Specificities of rat alkaline ribonucleases and cytochemical localization of pancreatic-like ribonucleases

Abstract Pancreatic-like ribonucleases differ, and may be distinguished, from other alkaline ribonucleases of the rat by their ability to hydrolyse uridine-3′-(α-naphthyl phosphate) (Up-naphthyl). This difference in specificity is not due to endogenous ribonuclease inhibitors; nor is it a steric effect since it is also valid for uridine-3/t'- phenyl phosphate (Up-phenyl), a new pancreatic ribonuclease substrate, the synthesis of which is described here. The specificity of this substrate is, however, limited by its susceptibility to spleen phosphodiesterase. Hog spleen alkaline ribonuclease was purified over 100-fold and shown to be inactive vs uridine-3′-(α-naphthyl phosphate) and uridine-3′-phenyl phosphate. However, storage of the purified enzyme, or of enzyme-enriched extracts of spleen and liver, led to the gradual appearance of a low activity against the two substrates. The activity of the purified spleen enzyme vs the α-naphthyl ester was associated with a labile fraction weakly bound by a cation exchanger. The ratio of the activities of this fraction vs RNA and uridine-3′-(α-naphthyl phosphate), was identical with that for pancreatic ribonuclease. With the aid of uridine-3′(α-naphthyl phospahte), pancreatic-like ribonuclease activity was found, and cytochemically localized, in the pancreas, the parotid gland and the kidney. It was also found in the duodenal fluid, the serum and the urine. The localization patterns in the pancreas and the parotid gland pointed to its extracellular nature. The occurrence of pancreatic-like ribonuclease activity in lysosome-like structures in kidney proximal tubule epithelial cells indicated that it is the extracellular enzyme adsorbed from the duodenal fluid into the blood stream and subsequently partially removed by kidney lysosomes.