Isozymes in the culture forms of Leishmania tarentolae.

Abstract

SYNOPSIS. Leishmania tarentolae grown in Trager's defined medium C, blood brain heart infusion broth and blood agar contained 2 forms of malic dehydrogenase (MDH) after zone electrophoresis in potato starch: one at the point of origin and the other migrating towards the anode. The pH optimum with oxaloacetate as substrate was ≃ 8.35 for the anodal form and 7.50 for the point of origin enzyme. The Michaelis constant (Km) with oxaloacetate was 1.8–2.8 × 10−5 M for the anodal form and 4.0 × 10−5 M for the nonmigratory form. At pH 7.4, both MDHs were inhibited by oxaloacetate concentrations greater than 3.75 × 10−4 M. Ratios of activity with different NAD analogs were dissimilar. A few of the non‐migratory enzyme ratios corresponded with those reported for mitochondrial MDH. There was no correspondence between the ratios shown by anodal MDH and ratios reported either for mitochondrial MDH or for cytoplasmic MDH. The thionicotinamide analog was not utilized by point of origin MDH; however, the anodal form did show greater activity with this analog which is a characteristic of cytoplasmic MDH. Anodal MDH was more stable than non‐migratory enzyme. Heat inactivation studies indicated 80% inactivation at 68°C for the anodal form and 100% inactivation at 37°C for the other form. The point of origin enzyme had a half life of about 48 hours at 4°C whereas anodal MDH was stable for at least one week at 4°C. Addition of enzyme stabilizing agents (Cleland's reagent, mercaptoethanol and gelatin) did not prevent breakdown of the non‐migrating enzyme. Phosphate buffer increased the activity of the point of origin enzyme but had no effect on anodal MDH. On the basis of the above results, non‐migratory enzyme is thought to be a variant of mitochondrial MDH. The characteristics of the anodal MDH do not readily indentify it as a typical mitochondrial or cytoplasmic type and it may be a modified type similar to those found in parasitic protozoa by other workers.