An analysis of intracellular and extracellular leptospiral enzymes was made by use of starch-gel electrophoresis with natural and synthetic substrates. Of 37 serotypes examined for extracellular exterase, all had activity of varying mobility and degree. All extracellular preparations were negative for catalase, phosphatase, and naphthylamidase. Intracellularly, five serotypes were examined, including Leptospira biflexa Patoc I, L. biflexa Waz, L. canicola Moulton, L. icterohaemorrhagiae RGA, and L. pomona S91. Among the enzymes detected by this electrophoretic technique were transaminase and catalase, confirming the results of previous investigators. Further, other enzymes heretofore unreported have been detected. These include esterases, phosphatases, lactic, malic, glutamic, succinic, a-glycerophosphate, and 6-phosphogluconic dehydrogenases, and a naphthylamidase. The presence of these enzymes suggests the existence of tricarboxylic acid, glycolytic, and pentose-related pathways in Leptospira. In addition, enzyme patterns show promise in leptospiral classification. Recent investigations have established the presence of the following enzymes in leptospires: catalase (14, 35), oxidase (13, 20), transaminase (27), and lipase (2, 23, 32). Phosphatase activity was determined by Dymowska and Zakrzewska (9) in culture filtrates of leptospires grown in Korthoff's medium, and results were equivocal. This study was concerned with a broad examination of leptospiral enzymes to (i) gain insight into metabolic sequences, (ii) relate enzyme patterns to classification and taxonomy as has been done for other bacteria (3, 25, 31), and (iii) look for enzymatic differences between avirulent and virulent serotypes of leptospires. MATERIALS AND METHODS Cultures. In this study, 37 strains of leptospires were used (Table 1). All were grown initially in Stuart's (38) medium supplemented with 10% rabbit serum and 0.15% agar. Prior to enzymatic studies, all cultures were grown for two transfers in albumin-Tween 80 liquid medium (10, 11). Before mass cultivation procedures were initiated, the cultures were washed and used as the inocula for extracellular and intracellular enzyme studies. Extracellular enzyme preparation. Cell-free culture 'This is a portion of a dissertation submitted by Stanley S. Green to the faculty of the University of Missouri Graduate School in partial fulfillment of the requirements for the Ph.D. degree in Micro

Extracts of 18 serotypes of the genus Leptospira were found to possess naphthylamidase activity, and differences in the pathogenic and saprophytic strains were noted. The former exhibited a preference for the leucyl naphthylamide substrate, whereas the latter demonstrated greater hydrolysis of alanyl naphthylamide. With the leucyl naphthylamide as substrate, pathogenic strains showed 10 to 20 times higher naphthylamidase activity than saprophytic strains. Optimal temperature and pH for enzymatic hydrolysis also differed between pathogenic and saprophytic strains. Maximal enzymatic activities for pathogenic and saprophytic naphthylamidases were 41 and 37 C, respectively, at pH 8.0 to 8.5. The pH and temperature optima suggested that the leptospiral enzyme activity was not leucine aminopeptidase.