Further Studies on myo-Inositol-1-phosphatase from the Pollen of Lilium longiflorum Thunb

Abstract

myo-Inositol-l-phosphatase has been purified to homogeneity from Lilium longiflorum pollen using an alternative procedure which includes pH change and phenyl Sepharose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis shows that the enzyme is a dimer (subunit molecular weight, 29,000 daltons). The enzyme is stable at low pH values and is inactivated only below pH 3.0. In addition to IL-and ID-myo-inositol-l-phosphate, it shows high specificity for IL-chiro-inositol-3-phosphate. As MI-I -phosphatase2 from lily pollen readily hydrolyzes the enantiomeric forms of MI-1-P (16) and in this respect resembles similar MI-1-phosphatases from yeast (4), rat testis (7), and bovine brain (11). It also hydrolyzes MI-2-P as does the MI-I-P phosphatase from chick erythrocytes (20). In so far as all these Mg2"-dependent, alkaline phosphatases have been examined, their molecular and catalytic properties are quite similar; the notable exception being specificity toward MI-2-P as substrate (16).Here, we describe an alternative method for purification to homogeneity of MI-I -phosphatase from lily pollen and.examine subunit structure, substrate specificity, response to inhibitors, and stability of enzyme to heat and pH. Studies on sulfhydryl involvement at the active site are also presented. MATERIALS AND METHODSChemicals. fl-Glycerol-P, i-glucose-6-P, i-glucitol-6-P, Dmannitol-6-P, phenyl Sepharose and polyethylene glycol (P-2139) were purchased from Sigma Chemical Co. PHMB, NEM, and 2-mercaptoethanol were purchased from Aldrich Chemical 'Supported in part by Grant GM-22427 Purification of MI-1-Phosphatase. The procedure previously described (16) was used through the heat treatment step that followed DEAE-cellulose chromatography to obtain a partially purified MI-I -phosphatase. The DEAE-cellulose step was slightly modified in that the enzyme-loaded DEAE-cellulose column was washed with 0.12 M NaCl in Tris-acetate buffer (300 ml) prior to gradient elution. This pregradient wash removed considerable inactive protein previously encountered in gradient fractions. The gradient was initiated with buffer containing 0.12 M NaCl instead of 0.10 M NaCl as previously described.Heat-treated enzyme (80 ml, 60°C, 20 min) was dialyzed against 10 mm citrate buffer (2 L, pH 3.5) overnight at 4°C. The dialyzed suspension was centrifuged (25,000g, 15 min, 4°C) to remove precipitated protein. NaCl was added to a final concentration of 0.5 M. The enzyme was loaded on a phenyl Sepharose column (8 x 0.6 cm) which had been preequilibrated with 0.5 M NaCl in Tris-acetate. The column was washed with 0.1 M NaCl in Tris-acetate buffer (25 ml) and then the enzyme was eluted with straight buffer. Active fractions were pooled, concentrated by osmodialysis with polyethylene glycol (average mol wt 8,000) to 5 ml, loaded on a column of Ultrogel AcA 34 (90 x 1.2 cm), and washed with 50 mM Tris-acetate, pH 8.0 (150 ml). The MI-I-phosphatase obtained from this column was homogenous by electrophoretic standards. Enzy...

VTC4 Is a Bifunctional Enzyme That Affects Myoinositol and Ascorbate Biosynthesis in Plants

“…In addition, Fru 1-P, Fru 1,6-bisP, Glc 6-P, D-a-glycerophosphate, sorbitol 6-P, and myoinositol 2-P are not good substrates for VTC4, as seen by little to no hydrolysis in assays (Table I). Together, these data indicate that VTC4 is a somewhat promiscuous enzyme and that the C1 phosphate position in a six-member ring substrate is important for catalysis, as was noted previously by others (Gumber et al, 1984;Laing et al, 2004;Islas-Flores and Villanueva, 2007). In contrast to what has been reported previously (Laing et al, 2004), we conclude that VTC4 has a minor (1.6-to 2.4-fold) difference in substrate hydrolysis of L-Gal 1-P as compared with D-Ins 3-P or D-Ins 1-P. We also note the difference between VTC4 and human IMP regarding D-Gal 1-P.…”

“…It has been reported that Mg 2+ is necessary for maximal activity of IMP and that a pH of 7.5 is optimal (Gumber et al, 1984;Laing et al, 2004;Islas-Flores and Villanueva, 2007). Selected concentrations from 0 to 50 mM MgCl 2 were added to reactions with D-Ins 3-P to determine the optimum MgCl 2 for enzyme activity (Fig.…”

Phosphatidyl inositol metabolism and its role in signal transduction in growing plants

“…With respect to Li +, no effect on InsP3 degradation was observed, but InsP2 dephosphorylation was inhibited at high concentrations [68]. The Li ÷ results are consistent with those of an inositol-1-phosphatase from pollen purified to homogeneity [35].…”

A highly specific l -galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis

“…In the myo-inositol pathway, inositol-1-P is derived from glucose-6-P. It is then converted to myo-inositol by a phosphatase (19)(20)(21) and, thence, to glucuronic acid by inositol oxygenase.…”