Measurement of Ribulose 1,5-Bisphosphate from Spinach Chloroplasts

Abstract

A technique was developed for the rapid and simple measurement of ribulose 1,5-bisphosphate from isolated spinach [cv. Viroflay] chloroplasts. The endogenous ribulose biphosphate was detected enzymically using 14CO2 and ribulose bisphosphate carboxylase/oxygenase released from the chloroplasts. Ribulose 5-phosphate kinase was inhibited with 0.4-0.6 mM 2,6-dichlorophenol-indophenol and 4 .mu.M carbonyl cyanide m-chlorophenylhydrazone. Phosphoenolpyruvate carboxylase activity was low with washed chloroplasts and its labeled product, [14C]oxalacetate, was destroyed by heating with 1.0 N HCl at 90.degree. C. The assay method was linear from 0.05-0.87 nM ribulose bisphosphate/ml. The latter value was determined with chloroplast material having 44 mg of chlorophyll/ml. This technique was simple and direct, used less chloroplast material, yet provided results comparable to a previously described enzymic technique in which ribulose bisphosphate was determined after the precipitation of chloroplast proteins by perchloric acid.