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Role of Acetylglutamate in Ureotelism

I. OCCURRENCE AND BIOSYNTHESIS OF ACETYLGLUTAMATE IN MOUSE AND RAT TISSUES

From the ¹ From the Department of Biochemistry, Chiba University School of Medicine, Inohana, Chiba, Japan

To elucidate the possible role of N-acetyl-l-glutamate known as the natural activator of the ammonia-dependent carbamyl phosphate synthetase in regulation of urea biosynthesis, studies were undertaken on mouse and rat.

1. Acetylglutamate was found to occur in livers of mouse and rat at a level of about 30 nmoles per g of tissue. Of the other abdominal organs of mouse tested, the compound was detected in the small intestine at a level of about 10 nmoles per g of tissue but practically none was found in kidney, spleen, and heart. It was further shown that the compound is localized in the mitochondrial fraction in liver cells. The intramitochondrial concentration of acetylglutamate is estimated to be of the order of 10^-4 m, which is close to the activation constant of the carbamyl phosphate synthetase for the compound. The tissue distribution and the subcellular localization of acetylglutamate approximately coincide with those of the enzyme thus far reported.

2. The acetylglutamate content in mouse liver increased from 15 to 40 nmoles per g of tissue in response to the increment of dietary protein content from 0 to 60% and a positive correlation was observed between the changes in acetylglutamate and urea content in liver.

3. The biosynthesis of acetylglutamate in mouse and rat tissues was shown both in vivo and in vitro with [¹⁴C]glutamate or [¹⁴C]acetate as tracer. Combined use of Dowex 1 column chromatography and paper chromatography permitted a complete separation of acetyl[¹⁴C]glutamate from other radioactive products formed from the labeled precursors. The experiments in vivo on acetylglutamate synthesis in mouse liver showed that the compound is subject to metabolic turnover with a half-life of a few hours. In experiments in vitro the synthetic activity was shown in liver slices and sectioned small intestine but not in slices of kidney and spleen. Of the activity in liver homogenate, a major part was recovered in the mitochondrial fraction. The localization of the activity coincided with that of acetylglutamate as observed.

4. The mitochondrial synthesis of acetylglutamate required the presence of both glutamate and acetate (or pyruvate) as substrates. The apparent K_m for glutamate was estimated to be 1 mm or less and the maximal synthetic rate obtained at a saturating concentration of glutamate was about 7 nmoles per g of tissue per hour. It is suggested that the intracellular concentration of glutamate may be a critical factor which can affect the tissue level of acetylglutamate.

The results are consistent with and support the postulated role of acetylglutamate in the regulation of urea biosynthesis mediated through its activating effect on carbamyl phosphate synthetase.

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