The Determination of Glutamic Acid Dehydrogenase in Tissue Homogenates

In 1938, two groups of workers published extensive studies of glutamic dehydrogenase in animal tissues.The enzyme preparation of von Euler, Adler, Giinther, and Das (1) was a dialyzed extract from acetone-dried pig or beef liver.The preparation of Dewan (2) was the precipitate obtained at pH 4.6 from an extract of acetone-dried pig liver or kidney.Both studies showed approximately the same results.The preparations with L-glutamate and diphosphopyridine nucleotide (DPN) reduced methylene blue or took up oxygen when a suitable carrier was present, The final reaction products were shown to be ammonia and a-ketoglutarate with cu-iminoglutarate as an intermediate product.The reaction was found to be reversible and the enzyme in question was shown to be highly specific for L-glutamic acid.The present paper deals with the adaptation of the known facts of this enzyme system to tissue homogenates. EXPERIMENTALMaterial and Methods-The animal tissues used in this study were obtained from young male rats of the Holtzman strain, approximately 3 to 4 months of age.The rats were killed by cervical dislocation and the tissue sample quickly taken, blotted, weighed, and placed in a cold homogenizing tube containing 0.5 ml. of water.The tissue was homogenized and sufficient cold glass-distilled water was added to give a 5 per cent homogenate.Aliquots of the homogenate were added to the properly fortified substrate in Warburg flasks with no side arms, which were kept cold until the tissue was added.The cold flasks were then attached to the manometers and transferred to the 38" bath and the measurement of oxygen uptake was begun after a 6 minute equilibration period.The glutamic acid and semicarbazide hydrochloride were obtained from the Eastman Kodak Company.The latter was further purified by dissolving in hot water and recrystallizing with the addition of 2 to 3 volumes