Studies on the activities, kinetic properties and subcellular localisation of cyclic AMP phosphodiesterase in human neutrophil leukocytes

Abstract

A one-step radioassay for cyclic AMP phosphodiesterase was optimised for human polymorphonuclear leukocytes. Kinetic studies indicated the presence of two forms of phosphodiesterase activity with apparent Km values of 0.015 mmol/l and 0.98 mmol/l for cyclic AMP. Control neutrophils were homogenised in isotonic sucrose and, after low speed centrifugation, the supernatant was subjected to analytical subcellular fractionation. Gradient fractions were assayed for principal marker enzymes and for cyclic AMP phosphodiesterase. Both forms of phosphodiesterase activity were located in the cytosol. Polymorphonuclear leukocyte homogenates were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. A portion of each homogenate was used for enzyme analysis and the remainder assayed for cyclic AMP content. The specific activity (mUnits/mg protein) of the low Km phosphodiesterase was reduced in both patient groups compared with control values, whilst that of the high Km phosphodiesterase was unchanged. Leukocytes from patients with chronic granulocytic leukaemia had only a fifth of the cyclic AMP content of control neutrophils, whilst leukocytes from patients in the third trimester of pregnancy had an elevated cyclic AMP content.