期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1974
卷号：71
期号：4
页码：1461-1465
DOI：10.1073/pnas.71.4.1461
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：When membrane vesicles prepared from a D-lactate dehydrogenase mutant of E. coli ML 308-225 are treated with a homogeneous preparation of D-lactate dehydrogenase, the enzyme binds to the vesicles and they regain the capacity to catalyze D-lactate oxidation and D-lactate-dependent active transport. Although membranebound enzyme increases linearly with addition of increasing quantities of enzyme, reconstituted transport activity and D-lactate oxidation are saturable functions of the amount of enzyme bound. The maximal specific transport activity obtained in the reconstituted system is similar in magnitude to that of wild type vesicles. Titration studies with 2-(N-dansyl)-aminoethyl-{beta}-D-thiogalactoside demonstrate that there is at least a 7- to 8-fold excess of lac carrier protein relative to D-lactate dehydrogenase. Hydroxybutynoate-inactivated enzyme does not bind to the vesicles, indicating that the coenzyme moiety is critically involved in binding. Conformational changes are also apparently involved since 0.6 M guanidine{middle dot}HCl is required for optimal binding and reconstitution. The relative unreactivity of reconstituted vesicles towards vinylglycolic acid suggests that D-lactate dehydrogenase is bound to the outer surface of the reconstituted vesicles.
