Immunogenetic analyses of several EMS induced alcohol dehydrogenase variants have led toward the elucidation of the mechanism of enzyme activity. Antisera raised against various allelic forms of the enzyme can precipitate any of the ADH allozymes; the precipitin formed retains enzyme activity. The single exception to this is anti U725, which precipitates antigen as well as the other antisera, but renders the ADH precipitin inactive. The enzyme can be protected from this inactivation by preincubation with its NAD cofactor, or with NADP, which cannot act as a coenzyme. This works both in double diffusion Ouchterlony when NAD or NADP is added to the agar, and in solution mixtures. Ethanol cannot perform such a protective role. These results suggest that the mechanism of enzyme activity involves first the binding of NAD, which causes a conformational change necessary for the actual transfer of protons, in a manner analogous to that of horse liver alcohol dehydrogenase (Biochemistry 9:4655, 1970). The prebinding of anti U725 may simply sterically prevent subsequent binding of NAD to ADH, or it may induce a conformational change in the enzyme so that it could not then bind NAD, or it may allow binding of NAD but not allow the conformational transition to the active state.
A number of Adh CRM+ nulls which have no detectable enzyme activity have also been studied immunologically. Simply precipitating these nulls by antisera raised against active forms of ADH does not restore enzyme activity. However, if the null extracts are preincubated with high levels of NAD or NADP, then immunoprecipitated, activity can be restored. Binding studies using the affinity, chromatography medium Blue Sepharose have shown that all seven of the CRM+ nulls are unable to bind NAD. One hundred-fold excess of NAD alone is not sufficient to restore spectrophotometrically detectable activity; it is the interaction among enzyme, high concentrations of coenzyme, and immunoglobulin that presumably molds and holds the enzyme in an active conformation.
Erin Irish and Drew Schwartz
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