Influence of Protein Factors on the Activation of NADP–malate Dehydrogenase by Dithiothreitol

Leaves of Phaseolus vulgaris L. contained malate dehydrogenase activity dependent on NADP (E. C. 1.1.1.82); it could be detected in extracts only when a dithiol such as dithiothreitol was present. After the addition of dithiothreitol, the activity increased with time, passed through a maximum and then diminished. The activation rate and/or the maximum level of activity were essentially dependent on dithiothreitol concentration, pH, and temperature. The presence of bovine serum albumine or glycerol in the medium decreased the inactivation rate; equivalent results were obtained at low temperatures. A mathematical model was established showing an apparent first-order rate for activation and inactivation only under conditions of alkaline pH near 8.3. These data allowed us to demonstrate that compounds other than dithiothreitol were necessary for the activation process. Evidence suggested that these compounds were protein factors of low molecular weights which increased the activation rate and the maximum level of activity when added to the incubation medium at pH 7.3. Their efficiency in the enzyme activation was higher at alkaline pH than at acid or neutral pH.