The presence of rotenone-sensitive NADH dehydrogenase in the long slender bloodstream and the procyclic forms of Trypanosoma brucei brucei.

The mitochondrial electron-transport chain present in the procyclic and long slender bloodstream forms of Trypanosoma brucei brucei was investigated by means of several experimental approaches. The oxidation of proline, glycerol and glucose in procyclic cells was inhibited 80-90% by antimycin A or cyanide, 15-19% by salicylhydroxamic acid, and 30-35% by rotenone. Cytochrom-c-reductase activity, with proline or glycerol 3-phosphate as substrate, in a mitochondrial fraction isolated from these cells was inhibited by antimycin and rotenone, but not by malonate, while cytochrome-c-reductase activity with succinate as substrate was inhibited by antimycin A and malonate, but not by rotenone. In addition, the reduction of dichloroindophenol by NADH was inhibited by rotenone but not by malonate, which suggests that rotenone-sensitive NADH dehydrogenase (complex I) is present in these mitochondria. The presence of three subunits of NADH dehydrogenase was observed in immunoblots of mitochondrial proteins with specific antibodies raised against peptides corresponding to predicted antigenic regions of these proteins, which provides further evidence for the presence of NADH dehydrogenase. In long slender bloodstream forms, the oxidation of glucose or glycerol was inhibited 100% by salicyhydroxamic acid, unaffected by cyanide or antimycin A, and inhibited 40% or 75%, respectively, by rotenone, which suggests that NADH dehydrogenase is present in these cells. In a mitochondrial fraction isolated from the bloodstream forms, oxygen uptake with glycerol 3-phosphate as substrate was inhibited 65% by rotenone. Low levels of rotenone-sensitive NADH-dependent reduction of dichloroindophenol and the presence of subunits 7 and 8 of NADH dehydrogenase provided additional evidence for the presence of NADH dehydrogenase in bloodstream forms of T. brucei.

Topographical organization of cytochrome b in the yeast mitochondrial membrane determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide.

In previous studies, we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in the cytochrome bc1 complex from yeast mitochondria and was bound selectively to cytochrome b. Extensive trypsin digestion of [14C]DCCD-labeled cytochrome b isolated from a cytochrome bc1 complex treated with DCCD yielded a single radiolabeled 7.0 kDa peptide with the N-terminus VTLWNVG, indicating that trypsin cleavage had occurred at arginines-110 and -178. This segment of cytochrome b contains one acidic residue, aspartate-160, localized in amphiphilic, non-membrane-spanning, helix cd. To explore the environment of amphiphilic helix cd, we employed a fluorescent derivative of DCCD, N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide (NCD-4). After incubation of NCD-4 with a cytochrome bc1 complex isolated from yeast mitochondria, a fluorescent compound was formed with a 340 nm excitation peak and a 441 nm emission peak. NCD-4 was selectively bound to cytochrome b and inhibited proton translocation with only a minimal inhibitory effect on electron transfer in the cytochrome bc1 complex reconstituted into proteoliposomes. Competition experiments and trypsin digestion of NCD-4-labeled cytochrome b indicated that NCD-4 and DCCD were bound to the same site on cytochrome b. The fluorescence of NCD-4 bound to the cytochrome bc1 complex was quenched equally by CAT-16, an amphiphilic spin-label that intercalates at the membrane surface, and 5-doxylstearic acid, a nitroxide derivative of stearic acid, and to a lesser extent by 7-doxylstearic and 12-doxylstearic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical evidence for the orientation of cytochrome b in the yeast mitochondrial membrane in the eight-helix model.

The topographical localization of the N-terminus of cytochrome b in the inner mitochondrial membrane was determined by mild proteolysis of the yeast mitochondrial cytochrome bc1 complex and identification of the proteolytic fragments derived from subunits of the complex with an established orientation in the inner membrane. The cytochrome bc1 complex was incorporated into proteoliposomes which were separated by cytochrome c affinity chromatography into two populations in either the mitochondrial or the submitochondrial orientation. Core protein I which protrudes from the matrix side of the inner membrane was digested by proteinase K only in proteoliposomes with the submitochondrial orientation and not in those with the mitochondrial orientation. By contrast, cytochrome c1 with protrudes from the cytoplasmic side of the inner membrane was digested by proteinase K only in proteoliposomes with the mitochondrial orientation and not in those with the submitochondrial orientation. Cytochrome b was digested by SV8 protease only in proteoliposomes with the mitochondrial orientation to yield two aggregating fragments of 25.6 and 24.5 kDa. These peptides were isolated by preparative gel chromatography and sequenced to establish that the cleavage of cytochrome b by SV8 protease occurred at glutamate residues 59 and 66. These residues are localized in the extramembranous loop between the two hydrophobic membrane-spanning helices A and B and thus face the cytoplasmic side of the inner mitochondrial membrane. These results indicate that the N-terminus of yeast cytochrome b protrudes from the matrix side of the inner membrane consistent with the eight-helix model for the orientation of cytochrome b in the membrane.

The effect of diabetes on mevalonate metabolism in the rat.

Previous studies have demonstrated a major difference in mevalonate metabolism by male and female rats by both the shunt and sterol pathways. This important sex difference was shown to be related to the presence of oestrogens and/or progesterone. In the present study we have investigated the effects of streptozotocin-induced diabetes on mevalonate metabolism. Firstly, insulin deficiency decreased the ability of the rats to oxidize mevalonate to carbon dioxide by the shunt pathway both in vivo and in vitro. This decrease was reversed by insulin treatment both in the intact animals as well as in tissue slices. Secondly, diabetes caused a marked increase in hepatic sterologenesis in the intact animal. This is the first demonstration that insulin plays significant regulatory role in both the shunt and sterol pathways of mevalonate metabolism.

The quantitative role of the kidneys in the in vivo metabolism of mevalonate.

The roles of the sterol and nonsterol pathways in the metabolism of circulating mevalonate have been estimated in the intact rat. On an average, the sterol pathway accounts for 74 per cent of the mevalonate metabolized, while the nonsterol, or shunt, pathway is responsible for 26 per cent of the mevalonate metabolized in the whole animal. The contribution of the kidneys to each of these processes was evaluated by two approaches. First, the localization of labeled sterols and sterol precursors derived from [14C]mevalonate was determined in each of the major tissues of the body and, second, the effect of nephrectomy upon mevalonate metabolism by the sterol and shunt mechanisms was examined. The results confirm our earlier conclusion that the kidneys represent the primary tissue site of conversion of circulating mevalonate to sterols and sterol precursors. In the present study, it was shown that by 6 h after administration of [14C]mevalonate, the major end product of mevalonate metabolism in the kidneys is cholesterol and that, moreover, the kidneys are responsible for most of the cholestreol synthesized in the intact animal from injected mevalonate. Following nephrectomy, the extrarenal tissues can readily assume the dominant role normally played by the kidneys in synthesizing cholesterol and other sterols from circulating mevalonate. The major observation of the present study is that the kidneys represent the primary site of mevalonate metabolism by the shunt pathway, in that nephrectomy results in approximately a 60 per cent decrease in the mevalonate metabolized by the shunt pathway. These studies, therefore, reinforce and expand the evidence that the kidneys represent the most important single tissue site for the metabolism of circulating mevalonate.