A serine protease from the keratin-degrading Streptomyces pactum DSM 40530 was purified by casein agarose affinity chromatography. The enzyme had a molecular weight of 30,000 and an isoelectric point of 8.5. The proteinase was optimally active in the pH range from 7 to 10 and at temperatures from 40 to 75؇C. The enzyme was specific for arginine and lysine at the P 1 site and for phenylalanine and arginine at the P 1 site. It showed a high stereoselectivity and secondary specificity with different synthetic substrates. The keratinolytic activity of the purified proteinase was examined by incubation with the insoluble substrates keratin azure, feather meal, and native and autoclaved chicken feather downs. The S. pactum proteinase was significantly more active than the various commercially available proteinases. After incubation with the purified proteinase, a rapid disintegration of whole feathers was observed. But even after several days of incubation with repeated addition of enzymes, less than 10% of the native keratin substrate was solubilized. In the presence of dithiothreitol, degradation was more than 70%.

SUMMARYHepatitis B virus-related DNA was detected in the chromosomal DNA of three out of seven hepatocellular carcinomas and two out of five cirrhosis samples examined, by means of the blot-hybridization technique, described by Southern (1975). The integration patterns were not identical but some similarities raise the question of whether there are some preferred sites of viral integration.

The nature of the protein kinase (PK) which phosphorylates the core protein of hepatitis B virus in vitro was studied. The PK copurified with the core particles during rate zonal centrifugation and gel chromatography. It showed the same size heterogeneity as the core particles, which consisted of a main fraction of 28-nm particles and a subfraction of 22- to 26-nm particles. DNA-containing heavy core particles with a density of 1.33 to 1.35 g/ml and less endogenous PK than did the light cores. The phosphorylation reaction had a rapid initial phase (several minutes) and a slow but long-lasting second phase (many hours). The PK had a high affinity for ATP (KM = 0.5 mumol/liter). Only few of the several hundred P21.9 subunits in one core particle were phosphorylated in vitro. The only amino acid which was phosphorylated in vitro was serine. The resistance of the introduced phospho group against alkaline phosphatase showed that the PK acceptor, and probably the enzyme itself, was located inside the core particle.

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Intestinal absorption of proteinases triggers the formation of TGF-beta binding species of alpha2-macroglobulin in blood. Mediated by this process high concentrations of TGF-beta might be reduced via enhanced clearance of alpha2-macroglobulin-TGF-beta complexes. Thus, proteinase therapy may have beneficial effects in treatment of fibrosis and certain cancers accompanied by excessively high TGF-beta concentrations.