Glutamate dehydrogenase (GDH) activity was determined in high‐speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low‐speed pellet (480 g for 10 min). Approximately 60% of the high‐speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similar Km values for the two GDH fractions for the substrates μ‐ketoglutarate, ammonia, and glutamate; however, there were small but significant differences in Km values for NADH and NADPH. Although the allosteric stimulation by ADP and L‐leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mM ADP, with apparent Ki values of 18.5 and 9.0 μM GTP for the soluble and particulate fractions, respectively. The HIll plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45°C and pH 7.4 in 50 mM phosphate buffer.
In a previous study it was shown that the acetyl moiety can be incorporated into the protein of purified synaptosomes (1). This process was inhibited by veratridine and the inhibitory effect was counteracted by tetrodotoxin. This suggested that the flux of Na+ may be related to the acetylation process. We now report that in a sodium free medium the amount of acetylation is increased and the inhibitory effect of veratridine (veratrine) is no longer evident. The addition of Na+ leads to a decrease in acetylation in the presence of veratrine. The presence of scorpion toxin has an effect similar to that of veratrine and the two are not additive. Hence, they appear to act on a common site. Molecular sieve chromatography shows four radioactively labeled peaks, two of which are particularly affected by veratrine. We also show that [3H]acetate incorporated into synaptosomal protein can be recovered as acetyldansylhydrazide. In addition, the concentration of free and bound acetate was measured in whole brain as well as in synaptosomes.
Incubation of synaptosomes with [3H]acetate results in rapid labeling of protein. Labeling is decreased in the presence of veratridine, and the effect of veratridine is blocked by tetrodotoxin. Most of the radioactivity can be removed by base or acid hydrolysis, and is probably incorporated as acetate; it is this fraction that is affected by the veratridine. The data suggest that veratridine stimulates deacetylation is involved in membrane function.
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