Protease inhibitors from jackfruit seed (Artocarpus integrifolia).

Protease inhibitory activity in jackfruit seed (Artocarpus integrifolia) could be separated into 5 fractions by chromatography on DEAE-cellulose at pH 7·6. A minor fraction (I) that did not bind to the matrix, had antitryptic, antichymotryptic and antielastase activity in the ratio 24:1·9:1·0. Fraction II bound least tightly to the ion exchanger eluting with 0·05 Μ NaCl and could be resolved into an elastase/chymotrypsin inhibitor and a chymotrypsin/trypsin inhibitor by chromatography on either immobilized trypsin or phenyl Sepharose CL-4B. Fractions III and IV eluted successively with 0·10 Μ NaCl and 0·15 Μ NaCl from DEAE-cellulose, inhibited elastase, chymotrypsin and trypsin in the ratio 1·0: 0·53:0·55 and 1·0:8·9:9·8 respectively. Fraction V, most strongly bound to the matrix eluting with 0·3 Μ NaCl and was a trypsin/chymotrypsin inhibitor accounting for 74% of total antitryptic activity. This inhibitor was purified further. The inhibitor with a molecular weight of 26 kd was found to be a glycoprotein. Galactose, glucose, mannose, fucose, xylose, glucosamine and uronic acid were identified as constitutent units of the inhibitor. Dansylation and electrophoresis in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. The inhibitor combined with bovine trypsin and bovine α-chymotrypsin in a stoichiometric manner as indicated by gel chromatography. It had very poor action on subtilisin BPN', porcine elastase, pronase, Streptomyces caespitosus protease and Aspergillus oryzae protease. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective on human and pig pancreatic extracts. Modification of amino groups, guanido groups and sulphydryl groups of the inhibitor resulted in loss of inhibitory activity. Reduction of disulphide bridges, reduction with sodium borohydride and periodate oxidation also decreased the inhibitory activity.

Comparison of biochemical characterisation of ICRC bacilli with M. leprae: effect of substrate alteration in the medium.

ICRC-bacilli strain C-44 when grown in Dubos medium of its equivalent, express M. avium taxonomic biochemical characters. Assuming that difference in characters of M. leprae and ICRC bacilli, could be due to 'in vivo' and 'in vitro' milieu, we altered the substrates in the medium. The bacilli grow well in the new medium containing selenium, ferric nitrate, magnesium chloride and deleting Tween 80. The ICRC strain C-44 grown in new medium expressed characters: 9/10 similarity with M. leprae. The 10 day tween hydrolysis reaction in weak but positive. It is probable that 'M. leprae culture isolate', may have acquired 'in vitro' growth potential by recombination with M. avium, an ubiquitous mycobacterium. The M. leprae culture isolate thus may express some characters of both M. leprae and M. avium.

Dopa-oxidase activity on ICRC bacilli.

Presence of O-phenoloxidase is regarded as M. leprae specific character. This enzyme activity was found to be present in ICRC bacilli, Strain C-44. Though this strain is cultivable 'in vitro', the expression of DOPA-Oxidase activity strongly suggests that it carries M. leprae genome. The ICRC bacilli, therefore, may thus from a group of M. leprae culture isolates, distinct from other known cultivable mycobacteria which do not possess this enzyme activity.

Effect of hydrocortisone on arginase activity in ICRC mouse mammary tumours in vitro.

Three fold increase of arginase activity was observed in hydrocortisone treated mammary tumour tissue when compared to the untreated tissue in vitro. No change in succinic dehydrogenase activity was observed. It is likely that arginase present in mammary tumour is due to the presence of mammary tumour virus and it is tempting to speculate that the increase in arginase activity by hydrocortisone may be due to sustained viral production in the presence of hydrocortisone.