Graduate Thesis Or Dissertation

 

Studies on glucose-6-phosphate dehydrogenase obtained from Vibrio marinus, an obligate marine psychrophile Public Deposited

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  • Four-fold partially purified glucose-6-phosphate dehydrogenase (G-6-PDH) from the obligately psychrophilic marine bacterium Vibrio marinus was effected by thermally induced leakage of the enzyme, ammonium sulfate fractionation and column chromatography on Sephadex G-200 gel. The effects of temperature, pressure, salinity, and pH were determined for the various degrees of enzyme purity. The intracellular enzyme was stable between 0 C and 32 C for one hour with an optimum temperature of activity at the organism's optimum temperature of growth (15 C). In cell free extracts the enzyme was cold-labile below 7 C and was heat-labile when exposed to temperatures above the organism's maximal growth temperature (20 C). There was a negligible amount of enzyme released from cells held for one hour at temperatures below 20 C, but above 20 C heat-treated samples leaked increasingly more enzyme with a maximal amount obtained at 31 C. The ammonium sulfate fractionated enzyme was most stable at 5 C in 26%,synthetic sea water at pH 7.4, and was activated slowly when held above 15 C. A hydrostatic pressure of 300 atm produced a seven-fold stimulation in the rate of activity over that observed at 1 atm. Pressures from 400 to 1000 atm inhibited enzyme activity but release of the pressure to 1 atm reversed the rate of activity equivalent to that observed at 300 atm. Four-fold partially purified enzyme was stable between 5 C and 26 C, with a stable optimum temperature of activity at 15 C. The salt-free enzyme displayed no activity, and the enzyme incubated in the presence of ammonium sulfate was labile below 10 C, but shifted the optimum temperature of activity from 15 C to 20 C Thermodynamic parameters were reported for the activated state for temperature activation and inactivation of G-6-PDH in the presence and absence of ammonium sulfate. Tris-HCl, KCl, NaCl, NaBr, CaCl₂, K₂SO₄, KF and (NH₄)₂SO₄, in decreasing order increased the rate of activity when the enzyme was incubated in the presence of these salts, but MgCl₂ inhibited by a competitive mechanism with complete reversal of inhibition after exposure to 40 C for 10 min. The Km determined was 8.5 x 10⁻⁴; the Vm, 0.083 and the Ki for MgCl₂, 0.10. NaC1, (NH₄)₂SO₄, and Rila marine salts, in decreasing order, protected the enzyme from thermal inactivation at 44 C. (NH₄)₂SO₄ reactivated, up to 130 percent, the 38 C-inactivated enzyme. A seven minute exposure to 17 C allowed 100 percent reactivation of the enzyme inactivated at 44 C in the presence of (NH₄)₂SO₄; in the presence of NaCl there was 20 percent reactivation and 50 percent in the absence of any added salts. When the enzyme was placed at pH 7.4 and under the desired pressure for 30 minutes, an optimum pressure of activity was observed at 400 atm and the amount of activity at 15 C was four-fold greater than that observed at 2 C. The amount of activity at both 2 and 15 C was negligible at pressures greater than 1000 atm.
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