Isolation, partial purification and biochemical characterization of chloroplastic L-myo-inositol-1-phosphate synthase from a macro alga Enteromorpha intestinalis under high salinity

Abstract

Induction of higher myo-inositol biosynthesis in a salinity driven macro-alga, Enteromorpha intestinalis (L.) Nees, as a model macrophyte, grown under natural conditions was established on the basis of a study on its prime enzyme, L-myo-inositol-1-phosphate synthase (MIPS) and the accumulation of free myo-inositol in chloroplasts. This enzyme was partially purified from isolated chloroplasts obtained from the plant grown under higher salinity to about 41-fold over homogenate following low-speed centrifugation, high-speed centrifugation, 0 to 80 % ammonium sulfate precipitation, successive chromatography through DEAE-cellulose, Sephadex G-200 and BioGel 0.5m columns. The apparent M r of the native enzyme was about 164 kDa. Temperature and pH optima were found to be 35 °C and 7.5 respectively. D-glucose-6-phosphate and NAD were its exclusive substrate and coenzyme, respectively, with K m value 0.1761 mM for D-glucsoe-6-phosphate and 0.1695 mM for β-NAD as determined by non-linear regression kinetics method. Among the important monovalent and divalent cations tested, K + had trivial stimulatory effect while Li + was sturdily inhibitory. Divalent cations recorded variable effects. Ca 2+ exhibited slight stimulatory effect and Cd 2+ reduced MIPS activity faintly. Salts of Cu 2+ and Hg 2+ were found to be potent inhibitors of this enzyme. The concentration of free myo-inositol was found to increase proportionately with salinity at least up to 12 PSU.