A comparison of mitochondria from Torulopsis utilis grown in continous culture with glycerol, iron, ammonium, magnesium or phosphate as the growth-limiting nutrient

1. Mitochondria prepared from Torulopsis utilis grown in a chemostat with iron-limited growth were found to lack energy conservation but not electron flow in that segment of the respiratory chain leading from intramitochondrial NADH to the cytochromes [i.e. the site 1 segment (Lehninger, 1964)]. 2. Site 1 energy conservation was present in mitochondria prepared from cells grown under conditions of limitation by glycerol, ammonium and magnesium. Phosphate-limited growth resulted in mitochondrial preparations without respiratory control. 3. Mitochondria from cells grown under conditions of iron limitation were insensitive to the respiratory inhibitor piericidin A, whereas sensitivity was present in mitochondria prepared from glycerol-, ammonium-, magnesium- or phosphate-limited cells. 4. These observations are considered to provide indirect evidence for a role of non-haem iron in the mechanism of energy conservation and also piericidin A sensitivity in T. utilis mitochondria. 5. A readily constructed and inexpensive pH-measuring and -controlling circuit is described for use with continuous-culture apparatus.     

A role of ubiquinone in energy conservation in mitochondria

Short chain ubiquinones (Q-3) uncouple oxidative phosphorylation in rat heart mitochondria, as shown by polarimetric experiments, and abolish P:O ratios in succinate driven oxidative phosphorylaton. The uncoupling is reversed by long chain ubiquinones (Q-7). Furthermore, short chain ubiquinones abolish oligomycin sensitivity of ATPase; the inhibition is restored by Q-7. The extraction of endogenous ubiquinone from mitochondria reversibly lowers oligomycin sensitivity of ATPase.     

On energy conservation and transfer in mitochondria

Structural and functional features of energy conservation and transfer in mitochondria have been examined in the light of recent developments, and hypothetical schemes for energy coupling and transfer have been presented.Supported by USPHS grant AM-08126 to Y.H. and San Diego County Heart Association Grant-in-Aid 97-71 to W.G.H.Recipient of USPHS career Development Award 1-K4-GM38291.     

Nucleotide sequence of leucine transfer RNA 1 from Candida (Torulopsis) utilis.

Two species of 32P-labelled leucine tRNA were highly purified from Candida (Torulopsis) utilis by successive column chromatographies. The purified major species of leucine tRNA 1 was completely digested with ribonuclease T1 [EC 3.1.4.8] and with pancreatic ribonuclease A [EC 3.1.4.22]. The resulting fragments were fractionated, and their nucleotide sequences were determined according to Barrell (1). The results of analyses of the two ribonuclease digests were consistent with each other, and indicated that this tRNA is composed of 85 nucleotide residues, including 14 modified nucleotides. A tentative total sequence has been derived on the basis of several features in the cloverleaf structure for tRNA.     

A modified nucleoside located in the anticodon of proline tRNA from Torulopsis utilis is 5-carbamoylmethyluridine

A modified nucleoside has been isolated from the first position of the anticodon of Torulopsis utilis tRNAPro. It was identified to be an uridine derivative, 5-carbamoylmethyluridine from analyses of its UV, 1H-NMR, and secondary ion mass spectra.     

Catalytic activity and EPR signals of DPNH dehydrogenase in relation to the acquisition and loss of piericidin sensitivity and of coupling site 1

The events accompanying the development of piericidin sensitivity and of energy coupling site 1 during the transition of Candida utilis cells from the log phase to the late stationary phase of growth have been investigated. There is a large increase in DPNH dehydrogenase (DPNHD) activity, and major increases in the EPR signals of iron-sulfur centers 1, 2, and 3 of the enzyme, as measured at 13°K. The increase in DPNHD activity, however, does not reflect the increased development of the same enzyme as is present in the log phase, for the enzyme being synthesized in the stationary phase is different from that present in the log phase, as judged by juglone reductase and DPNH oxidase activities, which declined during the transition, and by stability, kinetics, and in the type of EPR signal present, which are different in the log and stationary phases. On catabolite repression of stationary phase cells the converse occurs: the specific activities of juglone reductase and DPNH oxidase rise, DPNHD activity and EPR signals corresponding to centers 1 and 2 disappear. This process of catabolite repression by ethanol is prevented by cycloheximide.     

Piericiden A sensitivity, site 1 phosphorylation, and reduced nicotinamide adenine dinucleotide dehydrogenase during iron-limited growth of Candida utilis.

It has been reported that cells of Candida utilis, grown in continuous culture under iron-limited conditions, develop site 1 phosphorylation, without the appearance of piericidin sensitivity and without changes in the iron-sulfur centers of NADH dehydrogenase, on aeration in the presence of cycloheximide, as well as on increasing the supply of iron during growth. These findings were reinvestigated in the present study. The parameters and properties followed during these transitions were sensitivity of NADH oxidation to piericidin, presence or absence of coupling site 1, EPR signals appearing on reduction with NADH or dithionite, the specific activities of NADH oxidase, NADH-ferricyanide reductase, and NADH-5-hydroxy-1,4-naphthoquinone (juglone) reductase, and the kinetic behavior of NADH dehydrogenase in the ferricyanide assay. Monitoring the rates of oxidation of NADH in submitochondrial particles with artificial oxidants, observing the kinetics of the ferricyanide assay, and measuring the concentration of iron-sulfur centers elicited by EPR permitted ascertaining the type of NADH dehydrogenase present and its relative concentration in different experimental situations. It was found that on gradually increasing the concentration of iron during continuous culture (transition from ironlimited to iron- and substrate-limited growth), as well as on aeration of iron-limited cells, coupling site 1, piericidin sensitivity, NADH-ferricyanide activity, and iron-sulfur centers 1 and 2 increased concurrently, with concomitant decline of NADH-juglone reductase activity. Cycloheximide prevented all these changes. Iron-sulfur centers 3 plus 4 underwent relatively little increase during these transitions. It is concluded that in both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piercidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement. No evidence was found for the development of coupling site 1 without the appearance of piericidin sensir th