Dissociation and reconstitution of bovine seminal RNAase: Construction of a hyperactive hybrid dimer
- 1 December 1989
- journal article
- research article
- Published by Springer Nature in Protein Journal
- Vol. 8 (6) , 719-731
- https://doi.org/10.1007/bf01024897
Abstract
The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we describe the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.Keywords
This publication has 24 references indexed in Scilit:
- Reaction of (bromoacetamido)nucleoside affinity labels with ribonuclease A: evidence for steric control of reaction specificity and alkylation rateBiochemistry, 1987
- Selective deamidation and enzymic methylation of seminal ribonucleaseBiochemistry, 1986
- Site‐directed alkylation and site‐site interactions in bovine seminal ribonucleaseEuropean Journal of Biochemistry, 1986
- Bovine seminal ribonuclease: Non‐hyperbolic kinetics in the second reaction stepFEBS Letters, 1982
- Heterogeneity of bovine seminal ribonucleaseBiochemistry, 1981
- Transition-state analysis of the facilitated alkylation of ribonuclease A by bromoacetateBiochemistry, 1979
- Dissociation of bovine seminal ribonuclease into catalytically active monomers by selective reduction and alkylation of the intersubunit disulfide bridgesBiochemistry, 1975
- Interchain disulfide bridges in ribonuclease BS-1Biochemical and Biophysical Research Communications, 1973
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970
- Disk Electrophoresis of Basic Proteins and Peptides on Polyacrylamide GelsNature, 1962