Morton M. Weber

Respiratory control in mitochondria from Crithidia fasciculata.

Summary The trypanosomatid Crithidia fasciculata contains a single bifurcated mitochondrial organelle, and a method is described for its isolation as a relatively intact structure. These preparations carry out oxidative phosphorylation and show respiratory control with succinate as substrate. The oxidation and phosphorylation efficiency of succinate and α-glycerophosphate, as well as NAD+-linked substrates, are affected by antimycin A, mCl-CCP, and rutamycin.

Coenzyme Q9 (ubiquinone (45)) and ergosterol in Crithidia fasciculata.

Abstract 1. 1. A quinone has been isolated from the hemoflagellate Crithidia fascicidata and characterized as a coenzyme Q 2 (ubiquinone(45)) by its ultraviolet absorption spectrum, melting point, and Chromatographic behavior. 2. 2. Ultraviolet absorption spectra of solvent extracts of the organism indicated the presence of a sterol. It was identified as ergosterol by its characteristic ultraviolet and infrared absorption spectra, as well as by the melting points of the compound and its benzoate and acetate derivatives. 3. 3. Kinetic studies on the lysis of the organism by digitonin suggest that the sterol is located in the cell membrane.

Isolation, purification, and characterization of Crithidia fasciculata cytochrome c555☆

Abstract Cytochrome c555 was isolated from Crithidia fasciculata (Anopheles strain; A.T.C.C. No. 11745), and was purified by salt fractionation and chromatography on Amberlite CG-50 cation-exchange resin. The cellular content of cytochrome c555 was estimated to be 1.56 × 105 molecules per cell, accounting for about 25% of the hemin requirement of the organism. Absorbance maxima occurred at 413 mμ for the oxidized form, and at 420, 525, and 555.5 mμ for the reduced form, the extinction coefficient of the latter peak having been determined to be 29.7 m m −1 cm−1. The α-maximum of the pyridine hemochromogen was at 553 mμ. Acid acetone failed to remove the heme moiety from the cytochrome. The mono-heme protein had a minimum molecular weight of 12,051, calculated from the amino acid composition. The value calculated from ultracentrifugation data was 13,200. The mobility of the protein on Amberlite columns and upon electrophoresis in polyacrylamide gels was consistent with that which would have been expected from the amino acid composition. The isoelectric point was at pH 9.9, while the midpoint of the oxidation-reduction potential was +280 mV at pH 7.0. The cytochrome was not autooxidizable, nor did it combine with carbon monoxide at neutral pH. Comparison with other purified cytochromes c from a variety of sources showed that, in most of its properties, cytochrome c555 was similar to cytochromes from certain yeasts.

Oxidative phosphorylation in Crithidia fasciculata

Abstract Respiratory chain-linked phosphorylation has been demonstrated in cell-free extracts from the protozoan Crithidia fasciculata . The preparations were capable of coupling phosphate esterification to respiration, but were not “tightly coupled”. Oxidation and phosphorylation were sensitive to respiratory inhibitors and uncoupling agents.The P/O ratios observed with NADH and succinate as electron donors were lower than those obtained with mammalian mitochondria, but were typical for microbial systems.

Concerted inhibition of a NADP+-specific isocitrate dehydrogenase and the implications for metabolic regulation☆

Abstract A possible physiologic role for the concerted inhibition of a NADP + -specific isocitrate dehydrogenase by oxalacetate and glyoxylate is presented. The significance of this inhibition is also discussed with respect to the “induced fit” theory of enzyme-substrate interaction. However, since numerous structural analogues of these two compounds were without effect, it is believed that these two components are not acting as a “synthetic” substrate.

The relationship of soluble and mitochondrial isocitrate dehydrogenases in metabolic regulation

Abstract It has been shown that the soluble NADP+-specific isocitrate dehydrogenases from both prokaryotic and eukaryotic cells are inhibited by oxalacetate and glyoxylate in a concerted manner. We have now compared the mitochondrial and soluble isocitrate dehydrogenases from N. crassa and S. cerevisiae with respect to their inhibition by these compounds. The NAD+-linked mitochondrial enzymes were not inhibited, whereas the NADP+-linked enzymes were. The significance of this inhibition is discussed with respect to the regulatory role of these two enzymes.

Kinetic studies of a NADP+-specific isocitrate dehydrogenase from Salmonella typhimurium: Purification and reaction mechanism

Abstract The kinetics of a Mn2+-requiring, NADP+-specific isocitrate dehydrogenase from Salmonella typhimurium have been examined by the measurement of initial velocity rates in the presence and absence of the reaction products. The binding of each of the cosubstrates, isocitrate, and NADP+, is not independent of the other, and the isocitrate-Mn2+ complex is the kinetically important substrate species. All of the reaction products, α-ketoglutarate, CO2, and NADPH are competitive with both cosubstrates and the mechanism appears to be of the rapid equilibrium random type. The enzyme has been purified to homogeneity and has an isoelectric point at pH 4.0–4.2, and an apparent molecular weight of 102,000.

The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata

Abstract RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit of the trypanosomatid flagellate Crithidia fasciculata and analyzed by polyacrylamide gel electrophoresis at 4 °C consisted of one species with a molecular weight of 1.3 × 10 6 (relative to ribosomal RNA from E. coli MRE 600). When extracted with hot phenol (65 °C), the large ribosomal subunit gave rise to two components with molecular weights of 0.72 and 0.56 × 10 6 . On heating for 60 s, followed by rapid cooling, the single cold-phenol-extracted 1.30 × 10 6 -dalton species completely dissociated into two components of molecular weights 0.72 and 0.56 × 10 6 , present in equimolar amounts. When analyzed by polyacrylamide-agarose gel electrophoresis in the presence of SDS, RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit consisted of three components of molecular weights 1.3, 0.72, and 0.56 × 10 6 , present in apparently equimolar amounts. RNA from the small cytoplasmic ribosomal subunit consisted of one species with a molecular weight of 0.84 × 10 6 , independent of extraction or analytical conditions. It is proposed that under high salt and low temperature conditions, the large ribosomal RNA molecule is held together by its secondary structure, and that denaturing extraction or analytical conditions reveal an otherwise “hidden” lesion present in the molecule in vivo .

Chloramphenicol resistant mitochondrial protein synthesis in Crithidia fasciculata

Abstract Growth of Crithidia fasciculata , a heme-requiring flagellated protozoan, was not affected by the presence of chloramphenicol in concentrations as high as 4 mg/ml. Amino acid incorporation by a mitochondrial fraction, containing both mitochondria and kinetoplasts, was resistant to chloramphenicol and cycloheximide, but sensitive to puromycin. The postmitochondrial supernatant (27,000 g ) also incorporated [ 14 C]leucine into trichloroacetic acid insoluble material, but this incorporation was inhibited by cycloheximide. Chloramphenicol did not affect this system. Treatment of the mitochondrial fraction with digitonin enhanced the rate of [ 14 C]leucine incorporation, but did not render the preparation sensitive to chloramphenicol. Electron microscopic examination of the digitonin treated mitochondrial fraction revealed the presence of ghosts. Separation of kinetoplasts from mitochondrial vesicles was achieved by discontinuous sucrose gradient centrifugation. Both the kinetoplast and mitochondrial fractions incorporated [ 14 C]leucine at about the same rate. No inhibition of incorporation by chloramphenicol was observed with either fraction.