Purification and characterization of membrane-bound inositolpolyphosphate 5-phosphatase.
Membrane-bound inositolpolyphosphate 5-phosphatase was solubilized and highly purified from a microsomal fraction of rat liver. Its physiochemical and enzymological properties were compared with those of highly purified preparations of two types of soluble enzyme (soluble Type I and Type II) from rat brain. The molecular masses of the membrane-bound and soluble Type I enzymes were 32 kDa, while that of soluble Type II enzyme was 69 kDa, as determined by molecular sieve chromatography. The membrane-bound and soluble Type I enzymes showed similar broad peaks on isoelectric focusing (pI 5.8-6.4), while soluble Type II enzyme showed multiple peaks in the region between pI 4.0-5.8. All three enzymes required divalent cation for activity. Mg2+ was the most effective for both the membrane-bound and soluble Type I enzymes, while Co2+ enhanced soluble Type II enzyme activity about 1.5-fold relative to Mg2+ at 1 mM. The optimal pH of both the membrane-bound and soluble Type I enzymes was 7.8, while that of soluble Type II was 6.8. The Km values for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] of all three enzymes were similar (5-8 microM), but those for inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were quite different, the Km values of membrane-bound and soluble Type I enzymes being 0.8 microM, while that of soluble Type II was 130 microM. These similarities between the membrane-bound and soluble Type I enzymes suggest that these two molecules may be the same protein, and that concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, both of which are considered to play critical roles in the regulation of intracellular Ca2+-concentration, may be differently regulated by two functionally distinct enzymes.
Takimoto
K
K
Division of Protein Metabolism, Institute for Protein Research, Osaka University.
Okada
M
M
Nakagawa
H
H
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D000072181:Inositol Polyphosphate 5-Phosphatases; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D002413:Cations, Divalent; D007527:Isoenzymes; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases