Ornithine decarboxylase (ODC) was purified 6500-fold from NMRI mouse kidneys under conditions designed to inhibit degradation by proteinases. The enzyme was homogeneous by SDS/polyacrylamide-gel electrophoresis, and the specific activity was among the highest reported. The yield was 70%. A monoclonal antibody against this preparation was generated and used in studies to investigate the half-life of ODC in cultured rat hepatocytes labelled with [35S]methionine. This value was 39 +/- 4 min and was unchanged when either NH4Cl (as a lysosomotropic agent) or leupeptin (as a lysosomal proteinase inhibitor) was added to the culture medium. Thus the intracellular turnover of ODC in cultured hepatocytes occurs mainly in extra-lysosomal compartments. Arginylation of rat ODC was investigated in vitro by incubation with L-[3H]arginyl-tRNA, and the incorporation of the label was compared with that of total cytosolic proteins. Arginylated ODC had a specific radioactivity 8600 times that of the bulk of cytosolic protein. Edman degradation of this ODC showed that the post-translational arginylation occurred only at the alpha-amino end of the enzyme. The inhibitor of arginyl-tRNA:protein arginyltransferase (EC 220.127.116.11), L-glutamyl-L-valyl-L-phenylalanine, increased the half-life of ODC in cultured hepatocytes from 39 min to more than 90 min. The possible significance of the preferential post-translational arginylation of ornithine decarboxylase to its rapid turnover is discussed.
1. It has been found that cathepsin L is very susceptible to loss of activity through autolysis. When this is prevented by purification and storage of the enzyme as its mercury derivative, preparations are obtained with higher specific activity than previously. 2. Active-site titration shows, however, that even the new purification method does not give preparations in which the enzyme is 100% active. 3. Benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide has been discovered to be a very sensitive substrate for cathepsin L. Like all other known substrates for cathepsin L, however, it is also cleaved by cathepsin B. 4. Cathepsin L degrades insoluble collagen at pH 3.5 over 5-fold faster than at pH 6.0. The specific activity at pH 3.5 is 5-10-fold higher than that of cathepsin B (rat or human) or bovine spleen cathepsin N (‘collagenolytic cathepsin’). 5. Qualitatively, the action of cathepsin L on collagen is similar to that of cathepsins B and N, i.e. selective cleavage of terminal peptides leads to conversion of beta- and higher components mainly to alpha-chains.