Isoforms of baker's yeast transketolase

. .     

The influence of dyes on the respiration of baker's yeast

Résumé La respiration de la levure de boulangerie en milieu glucose-phosphate est inhibée par la trypaflavine, le bleu de methylène, le violet de cristal. Cette inhibition disparaît du moins partiellement par addition d'acide nucléique, d'acide adénylique et d'A.T.P.     

Inhibition of thiamine transport in baker's yeast by methylene blue

Summary Methylene blue was found to inhibit thiamine transport competitively (K_i=0.63 M) in baker's yeast. The dye was also effective in abolishing the growth inhibition ofSaccharomyces cerevisiae by pyrithiamine which is known to be taken up by a common transport system for thiamine in yeast cells. A possible mechanism for the inhibition by methylene blue of the thiamine transport system in baker's yeast is discussed.     

The number of coenzyme binding sites in transketolase from baker's yeast

Zusammenfassung Nachweis, dass 2 Mole Thiamindiphosphat pro Mol Apotransketolase gebunden werden.     

Observations on the alleged occurrence of vitamin A in baker's yeast

Résumé Traité au SbCl₃, l'extrait de lipide de levure de boulangerie prend en aérobiose une couleur bleue. Ce résultat n'est pas dû à l'action de la vitamine A ou des caroténoïdes.     

Presence of a specific uridine 5'-monophosphate pyrophosphorylase in baker's yeast.

Uridine 5'-monophosphate pyrophosphorylase was found to be present in baker's yeast. The enzyme preparation, purified about 30-fold, shows a strict specificity toward uracil and requires Mg++ for its activity.     

The influence of salts on the inhibition of the respiration of baker's yeast by basic dyes

Résumé MgCl₂ et d'autres sels à des concentrations définies protègent la respiration de la levure de boulangerie contre l'action inhibitrice de colorants basiques.     

Permeability of rat heart myocytes to cytochrome c

Rat heart myocytes undergoing progressive damage demonstrate morphological changes of shortening and swelling followed by the formation of intracellular vacuoles and plasma membrane blebbing. The damaged myocytes displayed impaired N,N'-tetramethyl-p-phenyldiamine (TMPD) ascorbate-stimulated respiratory activity which was restored by the addition of reduced cytochrome c to the cell culture medium. To clarify the role played by cytochrome c in the impairment of cell respiration, polarographic, spectrophotometric and fluorescence as well as electron microscopy imaging experiments were performed. TMPD/ascorbate-stimulated respiratory activity returned to control levels, at approximately 20 microM cytochrome c, establishing the threshold below which the turnover rate by cytochrome c oxidase in the cell depends on cytochrome concentration. Mildly damaged cardiac myocytes, as indicated by cell shortening, retention of visible striations and free-fluorescein exclusion, together with the absence of lactate dehydrogenase leakage and exclusion of trypan blue, were able to oxidize exogenous cytochrome c and were permeable to fluorescein-conjugated cytochrome c. The results, while consistent with an early cytochrome c release observed at the beginning of cell death, elucidate the role played by cytochrome c in the kinetic control of mitochondrial electron transfer under pathological conditions, particularly those involving the terminal part of the respiratory chain. These data are the first to demonstrate that the sarcolemma of cardiac myocytes, damaged but still viable, is permeable to cytochrome c.     

Determination of the binding constant of thiamine diphosphate in transketolase from baker's yeast by circular dichroism titration

Zusammenfassung Mit Hilfe des Circulardichroismus wurde die Bindungskonstante von Thiamindiphosphat in der Transketolase der Bäckerhefe bestimmt.     

Parameters influencing inactivation-dissociation/reactivation-association phenomena induced by variations of ionic strength of L (+)lactate: cytochrome c oxidoreductase (cytochrome b2) extract of the yeast Hansenula anomala

H-flavocytochrome b2, a tetramer enzyme, is inactivated, at low ionic strength and can be reactivated, increasing the ionic strength of the medium. The inactivation-reactivation process was structurally manifested by a dissociation-association phenomenon between subunits. It was clearly shown that the inactivation-dissociation process appeared independent of enzyme concentration whereas the reactivation-association phenomenon was enzyme concentration dependent. However, proteins protect H-flavocytochrome b2 from inactivation-dissociation, only when electrostatic interactions are possible between the two proteins: Horse heart cytochrome c was a good protector whereas serum albumin had no protector effect.     

A cytochrome c from the hepatopancreas ofSepia officinalis L.

