THE LIMITED PROTEOLYSIS OF BOVINE NEUROPHYSINS BY CATHEPSIN D

Abstract— The limited proteolysis of the bovine neurophysins at acid pH has been studied and the enzyme responsible has been characterized. Only 15 per cent of the catheptic activity in 4-year-old acetone-dried posterior pituitary lobe powder is soluble at pH 4.0. Solubility increases as the age of the powder decreases and the cathepsin is completely soluble in the presence of 1% Triton X-100. Acid proteinase activity in the neurohypophysis is not thiol activated and is inhibited by 3-phenylpyruvic acid. Bovine serum albumin was degraded at only 1 per cent of the rate of haemoglobin but with the same pH optimum (3.7). On this basis the enzyme was identified as cathepsin D. Neurophysin-I is degraded in two stages by cathepsin D; the first product (neurophysin-I′) runs faster and the second product (neurophysin-I″) runs slower than the native protein on starch-gel electrophoresis at pH 8.1. Neurophysin-II is also degraded in two stages; the first product has a higher electrophoretic mobility than the native protein and is identical in mobility with the faster-running component of the so-called neurophysin-M of Hollenberg and Hope (1967b). Prolonged incubation with the cathepsin gives rise to a slower-running component. Neurophysin-C is not attacked by the acid proteinase. Neurophysin-I′ and I″ have been isolated by ion-exchange chromotography. They have the same N-terminal amino acid (alanine) and C-terminal sequence (Ala-Phe-Ser) as the native protein and both bind 8-argininevasopressin. Neurophysin-I′ is identical in amino acid composition with the native protein but neurophysin-I″ has lost one leucine and two aspartic acid residues. Reduction, ¹⁴C-alkylation and separation of the fragments by starch-gel electrophoresis shows that the structural and functional integrity of neurophysin-I″ is maintained by the disulphide bonds, even though a tripeptide has been split out of the interior of the molecule. The low molecular weight material produced by catheptic digestion of neurophysin-I has been purified and shown to have a composition of one leucine and two aspartic acid residues. It is suggested that extensive in vivo proteolysis of neurophysin by lysosomal cathepsin, with consequent abolition of hormone-binding ability, is unlikely.