Mung bean nuclease I. 6. Physical, chemical, and catalytic properties

Abstract

A simplified purification procedure for mung bean nuclease has been developed yielding a stable enzyme that is homogeneous in regards to shape and size. The nuclease is a glycoprotein consisting of 29% carbohydrate by weight. It has a molecular weight of 39 000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme contains 1 sulfhydryl group and 3 disulfide bonds per molecule. It has a high content (12.6 mol %) of aromatic residues. Approximately 70% of the enzyme molecules contain a peptide bond cleavage at a single region in the protein. The two polypeptides, 25 000 and 15 000 daltons, are covalently linked by a disulfide bond(s). Both the cleaved and intact forms of the enzyme are equally active in the hydrolysis of the phosphate ester linkages in either DNA, RNA, or adenosine 3'-monophophate. The enzymatic activity of mung bean nuclease can be stabilized at pH 5 in the presence of 0.1 mM zinc acetate, 1.0 mM cysteine, and 0.001% Triton X-100. The enzyme can be inactivated and reactivated by the removal and readdition of Zn2+ or sulfhydryl compounds.

Analysis of Actinobacillus pleuropneumoniae and Related Organisms by DNA-DNA Hybridization and Restriction Endonuclease Fingerprinting

“…DNA-DNA hybridizations. Since some batches of S1 nuclease contain significant amounts of double-stranded DNase activity (14,16), the DNA hybrids were analyzed with mung bean nuclease [Pharmacia (Canada) Ltd.]. At low enzyme and high salt concentrations, mung bean nuclease will hydrolyze only single-stranded DNA (14,16).…”

“…Two tubes from each reassociation mixture were treated with 10 to 25 U of mung bean nuclease for 20 min at 50°C; the other two tubes were treated identically but without the addition of the enzyme. The nuclease activity was terminated by adding 50 y1 of 1.0 M Tris buffer (free base) and 40 ~1 of 4-mg/ml sheared herring sperm DNA (14). The double-stranded DNA was precipitated and collected on HAWP 02500 filters (Millipore Corp., Bedford, Mass.).…”

Conformational analysis of hairpin oligodeoxyribonucleotides by a single-strand-specific nuclease

“…Any deviations from this procedure are described in the legends. 1 unit of mung bean nuclease is the quantity which produces 1 pg acidsoluble material from denatured DNA/min at 37 "C; this amount is equivalent to 0.004 unit as described by Kowalski et al [13]. The reaction was stopped by adding 30 mM EDTA, pH 8.…”

Mutation detection using a novel plant endonuclease

“…Single-stranded nucleases such as S1 and mung bean nuclease nick DNA at single-stranded regions (1)(2)(3). However, the acid pH optima of these nucleases lead to DNA unwinding at A+T-rich regions and result in non-specific DNA degradation.…”