Riassunto Una emoproteina è stata estratta e parzialmente purificata dall'epatopancreas diSepia officinalis. Essa è ossidata dal ferricianuro e ridotta dall'acido ascorbico; gli spettri di assorbimento delle due forme, ossidata e ridotta, sono quelli tipici del citocromo c di mammifero.In comune con quest'ultimo il citocromo estratto dall'epatopancreas di sepia ha diverse altre proprietà come per esempio quella di non dare un emocromogeno con la piridina e di non perdere il gruppo prostetico dopo trattamento con acetone acido. A differenza del citocromo c di mammifero, il pigmento di sepia non resiste al calore e agli acidi; è precipitato dal solfato di ammonio al 75% di saturazione e sedimenta dopo prolungata centrifugazione a 105000×g. Non è ossidato dalla citocromossidasi di mammifero.

---

---

This work has been supported by a grant (RG-4548) of the U.S. Public Health Service.     

The ratio between the fast and slow forms of bovine cytochrome c oxidase is changed by cholate or nucleotides bound to the cholate-binding site close to the cytochrome a3/CuB binuclear centre

We determined the fraction of 'slow' and 'fast' conformations of bovine cytochrome c oxidase, following the kinetics of cyanide binding to the oxidized enzyme. We investigated whether treatment of heart mitochondrial particles with different commercially available types of cholate (standard and ultrapure) can affect the fraction of cytochrome c oxidase in the two states. Compared to standard cholate, the use of ultra-pure cholate for solubilization of heart mitochondrial particles significantly increased the fraction of the fast enzyme. Complete homogeneity (approximately 100% fast) was observed when cytochrome c oxidase was solubilized with ultra-pure cholate from heart mitochondrial particles pre-equilibrated with AMP; equilibration with ADP yielded a much smaller fraction of fast enzyme (approximately 35%). These observations are discussed on the basis of the structural relationships between the known cholate-binding site and the binuclear cytochrome a3-CuB site: variation in the occupancy of this binding site with cholate or nucleotides may modify reactivity of the oxidized binuclear centre towards cyanide.     

The precipitation of cytochrome C with a lipidic fraction from tissues and yeast

Riassunto Nel corso di ricerche sopra l'azione esplicata da alcune frazioni lipidiche sulla succinossidasi presente nei mitocondri di fegato di ratto, l'autore ha osservato che il citocromo C aggiunto al sistema precipita in presenza della frazione contenente lecitine cefaline e fosfatidi, estratta dal lievito o dal fegato di animali diversi.Di questo materiale è stata compiuta l'analisi elettroforetica su carta che non ha rivelato proteine migranti. L'analisi cromatografica su carta non ha mostrato aminoacidi liberi, ma dopo idrolisi 6–10 aminoacidi sono evidenziabili. Il precipitato risultante dalla unione della frazione lecitinica con il citocromo C è stato analizzato per il suo contenuto in P ed in N comparativamente ai materiali di origine. Detto precipitato è solubile solo in etere e la curva spettrofotometrica del citocromo C in queste condizioni appare modificata rispetto alla norma.La frazione lecitine non precipita in presenza di albumina di uovo e di emoglobina umana, mentre dà un forte precipitato in presenza di solfato di Fe e di citrato ferrico ammoniacale.L'attività biologica del citocromo C si annulla completamente con la precipitazione.     

The possible role of isoforms of cytochrome c oxidase subunit VIa in mammalian thermogenesis

A single cDNA of cytochrome c oxidase subunit VIa was characterised from liver, heart and the thermogenic organ of the partially endotherm tuna fish. The amino acid sequence revealed high identity with subunit VIa from carp and trout, but low identity to subunits VIaL (liver type) and VIaH (heart type) of mammalian cytochrome c oxidase. In reconstituted cytochrome c oxidase from bovine heart, the H ⁺/e⁻ stoichiometry is decreased from 1.0 to 0.5 at high intraliposomal ATP/ADP ratios via exchange of bound ADP by ATP at the matrix domain of the transmembraneous subunit VIaH. Reconstituted cytochrome c oxidase from bovine liver and kidney, containing subunit VIaL, revealed H ⁺/e⁻ ratios below 0.5, independent of the ATP/ADP ratio. The results suggest the evolution of three types of subunit VIa. Subunits VIaH and VIaL are postulated to participate in mammalian thermogenesis. Received 3 May 1999; received after revision 10 June 1999; accepted 29 June 1999