|Abstract or Summary
- 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.