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Biomedical subjects

G A Thompson

Publications and source records attributed to G A Thompson.

At least 199 records · Page 11Linked to original sources
PubMed1976

Molecular control of membrane properties during temperature acclimation. Membrane fluidity regulation of fatty acid desaturase action?

Further studies on the molecular mechanisms of temperature acclimation have been carried out using the ciliate Tetrahymena pyriformis. The most prominent change in lipid metabolism during acclimation to high temperature--depression of fatty acid desaturase activity--could be simulated by supplementing the growth medium of isothermally-grown cells with polyunsaturated fatty acids. Such cells resisted the membrane-fluidizing effect of the incorporated exogenous acids by increased use of de novo synthesized saturated acids in their phospholipids. The data support the conclusions arising from earlier experiments with temperature-shifted cells (Martin, C.E., Hiramitsu, K., Kitajima, Y., Nozawa, Y., Skriver, L., and Thompson, G.A., Jr. (1976), Biochemistry 15), showing that, when membrane fluidity increased to a superoptimal level, the activity of membrane-associated fatty acid desaturases was decreased. Since the reaction is controlled by membrane fluidity, rather than temperature per se, we postulate that it is the general mechnaism employed by cells adjusting to any fluidity-modifying factor, such as cations, drugs, etc.

Adaptation, Physiological↗
PubMed1976

Changes in membrane lipid composition during temperature adaptation by a thermotolerant strain of Tetrahymena pyriformis.

Experiments on temperature adaptation have been conducted using a thermotolerant clone of Tetrahymena pyriformis designated as strain NT-1. The strain was able to grow well at 39.5 and 15 degrees C and could adapt quickly when transferred from one of these temperatures to the other. Cells grown at the extreme temperatures differed markedly in their membrane lipid composition, particularly in the phospholipid polar head groups and hydrocarbon chains. The levels of fatty acid unsaturation increased at the lower temperature (e.g. 15 degrees C cells contained 31% gamma-linolenic acid vs. 25% at 39.5 degrees C) as did the content of alkyl glyceryl ether derivatives. Ethanolamine phosphoglycerides decreased by more than 10 mol % of the lipid phosphorus with the drop in temperature, the decrease being offset by a concomitant rise in 2-aminoethylphosphonolipid. These temperature-induced changes were noted in certain purified membrane preparations as well as in whole cells. Experiments with [14C]palmitic acid and sodium[14C]acetate showed that fatty acids are first incorporated into phospholipids predominantly in a saturated form. The membranes served as a reservoir of fatty acid substrate for desaturase activity. Tetrahymena pyriformis, strain NT-1, was proposed as a useful model system for studying the temperature adaptation process in eukaryotic cells.

Acclimatization↗
PubMed1976

Environmental effects on Tetrahymena membranes. Temperature-induced changes in membrane fatty acid unsaturation are indepedent of the molecular oxygen concentration.

The study was designed to show whether the increase in unsaturated fatty acids in Tetrahymena pyriformis membrane phospholipids with decreasing growth temperature is due to the sharp rise in the concentration of dissolved O2 found at lower temperatures. Increasing cell density in cultures growing at a constant temperature was found to have a more drastic effect upon the O2 level than shifting a culture from one temperature extreme to the other. However, the pattern of phospholipid fatty acid unsaturation did not vary over a 30-fold gradual decrease in O2 concentration measured in cultures during logarithmic growth at 39.5 degrees C, and unsaturation in 15 degrees C cells sampled over a 5-fold decrease in O2 concentration was also unchanged. Cells grown at 39.5 degrees C under a constant relatively high O2 concentration (5--6 mg O2/l) or low O2 concentration (0.3 mg O2/l) had nearly identical distributions of membrane fatty acids. Only under almost completely anaerobic conditions was it possible to measure via radioactive labeling experiments and inhibition of fatty acid desaturation. Thus the increased membrane fatty acid unsaturation induced by decreasing growth temperature is caused by some factor other than the rising levels of dissolved O2.

Acclimatization↗
PubMed1976

Leucine aminopeptidase (bovine lens). The relative binding of cobalt and zinc to leucine aminopeptidase and the effect of cobalt substitution on specific activity.

