Conditions for glutamate dehydrogenase activity in heart mitochondria.
Although heart mitochondria contain glutamate dehydrogenase, it has not been thought to play a role in their metabolism. We investigated this matter to define the conditions under which it is active. We found modest activity in the presence of glutamate and malate and a continuous source of ADP when pyruvate is added. This increases several fold as the osmolarity is increased from 296 to 370 mosM. At the higher osmolarity ammonia formation is brief, associated with a lower intramitochondrial alpha-ketoglutarate from citrate does not make up for the drop in glutamate conversion to alpha-ketoglutarate. Mitochondrial content of nucleotides and CoA compounds are not altered by pyruvate addition. The rate of glutamate deamination by GDH in sonicated heart mitochondria agrees with the rate of ammonia formation in intact mitochondria in the presence of pyruvate (20 nmol/min/mg of mitochondrial protein). We conclude pyruvate lowers mitochondrial oxalacetate which decreases alpha-ketoglutarate formation by transamination. The lower mitochondria alpha-ketoglutarate level permits glutamate deamination until alpha-ketoglutarate reaches a level that inhibits the forward reaction. Further proof of the key role of alpha-ketoglutarate is seen with aminooxyacetate which blocks transamination. In its presence ammonia formation occurs at the same rate (18 nm/min/mg of mitochondrial protein), is not dependent upon pyruvate, and does not stop after a couple of minutes. Leucine, which decreases alpha-ketoglutarate inhibition of GDH, also results in ammonia formation, further supporting the concept of regulation by alpha-ketoglutarate. The higher osmolarity increases GDH activity by increasing alpha-ketoglutarate transport from mitochondria.
VA Medical Center, Birmingham, Alabama.
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D009994:Osmolar Concentration; D010101:Oxygen Consumption / Q000502:physiology; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D051381:Rats; D017207:Rats, Sprague-Dawley
D005971:Glutamates; D008293:Malates; D011773:Pyruvates; D000244:Adenosine Diphosphate; D019289:Pyruvic Acid; D005969:Glutamate Dehydrogenase