Nicotinamide Deamidase from Rabbit Liver

Active-site studies on rabbit liver nicotinamide deamidase

“…Several changes made in scaling up the procedure did not affect the homogeneity of the isolated enzyme. The specific activity of the freshly prepared enzyme was occasionally 20-30% higher than that reported by Su et al (1969) but after several days frozen storage all preparations stabilized at 3.4-4.5 ^moles/mg hr. (a) Centrifugations were done at 16,000g for 30 min at the temperatures indicated previously, (b) The initial extraction involved 200 g of microsomal acetone powder and 4 1. of 0.2 m glycine buffer, pH 8.9, done in four 50-g batches, (c) The pH 5.0 precipitation step was omitted, (d) The deamidase activity was precipitated between 35 and 55% acetone (v/v) in the final step.…”

“…Two methods were used to prepare microsomal acetone powder from fresh rabbit livers. Method I has been described by Su et al (1969).…”

“…Purification of the enzyme was performed essentially according to the method of Su et al (1969). Several changes made in scaling up the procedure did not affect the homogeneity of the isolated enzyme.…”

“…Only preparations which were homogeneous by this criterion were used in the studies reported here. Su et al (1969) determined that fresh enzyme migrated as a single band in disc gel electrophoresis at various pH values and migrated as a single symmetric peak in the ultracentrifuge.…”

“…T Xhe widespread occurrence of the enzyme nicotinamide deamidase (Preiss and Handler, 1958a,b;Imsande, 1961;Joshi and Handler, 1962;Bernheim, 1967;Pallini et al, 1965;Petrack et al, 1963Petrack et al, , 1965Kirchner et al, 1966;Marki and Greengard, 1966) and the possibility that control of the Preiss-Handler pathway of NAD biosynthesis may occur in vivo at this reaction (Greengard et al, 1963(Greengard et al, , 1965(Greengard et al, , 1969 have stimulated investigations into the physical, kinetic, structural, and control properties of the enzyme (Su et al, 1969). Of particular interest was the finding that nicotinamide deamidase from rabbit liver also catalyzes the hydrolysis of a variety of ester substrates (Su et al, 1969). This dual activity of the enzyme is analogous to that of a number of other enzymes (e.g., chymotrypsin, trypsin, carboxypeptidase-A, f From the Department of Biochemistry and Biophysics, University cinnamic acid, and 3-indolepropionic acid).…”