Colorimetric assay of liver and Escherichia coli aspartate transcarbamylases in the presence of 2-mercaptoethanol

✍ Scribed by C. Richard Savage; Joan M. Schumer; Herbert Weinfeld

• Publisher: Elsevier Science
• Year: 1973
• Tongue: English
• Weight: 587 KB
• Volume: 53
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(73)90090-0

✦ Synopsis

A modification of the method of Prescott and Jones (1) for the colorimetric determination of carbamyl aspartate has been developed to permit the assay of aspartate transcarbamylases in the presence of 2-mercaptoethanol. Interference by this compound is eliminated by means of N-ethylmaleimide. The usefulness of the modified method is illustrated by examination of the contrasting properties of the Escherichk coli and rat liver enzymes.

Prescott and Jones (1) described a sensitive procedure for the colorimetric determination of CA.' The method failed in the presence of 1 mM 2-ME (1) as did (2) the Gerhart and Pardee modification (3) of the method of Koritz and Cohen (4). If this difficulty could be overcome, the method would be of value in a study of the ATCase of liver and of Escherichia coli. The liver enzyme was found by Bresnick to require the presence of 2 mM 2-ME for maximum stability (5). The liver enzyme has not yet been highly purified (6) and it may be anticipated that as purification proceeds, the need for a sulfhydryl environment will be crit,ical for a variety of experiments. The bacterial enzyme has always been prepared in contact with 1 mM or 2 mM 2-ME (3,7).

Ordinarily, liver ATCase is assayed using radioactive Asp or ['"Cl CAP as substrate (2,6) followed by the determination of radioactive CA (2,8,9). If radioactive Asp is used, the ion-exchange methods are tedious