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. 1974 Apr;71(4):1461–1465. doi: 10.1073/pnas.71.4.1461

D-Lactate Dehydrogenase Binding in Escherichia coli dld- Membrane Vesicles Reconstituted for Active Transport*

Steven A Short *, H Ronald Kaback *, Leonard D Kohn †
PMCID: PMC388249  PMID: 4598306

Abstract

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-β-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·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.

Keywords: guanidine·HCl, hydroxybutynoate, vinylglycolate, dansyl-galactoside

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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