The activity of ribonuclease after digestion with carboxypeptidase

Kalnitsky, George; Rogers, William I.

doi:10.1016/0006-3002(56)90299-2

Cited by 22 publications

(2 citation statements)

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“…Activation of RNase-A during digestion with carboxypeptidase was noted by Kalnitsky and Rogers (1956). The increase in activity was apparently related to the proteolysis.…”

Section: Discussionmentioning

confidence: 97%

The Action of Trypsin on Ribonuclease-S^*

Allende¹,

Richards²

1962

Biochemistry

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S-Protein and S-peptide, either separately or combined in the active enzyme RNase-S, can be digested by trypsin, whereas the native enzyme, RNase-A, is not attacked. In S-peptide the bond between residues 10 and 11 is hydrolyzed much more rapidly than the one between numbers 7 and 8. Cleavage of the first bond results in a loss of all demonstrable interaction with S-protein. Residue 11 appears to be a glutamyl (or glutaminyl) group, in agreement with the results of White and Anfinsen (1959). The tryptic attack on S-protein produces at least three chromatographically separable intermediates which all bind S-peptide and produce complexes with varying catalytic activity but in all cases less than that observed with S-protein itself. These intermediates appear to contain most, if not all, of the residues present in S-protein, but additional amino-terminal groups are found. The maximum number of bonds hydrolyzed in S-peptide is 2, in S-protein 7 to 8, but in RNase-S only 6 to 7. From this discrepancy and the kinetics of the activity loss, it is concluded that trypsin can attack RNase-S without dissociation of the parts, but that the course of proteolysis and the final products are not the same as in the sum of tbe digests of the separated components. S-Protein polymerizes in the neutral pH range but appears to be a monomer at pH 2, with a sharp transition between pH 2 and 3. The polymers are dissociated easily by S-peptide. The complex, RNase-S, does not show any tendency to polymerize. No evidence for polymerization of the tryptic digestion intermediates was found. In the very early stages of digestion of S-protein and RNase-S by trypsin, an increase in the RNase activity was observed. This was not seen with S-peptide or RNase-A. No correlation was established with extent of hydrolysis or amount of trypsin. Chromatograms of partial digests have not shown any material with specific activity higher than the starting material and the activity recoveries were low.No explanation has been found for this activation effect.

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