Carboxypeptidase G: purification and properties.

In such substrates RCOOH can represent not only substituted pteridines but a variety of organic acids, including many amino acids. However, hydrolytic activity seems to occur only when L-glutamate with a free a-carboxyl group is the donor of the amide nitrogen in the linkage. This specificity indicates that the enzyme may be thought of as a carboxypeptidase for glutamate. An enzyme with these properties might have a useful role in the elucidation of the structure and its relation to function of proteins and other complex biological compounds. Experimental Procedure.-(1) Materials and methods: Methotrexate (Lederle) was kindly provided by the Chemotherapy National Service Center of the National Cancer Institute. We are indebted to Dr. John E. Folk for gifts of the following peptides: Clac-Gly-Gln-Gly, Gln-Phe, Glu-Gly, Leu-Val-Glu,2 and Z-Glu-Val-ethyl ester. Other amino acid derivatives and dipeptides were obtained from commercial sources (Sigma, Mann, or Cyclo Chemical Corp.). Each compound tested as a substrate was subjected to high-voltage electrophoresis and staining with ninhydrin to assure the absence of any significant adventitious ninhydrin-reacting material. In addition, each compound was hydrolyzed chemically and the products were examined to assure the presence of only the expected amino acids. Products of hydrolysis obtained from peptides and amino acid derivatives were subjected to high-voltage electrophoresis at 50-60 v/cm in 7% formic acid (Savant model LT20A tank) at 240C on Whatman 3MM filter paper. Paper chromatography was used to separate the following compounds from their products of hydrolysis: Glu-Glu (system I); Z-Gln, Z-Glu, Gln-Phe, Phe-Glu, and Leu-Val-Glu (system II). System I was the organic phase of n-butyl alcoholacetic acid-water (2:1:1) by descending chromatography; system II was the organic phase of n-butyl alcohol-acetic acid-water (50:12:50) by ascending chromatography. Color of the peptides and amino acids was developed by dipping the chromatogram in 0.5% ninhydrin in acetone and heating the paper at 650C for 30 min. The photometric ninhydrin method of Troll and Cannan3 was used to determine the extent of amino acid release from peptides and acylated amino acids. For each compound tested, the ninhydrin color development of the reaction mixture was compared with that of a control (blank) in which boiled enzyme had been substituted for active enzyme. Hydrolysis, as indicated by an increase in ninhydrin color, was confirmed by chromatography of an aliquot of the reaction mixture. The reaction mixture contained 0.2 mmoles of ZnCl2, 25 lsmoles of Tris-HCl buffer, pH 7.3, enzyme, and 2.0 ;&moles of the compound to be tested in a volume of 1.0 ml. The incubation at 30'C for 1 hr was terminated by cooling the reaction mixture to 0C, and an aliquot of the reaction mixture was assayed for the development of ninhydrin color.