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A quantitative study on arginine synthesis from argininosuccinic acid and citrulline by crude enzymes of cattle kidney
Author(s) -
SULTANA Halima,
INADA Mikiko,
WADUD Shaila,
TAKAHASHI Toshihiro,
MORITA Tetsuo,
ONODERA Ryoji
Publication year - 2003
Publication title -
animal science journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.606
H-Index - 38
eISSN - 1740-0929
pISSN - 1344-3941
DOI - 10.1046/j.1344-3941.2003.00119.x
Subject(s) - argininosuccinate lyase , argininosuccinate synthase , arginine , chemistry , enzyme , biochemistry , incubation , lyase , citrulline , chromatography , tris , biosynthesis , urea cycle , amino acid
In vitro experiments were conducted to examine the synthesis of arginine (Arg) from argininosuccinic acid (ASA) and citrulline (Cit) by crude enzymes of cattle kidney cortex. Kidney samples, collected from Japanese black cattle, were homogenized in KCl solution (ice‐cold), and centrifuged at 27 000 × g for 20 min at 4°C, and the supernatant fluid was used as a crude enzyme solution. The enzyme solution was incubated at 39°C in Tris HCl buffer with 15 mmol/L ASA or with 10 mmol/L Cit in the presence of 10 mmol/L aspartic acid (Asp), 10 mmol/L ATP and 5 mmol/L MgCl 2 to examine the activities of two enzymes, argininosuccinate lyase and argininosuccinate synthetase, which work at the terminal steps of Arg biosynthesis. The production of Arg from ASA, or ASA and Arg from Cit by argininosuccinate lyase and argininosuccinate synthetase activities, respectively, were determined directly by the HPLC method. The optimum pH for argininosuccinate lyase activity was 7.85. Unfortunately, the optimum pH for argininosuccinate synthetase activity could not be determined because no inhibitor of argininosuccinate lyase was used in the Cit incubation, so the ASA produced from Cit spontaneously converted to Arg during incubation with Cit. The maximum production of ASA from Cit was found at pH 6.45 under these conditions. We observed the optimum pH for the synthesis of Arg from Cit at 7.7. The production of Arg from ASA or Cit was quantitatively determined as 14.4 or 8.83 µmol/g kidney tissue/h, respectively, at the optimal pH values. This suggests that the daily production of Arg from ASA or Cit by the kidney might be sufficient to cover the daily requirement of Arg in cattle.