Factors of activation and stabilization of DNA-methylases from Shigella sonnei 47 and Mycobacterium smegmatis butyricum cells.
A comparative study of factors of activation and stabilization of individual DNA-methylases from two bacterial strains--Shigella sonnei 47 and Mycobacterium smegmatis butyricum--isolated by isoelectrofocusing in a pH gradient has been carried out. Storage of enzymes at +4 degrees C (pH 7.5) is accompanied by periodic changes in the methylating activity. No such changes are observed when the enzymes are stored at pI of the protein. In this case the methylases with alkaline or close to neutral values of pI remain stable over a period of at least two weeks, whereas acidic proteins are irreversibly inactivated by the end of a two-week period. A stabilizing effect of BSA on DNA-methylases of Sso 47 and Mbu strains has been demonstrated. A direct correlation between the stabilizing effect of BSA and the degree of enzyme purity has been established. Ca2+ appear to be a universal activator of methylases of the above strains; these cations produce a potent, although a short-term effect and can be used in the production of enzyme preparations with a high specific activity in DNA recombinant technology. Protease inhibitors do not exert any appreciable effect on the methylase activity upon storage. Storage at -20 degrees C and at neutral pH leads to complete inactivation of all DNA-methylases within 24 hours. In this case glycerol is fairly ineffective as a stabilizing agent.
Nikol'skaya
I I
II
Institute of Medical Enzymology, USSR Academy of Medical Sciences, Moscow.
Sharkova
E V
EV
Suchkov
S V
SV
Lopatina
N G
NG
Habar
K
K
Somodi
P
P
Földes
I
I
Debov
S S
SS
D000468:Alkalies; D002118:Calcium / Q000494:pharmacology; D003080:Cold Temperature; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis*; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D009161:Mycobacterium / Q000201:enzymology*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D012764:Shigella sonnei / Q000201:enzymology*
D000468:Alkalies; D012710:Serum Albumin, Bovine; D004248:DNA (Cytosine-5-)-Methyltransferases; D002118:Calcium