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A membrane-bound D-glucose dehydrogenase [E.C. 1.1.99.a] was solubilized from the membrane of Pseudomonas sp. and purified to a nearly homogeneous state. The solubilized enzyme was monomeric in the presence of 1 % Triton X-100 but aggregated after removing the detergent.The enzyme was a single peptide having a molecular weight of about 90 ,000. The enzyme reacted with various artificial electron acceptors such as phenazine methosulfate, 2,6-dichlorophenolindophenol, Wurster's blue, coenzyme Q1, and ferricyanide . The enzyme had a dual optimum pH depending on the electron acceptor. Reductase activities of the enzyme for 2,6-dichlorophenolindophenol, ferricyanide and coenzyme Q1, were found in more acidic pH region, whereas its activities for phenazine methosulfate and Wurster's blue were observed in more alkaline region. p-Benzoquinone inhibited phenazine metho sulfate reductase activity non-competitively but it inhibited 2,6-dichlorophenolindophenol reductase activity competitively against the acceptor.The enzyme possessed fairly broad substrate specificity, and the reaction product was a gluconolactone.

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D-Glucose dehydrogenase purified from the membraneof Pseudomonasfluorescens was shown to be highly hydrophobic in amino acid analysis, with a polarity of 39.7%. The purified enzyme was inactivated upon removal of detergent by acetone treatment. The detergent-depleted enzyme was activated partially with Triton X-100, and the activity was restored almost completely upon addition of both phospholipids and Triton X-100, followed by sonication. The purified enzyme, in spite of being a single polypeptide dehydrogenase, directly reduced not only short-chain ubiquinone but also long-chain homologs. It should be noted that coenzyme Q-6 or Q-9 incorporated in phospholipid vesicles was efficiently reduced with the enzyme. These results show that, in the cytoplasmic membraneof Pseudomonasfluorescens, the glucose dehydrogenase may be linked to an electron transport chain via ubiquinone. Oxidative bacteria, such as Pseudomonas, Klebsiella, Serratia and acetic acid bacteria, oxidize D-glucose to D-gluconate and then to 2keto-D-gluconate. The oxidizing system is constructed of D-glucose and D-gluconate dehydrogenases located in the cytoplasmic mem-brane1'2) of the organisms and coupled to the respiratory chain.3) Wehave already reported the purification and some properties of the membrane-bound glucose dehydrogenase from Pseudomonas fluorescens.A) The enzyme was solubilized with Triton X-100 in the presence of KC1 and purified to near homogeneity. The purified enzyme was a monomerin the high con