Glucose oxidase from Aspergillus niger was purified by ammonium sulfate fractionation and chromatography on DEAE-cellulose. The purified enzyme was homogeneous on ultracentrifugation, density-gradient centrifugation, and starch-gel and paper electrophoresis. The enzyme was capable of oxidizing D-aldohexoses, monodeoxy-D-glucoses and O-methyl-D-glueoses at varying rates. Differences in the rates of oxidation of these compounds have been interpreted to indicate that the structural features of the substrate of particular importance in the enzymatic reaction are a pyranose ring in the chair or Cl conformation, an equatorially orientated hydroxyl group at position 1, and an equatorially orientated hydroxyl group at position 3. These structural features are probably involved in the formation of the enzyme-substrate complex.* This investigation was supported in part by a grant from the Miles Laboratories, Inc., Elkhart, Ind., and is published with the approval of the Director as Paper No.

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The activity of T4 polynucleotide kinase (EC 2.7.1.78) was found to be greatly stimulated by salts, such as NaCl and KCl, and polyamines such as spermine and spermidine. Up to a sixfold increase in initial rates was observed with a variety of different single-stranded DNAs and mono- and oligonucleotides. The optimal concentrations of salts were 0.125 M, corresponding to a total ionic strength of mu equals 0.19. For polyamines the optimal concentrations were found to be at approximately 2 mM. With low enzyme concentration and in the absence of activators complete phosphorylation was not achieved for a number of substrates. In the presence of salts or polyamines or high concentration of enzyme the phosphorylation proceeded to completion. Addition of salt led to an increase in both the apparent V-max and the Michaelis constant for the DNA substrate whereas the Michaelis constant of ATP remained unchanged. Polyamines had a similar influence on the kinetic constants for the DNA substrate whereas a decrease was found for the apparent Michaelis constant for ATP. The overall mechanism in the presence of activators was found to be sequential but probably of a rapid equilibrium random type. Of the inorganic anions tested both P-i and PP-i inhibited the enzyme in a competitive manner with both substrates.