Starch Gel Electrophoresis of Lactic Dehydrogenase from Rat Kidney.

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Abstract
Whole kidneys homogenized in water were frozen and thawed, centrifuged and the supernatant subjected to starch gel electrophoresis at pH 8.6. The starch columns were incubated in a standard medium containing lactic acid, DPN, Nitro BT, phosphate buffer (pH 7.4), potassium cyanide, and phenazine methosulfate (PMS) at 37[degree] C for about 30 minutes. Five distinct bands exhibited lactic acid dehydrogenase (LDH) activity. Four of these migrated toward the anode while one moved toward the cathode. The reduction of Nitro BT was substrate dependent and enzymatic activity was completely abolished by heat. The standard substrate, was varied by omitting either DPN, PMS, or both. By this method it was demonstrated that the two lower migrating bands contained, at least, LDH and DPN. The two faster migrating bands contained LDH but not DPN since they were demonstrable only when phenazine methosulfate and DPN or exogenous diaphorase and DPN were present. In addition to the LDH fractions, two DPN diaphorase fractions were demonstrated (oxidation of DPNH).