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. 1992 Jul 15;285(Pt 2):489–494. doi: 10.1042/bj2850489

Active-site characterization of S1 nuclease. I. Affinity purification and influence of amino-group modification.

S Gite 1, G Reddy 1, V Shankar 1
PMCID: PMC1132814  PMID: 1637340

Abstract

A simple procedure, involving heat-treatment, DEAE-Sephadex, AMP-Sepharose and Bio-Gel P-60 chromatography, was developed for the purification of S1 nuclease to homogeneity from commercially available Takadiastase powder. Chemical modification of the amino groups of purified S1 nuclease revealed that lysine is essential for single-stranded DNAase, RNAase and phosphomonoesterase activities associated with the enzyme. The kinetics of inactivation suggested the involvement of a single lysine residue in the active site of the enzyme. Additionally, lysine modification was accompanied by a concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of single-stranded DNA, RNA and 3'-AMP. Substrate-protection and inhibitor-binding studies on enzyme modified with 2,4,6-trinitrobenzenesulphonic acid showed that lysine may be involved in the substrate binding.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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