The purification and properties of detergent-solubilized rat liver nucleotide pyrophosphatase.

Nucleotide pyrophosphatase was solubilized from the microsomal fraction of rat liver with Triton X-100 and was purified by ion exchange chromatography on DEAE-cellulose, gel filtration through Sepharose 4B and preparative disc electrophoresis in the presence of 0.1% sodium dodecyl sulfate. This procedure resulted in a final purification of 176-fold. Maintenance of enzymatic activity through the various stages of purification depended on the presence of detergent. When detergent was removed, enzymatic activity was lost and aggregation of protein was observed. The Trlton X-100 solubilized enzyme appeared to reside in relatively large membrane fragments. These were reduced in size by treatment with sodium dodecyl sulfate. Disc electrophoresis in the presence of sodium dodecyl sulfate resolved these fragments into a number of components but enzymatic activity was decreased. When Triton X-100 was included, less resolution was achieved but enzyme activity was preserved. The hydrolysis of NADH catalyzed by the enzyme was competitively inhibited by UDP-glucose and ADP-ribose and the values of Ki for these materials as inhibitors agreed with their apparent K, values as substrates. In addition, the ratio of NADH pyrophosphatase activity to that using other substrates remained constant over several stages of purification. Segments of analytical disc gels which had been run in the presence of sodium dodecyl sulfate were incubated with various substrates and the locations of pyrophosphatase activity using NAD, NADPH, ADP-ribose, UDP-N-acetylglucosamine, and UDP-glucose were each compared to that using NADH as a reference substrate. All activities tested were coincident with that employing NADH. It is concluded on the basis of the experiments described that a single enzyme with broad specificity for nucleotide pyrophosphates is responsible for hydrolysis of all the nucleotide pyrophosphates tested.