258 Sharp and Slaterstored in 50% (w/v) glycerol, has been shown to be stable for at least 6 mo
at 20°C. Johnson and Laskowski (3) found that the enzyme, lyophilized
and kept in a deep-freeze (temperature not specified), lost 50% of its
specific activity in 1 yr.
Stability is affected by surface adsorbtion of the enzyme to the
container and by the composition of the container. Stability was found
to improve by storage at higher enzyme concentrations (9).
2.9. Inhibitors
Dialysis of mung-bean nuclease 1 against 0.05M sodium acetate
(pH 5.0) containing 0.001% Triton X-100 results in 70-80% inhibi-
tion of the enzyme. This inhibition is reversible on addition of 0.1 mM
Zn 2÷ and 1.0 mM cysteine with incubation at 23°C for 20-30 min.
Co 2÷, Mg 2÷, Mn 2÷, Ca 2÷, Fe 2÷, and Cu 2÷ are incapable of reactivating
the enzyme under the same conditions. Dialysis against 0.001 M EDTA
will remove the Zn portion of the metalloenzyme resulting in irrevers-
ible inactivation. Adjustment of the pH to 8.0 results in 99.99% inhi-
bition of the activity as compared with pH 5.0. The addition of 0.01%
SDS at pH 5.0 will inactivate the enzyme completely (9).
2.10. Uses of Mung-Bean Nuclease 1
Mung-bean nuclease 1 shows differing activities under different
reaction conditions, and, as such, it has the potential to be used for a
variety of purposes and techniques. One of the main uses of mung-
bean nuclease 1 is the conversion of protruding termini to blunt ends
(10,18, 19) (see Fig. 1). Mung-bean nuclease 1 may be the enzyme of
choice for this technique over nuclease S 1, since it may be less vigor-
ous in its actions with respect to cleaving double-stranded DNA.
Ghangas and Wu (10) found the terminally directed or exonucleolytic
"trimming" activity to be specifically 5'-3'. Proper trimming was
obtained when the final blunt end contained a GC base pair at its
terminus. This was not always the case if an AT base pair occupied this
position. The nucleolytic composition of the overhang did not seem to
affect the efficiency or quality of the digestion.
Mung-bean nuclease 1 has also been used: as a structural probe in
the detection of complexes of DNA hairpins (19); in high-resolution
mapping of termini in RNA transcripts (20); as a probe for DNA sec-
ondary structure (8); as a nuclease for gene isolation (5,21); and for the
linearization of super-coiled DNA (12).