2′,3′‐CYCLIC NADP AS A SUBSTRATE FOR 2′,3′‐CYCLIC NUCLEOTIDE 3′‐PHOSPHOHYDROLASE

Abstract

Abstract– 2′,3′‐Cyclic NADP has been prepared by cyclization of NADP at pH 6 in the presence of l‐ethyl‐(3‐dimethylaminopropyl)‐carbodiimide. The NADP derivative is readily hydrolyzed to NADP by the enzyme in brain and nerve that hydrolyzes 2′,3′‐cyclic nucleotides to 2′‐phospho esters. The K m for this substrate is the same as that for 2′,3′‐cyclic AMP (0.22 m m ) at pH 6 and 25°C. The two substrates are hydrolyzed by the phosphohydrolase at similar maximum velocities. The nicotinamide moiety in cyclic NADP thus has little effect on the enzyme‐substrate interaction. This synthetic substrate can be used in a rapid (2 min) and sensitive (10 ng of 31‐fold purified enzyme) spectrophotometric coupled enzyme assay for 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase; in this assay the hydrolysis proceeds in the presence of glucose‐6‐phosphate dehydrogenase and its substrate and the NADPH formed is measured by the increase in absorbance at 340 nm. The assay is applicable to tissue extracts as well as to purified preparations of the enzyme. There is no interference from nucleases of the pancreatic RNase A type.