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1. A procedure is described for the isolation of d-galactose dehydrogenase from Pseudomonas fluorescens, grown on d-galactose as carbon source. A 750-fold purification was achieved with an overall yield of about 40%. The final preparation had a specific activity of about 850 μmol NADH formed per min per mg protein. 2. The enzyme has been shown to be homogeneous by ultracentrifugation, by disc gel electrophoresis, by gel isoelectric focusing and by dodecylsulfate gel electrophoresis. 3. The molecular weight of the native enzyme has been determined to be 64000 by sedimentation equilibrium studies and by gel permeation chromatography. 4. The enzyme dissociates in the presence of 0.1% sodium dodecylsulfate to polypeptide chains, whose molecular weight was determined as 32000 by electrophoresis, indicating that the native enzyme is composed of two subunits. 5. The amino acid composition of d-galactose dehydrogenase has been determined. 6. The pI of the enzyme has been shown to be 4.28. The pH optimum is between 9.1 and 9.5. At pH 9.1 and 30 °C the limiting Michaelis constant for NAD+ is Ka= 0.24 mM, for NADP+Ka= 2.3 mM and for d-galactose with NAD+ and with NADP+ as cosubstrate Kb= 0.7 mM. The dissociat on constant for NAD+ is Kia= 0.54 mM and for NADP+Kia= 6.2 mM. 7. d-Galactose dehydrogenase from Ps. fluorescens catalyzes the dehydrogenation of d-galactose by either NAD+ or NADP+. The enzyme oxidizes the following sugars arranged according to relative rates in decreasing order: d-fucose > d-galactose > l-arabinose > 2-deoxy-d-galactose ≫ 4-deoxy-d-galactose > 2-deoxy-2-amino-d-galactose. 8. The correspondence between amino acid composition, subunit size and structure and between specificity of d-galactose dehydrogenase from Ps. fluorescens and from Ps. saccharophila is discussed.

1 d-Galactose dehydrogenase from Pseudomonas saccharophila has been purified over 700-fold with an over-all yield of 27%. The purified enzyme exhibits a specific activity of about 200 μmol NADH formed × min−1× mg protein−1 and has been shown to be homogeneous by ultracentrifugation and by gel electrophoresis. 2 The amino acid composition of the enzyme has been determined. 3 The native enzyme has a sedimentation coefficient (S20,w0) of 5.94 S and a diffusion coefficient (D20,w0) of 5.60 F. The experimentally determined partial specific volume (v) is 0.744 ml/g. The molecular weight calculated from these data is 103000, in good agreement with the value of 102000 estimated by gel permeation chromatography. 4 The enzyme dissociates into polypeptide chains in the presence of 5 M guanidine hydrochloride or 0.1% sodium dodecylsulfate. The molecular weight of the polypeptide chains has been found to be 25000 by ultracentrifugation (S20,w0= 1.35 S, D20,w0= 5.28 F) and by dodecylsulfate-polycrylamide electrophoresis. The native enzyme is composed of four polypeptide chains. 5 The pI of d-galactose dehydrogenase is 5.1. The pH-optimum is between pH 8–9 in Tris-HCI buffer. AT PH 8.6 and 30°C the limiting michaelis constant for NAD+ is Ka= 0.14 mM, the limiting michaelis constant for d-galactose is Kb= 1.0 mM and the dissociation constant for NAD+ is Kia= 0.48 mM. 6 The enzyme is specific for NAD and uses NADP only to a minor extent as a coenzyme. It oxidizes the following sugars arranged according to relative rates, in decreasing order: d-FUCOSE > d-galactose > 3-deoxy-d-galactose > 2-deoxy-d-galactose > 4-deoxy-d-galactose > l-arabinose > 2-deoxy-2-amino-d-galactose.