro-ProDiCon concentrator (BiomolecularDynamics, Beaverton, OR)
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