[77] Aldehyde Dehydrogenase from Baker's Yeast

Shelby L. Bradbury, Julia F. Clark, Charles R. Steinman, William B. Jakoby

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This chapter describes the assay method, purification procedure, and properties of aldehyde dehydrogenase from the baker's yeast. The reaction is conveniently monitored by measuring the production of DPNH spectrophotometrically at 340 nm and 25° with benzaldehyde as the aldehyde substrate. Benzaldehyde was selected because it did not inhibit the reaction under standard assay conditions. However, by limiting the aldehyde concentration, the method described in the chapter can also be used as an assay for a wide variety of aldehydes some of which are inhibitory. Early studies with the aldehyde dehydrogenase from yeast resulted in a crystalline preparation of apparent homogeneity. However, analysis of the amino terminus revealed the presence of a spectrum of amino acids in the N terminal position. That preparation has been obtained by autolysis, and despite the use of a serine esterase type of protease inhibtor, PMSF, had undergone degradation. The purification procedure detailed in the chapter also makes use of PMSF and produces three species of aldehyde dehydrogenase designated as A, B, and C, of which species A is homogeneous; only serine is found as the N-terminal amino acid. A second method of preparation is also presented in which DFP serves as the major inhibitor of proteolysis. The product consists almost entirely of species A. Yeast aldehyde dehydrogenase, a tetramer of about 200,000 daltons, consists of four apparently identical subunits, which may be reversibly dissociated in 3 M guanidine hydrochloride. As is the case with all aldehyde dehydrogenases, the enzyme from yeast is inhibited by trivalent arsenicals.

Original languageEnglish
Pages (from-to)354-360
Number of pages7
JournalMethods in Enzymology
Issue numberC
StatePublished - 1 Jan 1975


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