A specific nucleoside diphosphatase was purified from the plant portion of soybean (Glycine mar L.) root nodules. This enzyme is highly specific for nucleotide diphosphates; it is unable to hydrolyze nucleotide tri-and monophosphates or a variety of other phosphorylated compounds. It will, however, hydrolyze any nucleotide disphosphate tested. The pH optimum of the enzyme is about 7.5; it requires a divalent cation for activity; and it is neither inhibited nor activated by any of the metabolites tested. It a,ppears that in vivo this enzyme would be very active, but its function is not clear.Nearly all reports of specific nucleoside diphosphatases, whether from animal (8, 9, 12), algal (2), or higher plant (3, 10) sources, describe membrane-bound enzymes which are essentially unable to hydrolyze ADP. The one exception is a brief report of the detection in soybean and alfalfa nodule extracts of an NDPase3 which could hydrolyze any NDP tested (11). In the previous paper (5), we reported confirmation of this work and assayed a variety of tissues for this NDPase activity. Soybean nodules were found to have by far the highest levels of this activity. Here, we report the purification and characterization of the soybean nodule NDPase. Affligel Blue Chromatography. The dialyzed extract was loaded on a 10 ml column of Affigel Blue (100-200 mesh, Bio-Rad Laboratories) equilibrated with the dialysis buffer. The column was eluted stepwise with four 10-ml aliquots of 30 mM Hepes-KOH (pH 7.5), followed by three 10-ml aliquots of 30 mM Hepes-KOH (pH 7.5), 1 M NaCI, 1 M AMP. NDPase activity was recovered primarily (80%) in the first two high-salt fractions; these fractions contained about 5% of the protein loaded on the column. They were pooled and dialyzed overnight against buffer B. The dialyzed enzyme was then concentrated by filtration (Minicon, Amicon Corporation).
MATERIALS AND METHODSDEAE-Sepharose Chromatography. The concentrate was loaded onto a 40 ml column of DEAE-Sepharose CL6B equilibrated with buffer B. The column was eluted first with 30 ml of buffer B, followed by a 150 ml gradient of 0 to 500 mM NaCL in buffer B. Fractions of 3 ml were collected and assayed for protein and NDPase activity.Gel Filtration Chromatography. A Superose-12 column (Pharmacia) attached to an FPLC system was equilibrated with 30 mM Hepes-KOH (pH 7.5), 100 mm NaCl. A 200 IlI aliquot of the NDPase peak from DEAE-Sepharose chromatography was loaded onto the column, which was eluted isocratically at a flow rate of 0.2 ml/min. Fractions (0.4 ml) were collected and assayed for NDPase activity. A standard curve for mol wt estimation was produced by injecting and running standards in a similar manner, with detection by UV absorbance.SDS Gel Electrophoresis. Fractions were analyzed by SDS-PAGE on 13% acrylamide gels using the discontinuous buffer system of Laemmli (13).Enzyme Assays. Routine NDPase assays were as given in the preceding paper (5); specific details are given in the test where appropriate.For product identification studies, t...