Mammalian Galactose Dehydrogenase: II. PROPERTIES, SUBSTRATE SPECIFICITY, AND DEVELOPMENTAL CHANGES

Abstract

Abstract The properties of a 100-fold purified preparation of rat liver galactose dehydrogenase are presented. Optimum activity occurs at pH 8.3. The enzyme is relatively stable to dialysis, heat treatment, and storage at -30°, but is quite unstable below pH 5. Nicotinamide adenine dinucleotide, but not galactose, protects from heat inactivation. Heat inactivation kinetics suggests that one enzyme is responsible for galactose oxidation. Activity is strongly inhibited by sulfhydryl group inhibitors, Cu++, Zn++, and Fe++, yet sulfhydryl reagents do not enhance activity or protect from inactivation. Activity was not inhibited by cyanide, arsenate, or ethylenediaminetetraacetate. The Km for galactose is 2.6 x 10-2 m and for NAD is 1.7 x 10-4 m. The Vmax is 0.57 µmole per min per mg of protein. The reaction product, NADH, is a strong competitive inhibitor of galactose dehydrogenase (Ki, 3.4 x 10-5 m). The cofactor NADP is less than 1% as effective as NAD, and it competitively inhibits (Ki, 6.6 x 10-3 m). NADPH is also a competitive inhibitor (Ki, 1.2 x 10-3 m). The enzyme preparation has a very broad specificity for sugar substrates, reduction of NAD (but not of NADP) occurring in the presence of various aldohexoses, aldopentoses, aldoheptoses, and disaccharides. The specificity patterns suggest the presence of at least three aldose dehydrogenases with very specific steric requirements for substrates. Studies with combinations of sugars, heat inactivation, and starch gel electrophoresis were more consistent with the presence of a single enzyme of unusual substrate specificity. Heat inactivation kinetics suggests two maltoseoxidizing enzymes. The NAD-reduction reactions occurring with l-aldohexoses, aldoheptoses, maltose, and cellobiose suggest novel reactions hitherto unrecognized in mammalian tissues. Some keto sugars behave as substrates, probably by isomerization to corresponding aldo sugars, but an unusual direct oxidation was not excluded. Developmental changes in rat liver galactose dehydrogenase show that activity is barely detectable until birth, when a sudden increase occurs, reaching a maximum in animals 5 days old and falling slowly to the adult level in animals 30 days of age.