Dissociation and electrophoretic separation of dextranase and dextranase inhibitor from a tightly bound enzyme-inhibitor complex of Streptococcus sobrinus.
Endodextranase was separated from dextranase inhibitor in culture filtrates of Streptococcus sobrinus by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in gel slabs containing blue dextran. Sample preparation included dissociation of the enzyme from its inhibitor by boiling for 1 min in SDS. During subsequent incubation of the gel, dextranase was located as clear bands on a blue background, and dextranase inhibitor appeared as blue zones on a clear background following incubation in dextranase solution. The enzyme and the inhibitor existed in multiple forms, and the range of molecular masses for dextranase (223-132 kDa) permitted an excellent separation from dextranase inhibitor (49-25 kDa). Although dextranase-negative mutants, and wild type strains grown at low dilution rate in the chemostat, were devoid of free dextranase activity, the enzyme was easily located by analytical SDS-PAGE. Likewise, analysis of filtrates from wild type strains, which contained no free inhibitor activity when growth occurred at high dilution rate, revealed dextranase inhibitor activity on the gels. The total production (free + combined) of dextranase and inhibitor by S. sobrinus was determined by dissociation of enzyme-inhibitor complexes in concentrated cell-free filtrates, their separation by preparative SDS-PAGE and electroelution from the gels, followed by renaturation of protein activity. From a comparison of activity tests of free dextranase and free inhibitor in untreated filtrates with the results of similar tests on renatured electroeluates, the proportion of each constituent bound into a complex under each growth condition could be deduced.
Institute of Dental Research, United Dental Hospital, Sydney, NSW, Australia.
J E
JE
Wellington
J M
JM
Shaw
G J
GJ
Walker
D003910:Dextranase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009154:Mutation; D016986:Streptococcus sobrinus / Q000201:enzymology* / Q000235:genetics
D003910:Dextranase