Identification of the Terminal Respiratory Chain in Kinetoplast

Kinetoplast-mitochondrial vesicles from Leishmania tropica promastigotes were prepared by subjecting the particulate fractions from cell lysates to floatation ultracentrifugation in renografin gradients. They formed a distinct band with a density of 1.19 to 1.22 g/ml and appeared homogeneous as judged by phase microscopy. They showed succinate-cytochrome c reductase activity as well as the incorporation of [3H]thymidine into DNase-resistant DNA. As would be expected of intact mitochondria, these vesicles were impermeable to NADH and failed to show any stimulation of oxygen uptake with the addition of exogenous cytochrome c. Succinate oxidation by these vesicles was partially inhibited by oxalacetate, but sonication resulted in almost complete inhibition further indicating the intactness of these vesicles. Simple difference spectra from these kinetoplast-mitochondrial vesicles after enzymatic and/or chemical reduction showed the predominance of cytochrome b with only small amounts of cytochromes a and c. However, by manipulating the assay conditions, the expression of cytochromes a + a3 and c was enhanced considerably. The presence of cytochromes a + a3 and c was further corroborated by the identification of heme a and heme c from pyridine hemochromogen difference spectra. Carbon monoxide difference spectra demonstrated the presence of cytochrome a3 and a cytochrome o-like pigment. Respiration by the kinetoplast-mitochondrial vesicles was inhibited 100% by KCN (below 1 mu) and no evidence was obtained for the presence of an a-glycerophosphate oxidase. By using site-specific inhibitors with substrate oxidations, a possible scheme of the terminal respiratory chain in L. tropica promastigotes is postulated.