Reconstitution of fully active RNase S by carboxypeptidase-degraded RNase S-peptide.

In attempting to relate the covalent structure of ribonuclease to its enzymic activity, controlled proteolytic degradation is a useful approach. Although native ribonuclease is markedly resistant to a variety of proteolytic enzymes, the separated components of ribonuclease S (1, 2), S-peptide, and S-protein, are extensively degraded by carboxypeptidase. Previous studies (3) on the sequence of amino acids in RNase S-peptide (residues 1 to 20, inclusive, in the proposed primary structure of ribonuclease (4))) indicated that carboxypeptidase completely removes five amino acids from the COOH-terminal region of the peptide. The present communication summarizes experiments showing that the removal of these five amino acids from S-peptide does not alter its ability to effect reconstitution of a fully active RNase S on combination with S-protein at molar equivalence. The ribonuclease used in this study was obtained from the Sigma Chemical Company (lot R-BOB-204) and shown to be 95 % RNase A (3). The preparation and purification of RNase S and the separation of RNase S-peptide were accomplished employing the methods of Richards (1, 2). Carboxypeptidase (a single preparation from Worthington Biochemical Corporation) was solubilized in 2.0 M ammonium bicarbonate at a concentration of 10 mg per ml, treated with a 5-fold molar excess of diisopropylfluorophosphate (Mann Research Laboratorics), and kept frozen in separate aliquots until used (3). Carboxypeptidase digestion was performed by incubating S-peptide, 10 mu (uncorrected for moisture and residual salt), with carboxypeptidase at a concentration of 1 mg per ml in a total volume of 1 ml of 0.2 M ammonium bicarbonate containing glycine (4 mM as an internal standard). A 200.~1 aliquot was removed as control at zero time and diluted with an equal volume of acetic acid. After 9 hours of incubation at 25”, a 200.~1 aliquot of the digest was removed and analyzed on the Beckman/Spinco automatic amino acid analyzer (5). The analysis revealed the following micromolar quantities of amino acids: Gly (added as an internal standard), 0.89 pmole (110% of theoretical recovery) ; Ala, 3.24 pmoles; Ser, 3.27 wmoles; Thr, 1.67 pmoles (81 to 54% of theoretical recovery, on a weight basis, for the expected 1 threonine, 2 alanine, and 2 serine residues). The remaining solution was diluted, exactly as the control aliquot, with an equal volume of glacial acetic acid. The digested and control peptide solutions were kept frozen before enzymic assay. To measure enzymic activity, various aliquots of these diluted