Subunit hybridization of higher plant lactate dehydrogenases

developed a procedure, based on the affinity of all three enzymes for Cibacron Blue 3GA, which allows their simultaneous purification from a cell extract. E. coli K 12, EMG-2 was grown, harvested and disrupted as described by Miller & Stadtman (1972). After treatment with 1% (w/v) streptomycin sulphate, the cell extract was subjected to ammonium sulphate fractionation. The protein fraction precipitating between 30 and 60% saturation was found to contain the desired enzymes. This fraction was collected, resuspended in and dialysed against 5 0 m ~ potassium phosphate buffer, pH6.0, containing 1 mM2-oxoglutarate, 1 mM-EDTA and 5 m~-2-mercaptoethanol (buffer A). The dialysed material was clarified by centrifugation and applied to a column (I.Ocm x 5.Ocm) of Blue Dextran-Sepharose (Ryan & Vestling, 1974). equilibrated with buffer A. A column of these dimensions bound all of each of the three enzymes from log of cells. The column was washed with 20 column vol. of buffer A and was then eluted with lOml of I.OM-NaCI in buffer A. G D H and GOGAT were eluted by the salt wash but GS was not. GS was eluted by washing the column with lOml of ~ ~ M A D P in buffer B (50mM-potassium phosphate, pH 7.5, containing 1 mM2-oxoglutarate, I mM-EDTA and 5 m~-2-mercaptoethanol). This behaviour of GS has been shown previously by Burton & Eisenberg (1980). Electrophoresis on non-denaturing gradient gels and on sodium dodecyl sulphate/polyacrylamide gels (Laemmli, 1970) of the fractions eluted by salt and by ADP indicated that the GS was homogeneous. However, the salt-eluted material contained many proteins in addition to G D H to GOGAT. Further purification of these two enzymes was carried out by desalting them on a small Sephadex G25 column and applying the eluate to a column (0.7cm x 5cm) of 2’,5’-ADP-Sepharose. This material binds both GDH and GOGAT (Schmidt & Jervis, 1980) and the two enzymes can be eluted with a pulse of 1 0 0 ~ ~ NADPH plus 1 mM-L-glutamine in buffer A. Electrophoresis of the eluted material indicated that GDH and GOGAT were the major proteins present. The material was concentrated with an Amicon Centricon 10 microconcentrator and the concentrate was subjected to gel filtration on Sephacryl S-300 to resolve the G D H and GOGAT on the basis of the difference in their M , values (300000 and 800000 respectively). The purification of GS, G D H and GOGAT is summarized in Table 1.