| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1989: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1988: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
Activity expression, activity change, enzyme purification and properties of L-myo-inositol- 1-phosphate synthase(MIPS) were analyzed employing rice cells in suspension-culture. myo-Inositol(MI) biosynthesis and a role in controlling metabolism also were investigated. It was shown that in MI deficient medium at the early logarithmic stage, the activity of MIPS is prominent, but in medium to which MI was, is suppressed. Sigmoid curve of the enzyme activity with critical point, 20 mg MI/1, was observed. Furthermore, the activity change at MI concentration was examined, Km was unchanged but Vmax varied in proportion to the activity. It was, therefore, suggested that the activity regulation by MI was not inhibition of enzyme activity but synthetic regulation of enzyme molecules.
Using 20 mM Tris-HC1 buffer(pH 7.5) containing 20%(V/V) glycerin, 2 mM 2-mercaptoethanol, the purification of enzyme was performed at 3ﾟC. The enzyme was isolated from the cell extract and purified 600-fold with a 6% yield by ammonium sulfate fractional precipitation, and chromatographies on DEAE-Toyopearl, butyl-Toyopearl, Hydroxylapatite, Cellulofine GCL-2000 and chromatofocusing Attempts to obtain the homogeneous enzyme were unsuccessful.
On the other hand, due to the presence of the powerful non-specific acid phosphatase in the enzyme, periodate oxidation-malachite green method was adopted instead of bioassay method, and subsequent assay was simplified. NH^+_ accelerates the rate of reaction 4-fold. The enzyme showed pH optimum at 8.0 with Km value of 0.79 mM. The activity was about 15 - 16% NAD^+-independent. The activity was remarkably inhibited by a number of pentose phosphate pathway: 6-phosphogluconate, riburose-5-phosphate, ribose-5-phosphate and fructose-6-phospha-Le. The enzyme activity was inhibited by Ag^+, Cu^<2+>, Zn^<2+>, Ba^<2+> and Fe^<3+>. Some sulfhydryl reagent and nucleotide-coenzyme used also were inhibitory.
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