Abstract
The preparation, characterization, and purification of antibody against the membrane-bound D-lactate dehydrogenase solubilized and purified from Escherichia coli ML 308-225 are described. The antibody is highly specific for the flavin-linked D-lactate dehydrogenase, and incubation of the enzyme with antiserum results in marked inhibition of enzymatic activity. By means of a radioimmune assay, it is demonstrated that membrane vesicles prepared from E. coli ML 308-225dld-3 contain catalytically inactive material which cross-reacts with native D-lactate dehydrogenase. In the following paper, the effects of this antiserum on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native and reconstituted membrane vesicles are examined.
Highlights
The preparation, characterization, and purification of antibody against the membrane-bound n-lactate dehydrogenase solubilized and purified from Escherichia coli ML 308-225 are described
By means of a radioimmune assay, it is demonstrated that membrane vesicles prepared from E. coli ML 308-225&d-3 contain catalytically inactive material which cross-reacts with native n-lactate dehydrogenase
Active transport of many solutes in Escherichia coli membrane vesicles is coupled to the oxidation of n-lactate to pyruvate catalyzed by a membrane-bound n-lactate dehydrogenase which functions in the absence of pyridine nucleotides
Summary
Escherichiu coli ML 308-225 (i-z-y+a+) and ML (i-z-y+a+dld-) [10] were grown on minimal medium succinate as a sole source of carbon [11]. Prior to application on Sepharose-n-lactate dehydrogenase (1 x 9 cm) column prepared as described above, y-globulin was purified from the pooled antiserum by 50% ammonium sulfate precipitation and DEAE-chromatography [22], concentrated to 3.5 mg of protein per ml using an Amicon concentrating apparatus with an XM-50 filter, and dialyzed overnight against PBS, pH 7.4. Ten milliliters of this preparation were allowed to percolate slowly into the column at room temperature and allowed to equilibrate with the gel for 2 hours at room temperature and 10 hours at 2 to 4”. The cenkr well contains purified n-lactate dehydrogenase and Wells 4, 5, and 6 contain preimmune serum, normal rabbit serum, and normal rabbit y-globulin, respectively
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