Biomedical subjects
Publications and source records attributed to M Höfer.
Strains of Pasteurella multocida use L-aspartate, L-malate and furmarate, respectively, as substrates for production of succinic acid which accumulates in the medium. As was established by studies with 14C and 3H labelled substrates, the degradation of these substances proceeds analogous via the citric acid cycle.
The effect of the polyene antibiotic nystatin on the energy-requiring transport of substrates in the obligatory aerobic yeast Rhodotorula glutinis (gracilis) is restricted to its interaction with the plasma membrane, whereas the energy metabolism in mitochondria remains unaffected by the antibiotic. A method for preparation of the mitochondrial fraction from this yeast is described.
Analysis of the temperature dependence of the monosaccharide transport system in the yeast Rhodotorula gracilis (ATCC 26194, CBS 6681), as tested with D-xylose, revealed that the apparent affinity of the transport system, measured as the reciprocal of the half-saturation constant KT, increased when transport velocity was stimulated by temperature (15--30 degrees C) and decreased when the rate of uptake was reduced at temperatures aboce 30 degrees C. Breaks in Arrhenius plots were accompanied by corresponding breaks in van't Hoff plots. Whereas untreated cells exhibited in the van't Hoff plot a discontinuity at 28--30 degrees C this was not observed in heat-treated cells (at either 37 or 45 degrees C). In heat-treated cells the maximum transport velocity was always lower and the apparent affinity higher than in untreated cells at the same temperature; the optimum temperature for both transport velocity and apparent affinity was shifted to higher values. The data are interpreted in terms of a reversible phase transition of membrane lipids effecting an irreversible alteration of membrane structure. The temperature-induced reversible alkalinization of unbuffered yeast suspensions supports this interpretation.
1. The uptake of monosaccharides and polyols in the obligatory aerobic yeast Rhodotorula gracilis (glutinis) was accompanied by proton uptake. 2. The half-saturation constant of transport, KT, depended on pH, changing from about 2mM at pH 4.5 to 80mM at pH8.5 for D-xylose; this change of the effective carrier affinity was reversible. 3. The apparent dissociation constant of the monosaccharide carrier was estimated at pKa 6.75. 4. At pH8.5, when the pH gradient across the cell membrane vanished, no sugar accumulation was demonstrable. 5. The half-saturation constants of sugar uptake and H+ co-transport were very similar to each other, the latter obviously being controlled by the former. 6. The H+/sugar stoicheiometry remained constant under various physiological conditions; it amounted to one H+ ion per sugar molecule taken up. 7. The data are interpreted as a strong piece of evidence in favour of the active monosaccharide transport in R. gracilis (glutinis) being an H+-symport energized by the electrochemical gradient of H+ across the plasma membrane of the yeast.
Two culturing recipes were suggested for Pasteurella multocida and Pasteurella haemolytica, one partialy defined nutritive defined nutritive medium with 0.5 per cent yeast extract and one chemically defined medium. Both had been composed on the basis of studies into nutrient demand and into the effects of complex nutritive substrates. Both nutritive media are applicable also to culturing other bacterial species. (Among the strains tested so far were streptococci, staphylococci, Escherichia coli, pseudomonas, protest, Salmonella,, Shigella, and Haemophilus.)
The obligate aerobic yeast Rhodoturula gracilis was found to take up the alditols D-glucitol, D-mannitol, ribitol, xylitol, D-arabinitol, L-arabinitol and erythritol by means of a constitutive mobile membrane carrier. This uptake involved active transport, that is, it was dependent on the supply of metabolic energy, leading to the accumulation of alditols inside the cells. The accumulation ratio (intracellular concentration to extracellular concentration, Si/SO) was much lower for alditols than for monosaccharides. As for monosaccharides, this ratio decreased with increasing extracellular concentration, even to values below 1. The kinetic data showed that the carrier system for alditols was identical to that for monosaccharides, though it had a much lower affinity and maximum velocity for alditols. Hence the uptake of alditols was blocked in the presence of monosaccharides. Only ribitol and L-arabinitol were catabolized following enzyme induction. The other alditols were not broken down.
Although alditols reached the interior of the cells of Rhodotorula gracilis by means of the constitutive monosaccharide carrier, a second carrier of pentitols was found to be inducible. Ribitol and L-arabinitol, entering the cells by the constitutive carrier, induced a single catabolic enzyme system, including a carrier of pentitols. This induction depended on de novo protein synthesis. In addition to ribitol and L-arabinitol, the induced catabolic enzyme system was also specific for xylitol and D-arabinitol; these two alditols were not inducers. Hexitols, erythritol and pentoses were not transported or catabolized by the induced system. The induced carrier had a lower half-saturation constant, KT, and higher maximal velocity, V, than the constitutive carrier of penitols. Kinetic studies of induced transport indicated, that this was an active process which appeared to be coupled with the simultaneous uptake of H+-ions (H+-substrate symport). The induced transport of pentitols was strongly inhibited by monosaccharides, although not competitively. The enzyme synthesis induced by ribitol or L-arabinitol was repressed by monosaccharides.
The nutrient demand of S-dublin strains was investigated. Media on chemically defined basis for culturing of S. dublin are recommended with reference to the above studies. The definition includes Na2HPO4, KH2PO4, NaCl, glucose, the amino acids L-asparaginic acid, L-cystine, L-glutamic acid, serine, and L-threonine, and nicotinamide as vitamin component. Such media will be quite favourable for certain culturing problems, but complex substrate (yeast extract, peptone, tryptone) should be added for high yields.
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The temperature dependence of the active monosaccharide transport across the cell membrane of the yeast Rhodotorula gracilis has been studied between 0 and 55 degrees C with D-xylose as the transported substrate: (i) Between 0 and 10 degrees C there is virtually no transport. (ii) The initial velocity of transport increases exponentially from 15 to 30 degrees C (deltaE equal to 32 plus or minus 2 kcal/mol). (iii) At 30 degrees C a sharp "break" occurs in the Arrhenius plot and with increasing temperature the transport becomes inactivated, with a positive slope of the corresponding straight line ("deltaE equal to minus 15 kcal/mol"). (iv) In the temperature range of 50-55 degrees C, both the transport and the metabolic activity cease. In order to account for the abrupt changes of the membrane permeability, we attempted to ascribe them to phase transitions in the membrane structure: the first one, between 10 and 15 degrees C, to the crystalline: liquid-crystalline phase change; the second one, around 30 degrees C, to a change from highly ordered (low entropy) to less ordered (high entropy) membrane structure. Whereas the former phase transition is reversible, the latter appears to be irreversible. Arrhenius plots of the cell respiration exhibit a "break" at 30 degrees C, as well. However, at higher temperatures there is no thermal inactivation of the respiratory activity. The importance of a proper organization of the cell membrane constituents for the efficient transport function is discussed.
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