80] l-Ribulokinase

Abstract

This chapter reveals that the L-Ribulokinase can be assayed by several methods: (1) by measuring CO 2 evolution from bicarbonate manometrically; (2) by coupling the reaction with pyruvate kinase and lactic dehydrogenase and following the disappearance of reduced nicotinamide adenine dinucleotide (NADH) spectrophotometrically; and (3) by using radioactive L-ribulose and precipitating and assaying the radioactive L-ribulose 5-phosphate formed. The second method cannot be used to assay crude extracts because of the presence of interfering substances, however it is convenient for partially purified preparations and gives identical results as with the third assay method. One unit of L-ribulokinase is defined as that amount which phosphorylates 1 micromole of L-ribulose per minute. Specific activity is in units per milligram of protein. The steps discussed in the purification procedure includes growth of culture and preparation of crude extract; removal of nucleoproteins; ammonium sulfate fractionation; pH 7.6 diethylaminoethyl (DEAE)-cellulose column chromatography, pH 6.5 DEAE-cellulose column chromatography; sephadex G-200 column chromatography; and crystallization.