Carboxymethylation of a Minor Ribonuclease from Aspergillus saitoi
1979 Volume 86 Issue 1 Pages 35-44
Details
1979 Volume 86 Issue 1 Pages 35-44
(1) RNase Ms was inactivated by iodoacetate. The inactivation was most rapid at pH 6.0, and was inhibited in the presence of a denaturant such as 8 M urea or 6 M guanidine-HCl.
(2) Competitive inhibitors protected RNase Ms from inactivation by iodoacetate; the effect was in the order 2', (3')-GTP>2', (3')-AMP, 2', (3')-UMP_??_2', (3')-CMP. The order is not consistent with that of the binding constants of the 4 nucleotides towards RNase Ms (A>C>G>U).
(3) RNase Ms was inactivated with the concomitant incorporation of one molar equivalent of carboxymethyl group. The following evidence indicated that the carboxymethyl group was incorporated into the carboxyl group of an aspartic acid or glutamic acid residue. (i) The carboxymethyl group incorporated into RNase Ms was liberated by treatment with 0.1 N NaOH or 1 M hydroxylamine. (ii) The amino acid composition of carboxymethylated RNase Ms (CM RNase Ms) after acid hydrolysis is similar to that of RNase Ms.
(4) 14C-Labeled CM RNase Ms was digested successively with alkaline protease and amino-peptidase M. The radioactive amino acid released was eluted just before aspartate on an amino acid analyzer. After hydrolysis with 6 N HCI, glutamic acid was produced exclusively from the radioactive amino acid. The specific radioactivity of this amino acid calculated from the radioactivity and glutamic acid formed was practically the same as that of CM RNase Ms. Thus, it was concluded that a carboxymethyl group was incorporated at the carboxyl group of a glutamic acid residue of RNase Ms.
(5) CM RNase Ms bound with 2'-AMP to the same extent as native RNase Ms, but bound to a lesser extent with 2', (3')-GMP.
(6) Although the conformation of CM RNase Ms as judged from the CD spectrum was practically the same as that of native RNase Ms, the reactivity of CM RNase Ms towards dinitrofluorobenzene was different from that of native RNase Ms, indicating some difference in the conformation.
(7) These results indicate that one glutamic acid residue is involved in the active site of RNase Ms.