Prolonged incubation of zinc-zinc leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with 0.05 M CoCl2 and M KCl in 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees yields an active enzyme in which 2 g atoms of Co2+ per 54,000 dalton subunit have replaced the Zn2+. Incubation of cobalt-cobalt leucine aminopeptidase with various AnCl2 concentrations or zinc-zinc leucine aminopeptidase with various CoCl2 concentrations in M KCl and 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees demonstrates that Co2+ and Zn2+ compete reversibly for two independent binding sites per subunit for which the ratio of the association constants for Zn2+ and Co2+ (1KZn:1KCo = 1KZn/Co; 2KZn:2KCo = 2KZn/Co) are 115 and 15.9 for sites 1 and 2, respectively. The specific activities of the various species of enzyme with 2 mM L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl and 0.01 M NaHCO3 at pH 7.5 are estimated to be (in micromoles per min per mg) 0.043 for the zinc-zinc. 0.039 for the zinc-cobalt, 0.541 for the cobalt-zinc, and 0.536 for the cobalt-cobalt forms, which implies that activity is affected only when cobalt is substituted at site 1, the "activation site." The site, at which cobalt substitution has no effect on activity, is designated the "structural site." The value of Km for cobalt-cobalt leucine aminopeptidase with L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl at pH 7.5 containing 0.01 M NaHCO3 at 30 degrees is 0.52 mM while Vmax is 0.90 mumol per min per mg. In the additional presence of 1 M KCl, Km is 0.19 mM while Vmax is 0.68 mumol per min per mg.

Animals↗
PubMed1976

Leucine aminopeptidase (bovine lens). Effect of pH on the relative binding of Zn2+ and Mg2+ to and on activation of the enzyme.

Incubation of leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with various concentrations of Mg2+ at various pH values in 1 M KCl and 0.155 M trimethylamine-HCl at 37 degrees confirms that Mg2+ competes with Zn2+ for binding only 1 site per 54,000-dalton subunit. The ratio of the apparent association constants (1KZn:1KMg = 1KZn/Mg) at this site (site 1) was estimated to be 20,720 at pH 8.16, 10,570 at pH 8.44, 3,590 at pH 8.78, and 660 AT PH 9.14. The decrease in values of 1KZn/Mg with increasing pH in the activation of leucine aminopeptidase by Mg2+ is attributed to the lowering of the free Zn2+ concentration relative to that of free Mg2+ caused by the formation of ZnOH+ and Zn(OH)2 complexes with increasing OH- concentration. When corrections are made for the binding of Zn2+ by OH- ions, the pH-independent ratio of association constants (1KZn:1KMg = 1KZn/Mg) for the relative binding of Zn2+ and Mg2+ at site 1 of leucine aminopeptidase in 29,800. From the effect of pH on the relative binding constant, a value (beta2) for the product of the two stepwise association constants for the formation of Zn(OH)2 from Zn2+ and OH- (Zn2+ + OH- in equilibrium ZnOH+; ZnOH+ + OH- in equilibrium Zn(OH)2) was estimated to be 4.42 X 10(10) M-2 at 37 degrees. Values of Km at pH 7.5 AND 30 degrees with L-leucine p-nitroanilide as substrate in the presence of 0.01 M NaHCO3 are 4.13 and 2.01 mM for the zinc-zinc and magnesium-zinc enzymes, respectively. Values for Vmax are 0.2 and 2.49 mumol/min/mg, respectively.

Animals↗
PubMed1975

Utilization of L-cystine by the gamma-glutamyl transpeptidase-gamma-glutamyl cyclotransferase pathway.

Cystine is a good acceptor of the gamma-glutamyl group of gamma-glutamyl donors in the reaction catalyzed by gamma-glutamyl transpeptidase. The product of the enzymatic reaction and an authentic sample of gamma-glutamylcystine were shown to exhibit identical chromatographic and electrophoretic behaviors; acid hydrolysis gave equimolar amounts of cystine and glutamate. In studies with two gamma-glutamyl donors, apparent Km values in the neighborhood of 0.3 mM were found for L-cystine; these values are not far from the concentrations of L-cystine in mammalian blood plasma. At an amino-acid acceptor concentration of about 0.5 mM, L-cystine is somewhat more active than L-glutamine, and much more active than L-cystein. L-gamma-Glutamyl-L-cystine was found to be a good substrate of gamma-glutamyl cyclotransferase. These observations thus indicate that L-cystine is a very active substrate of the gamma-glutamyl transpeptidase-gamma-glutamyl cyclotransferase pathway. In relation to the hypothesis that the gamma-glutamyl cycle functions in animo-acid transport, it may be significant that glutathione (which is the most abundant intracellular form) is a much better gamma-glutamyl donor than glutathione disulfide, while the predominant extracellular form-cystine-is a much better gamma-glutamyl acceptor substrate than cystein.

Acyltransferases↗