Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties

Abstract

Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.

Relationships amongst some bacterial and yeast lactate and mandelate dehydrogenases

“…Overall, the D( -)-mandelate dehydrogenase and D( -)-lactate dehydrogenase from A . calcoaceticus are similar to each other and to the D(-)-lactate dehydrogenase from E. coli (Allison et al, 1985). All these mandelate and lactate dehydrogenases are membrane-bound enzymes.…”

“…All three enzymes contain FAD and have a similar MI. (Allison et al, 1985). Perhaps surprisingly, the two lactate dehydrogenases are not very similar over the sequences examined and it is unfortunate that repeated attempts failed to extend the sequence of the enzyme from A .…”

“…delate dehydrogenase and D( -)-lactate dehydrogenase were purified from A. calcoaceticus strain C1219 by the methods of Hoey et al (1987), Allison et al (1985) and Allison & Fewson (1986). L(+)-Mandelate dehydrogenase was purified from P. putida NCIB 9474 by a similar method to that used for the equivalent enzyme from A .…”

“…Micro-organisms were generally maintained as described previously (Allison et al, 1985;Baker & Fewson, 1989) and they were grown and harvested by conventional methods. Extracts were prepared by disruption with an ultrasonic probe or by passage through a French press (Baker & Fewson, 1989).…”

“…calcoaceticus is larger ( M , = approx. 60000) and contains FAD as cofactor (Allison et al, 1985). Overall, the D( -)-mandelate dehydrogenase and D( -)-lactate dehydrogenase from A .…”