Cyclic nucleotide phosphodiesterase of normal and leukemic lymphocytes. Kinetic properties and selective alteration of the activity of the multiple molecular forms.

The multiple forms of cyclic AMP and cyclic GMP phosphodiesterase of normal and leukemic lymphocytes were separated by gel electrophoresis and characterized by measuring their kinetic properties and their response to activators and inhibitors of phosphodiesterase. The pattern of the cyclic AMP phosphodiesterases of normal and leukemic cells was similar: both evidenced 4 forms of the enzyme, designated as I, IA, III and IV. Peaks I, IA and IV had classic Michaelis-Menten kinetics with apparent K m values of 10, 10 and 70 µM respectively. Peak III, a major form of phosphodiesterase activity, had anomalous kinetic behavior, suggestive of an allosteric enzyme displaying positive cooperativity. By contrast, the pattern of the cyclic GMP phosphodiesterases of leukemic lymphocytes was different from that of the normal lymphocytes. Normal lymphocytes had 3 forms of cyclic GMP phosphodiesterase corresponding to peaks IA, III, and IV of cyclic AMP phosphodiesterase. However, leukemic lymphocytes showed only 2 forms of cyclic GMP phosphodiesterase; these corresponded to peaks III and IV of cyclic AMP phosphodiesterase. The apparent K m values for peak III (the major peak) was similar in the two types of cells (100 µM). Peak IV showed K m values of 10 µM for normal lymphocytes and 40 µM for leukemic lymphocytes. Peak IA was present only in normal lymphocytes and had an apparent K m of 16 µM. A study of the cyclic AMP phosphodiesterase activities of mouse cerebrum and salivary gland showed that the major form of phosphodiesterase in these tissues did not correspond to that found in leukemic lymphocytes. Calmodulin increased the activity of the major form of phosphodiesterase isolated from cerebrum but had no effect on the forms of phosphodiesterase isolated from lymphocytes or several other tissues. Cyclic GMP (5 µM) increased the activity of the major form of cyclic AMP phosphodiesterase from normal and leukemic lymphocytes but had no effect on other forms of phosphodiesterase of lymphocytes. This selective activation by cyclic GMP of the phosphodiesterase of lymphocytes was found to normalize the anomalous kinetic behavior of this form of the enzyme. The various forms of phosphodiesterase from leukemic lymphocytes and normal tissues of the mouse could be selectively inhibited by drugs. Thus, isobutylmethylxanthine, a competitive antagonist, was a more potent inhibitor of the form of the enzyme with high Michaelis constants ( K m ) than those with low K m values. Chlorpromazine, a drug that interferes with the calmodulin-induced activation of phosphodiesterase, was a potent inhibitor of the activable form of phosphodiesterase from cerebrum but a weak inhibitor of the other forms of the enzyme. Papaverine, which has a mixed mechanism of action, was an effective inhibitor of all forms of phosphodiesterase examined. Dipyridamole was a potent inhibitor of the major form of phosphodiesterase from normal and leukemic lymphocytes but was less effective in inhibiting the major form of phosphodiesterase from cerebrum. When these drugs were tested on the phosphodiesterase activity of crude homogenates, their order of potency reflected their potency as inhibitors of the major form of the enzyme isolated by gel electrophoresis. These studies demonstrate that the phosphodiesterases isolated from normal and malignant tissues of the mouse could be selectively activated and inhibited. Since an abnormal metabolism of cyclic nucleotides may be associated with malignancy, the data suggest the possibility of developing chemotherapeutic agents that act by selectively altering the metabolism of cyclic nucleotides in malignant tissue.