Cyclic AMP-dependent protein kinases and binding sites for cyclic AMP in rat erythrocytes

Abstract

In red cell preparations from reticulocyte-poor (untreated animals; approximately 2% reticulocytes) and reticulocyte-rich blood (animals pretreated with acetylphenylhydrazide; approximately 60% reticulocytes) of rats, cAMP binding sites and cAMP-dependent protein kinase activities were determined. High affinity binding sites for cAMP were present both in membrane and cytoplasmic preparations; while the apparent binding constants determined in both cell fractions (approximately 3 x 10(-9) M for membrane, approximately 2 x 10(-8) M for cytoplasmic fractions) were independent of the reticulocyte content of the preparations, the respective numbers of sites were about twice as high in the reticulocyte-rich as in the reticulocyte-poor preparations. In membrane preparations, significant cAMP-dependent protein kinase activity could be detected only in membrane fractions from reticulocyte-rich blood which were considerably contaminated by intracellular components ("haemoglobin-containing membranes') while in washed ("haemoglobin-free') membranes no cAMP-dependent protein kinase activity was found. In cytoplasmic preparations both from reticulocyte-poor and reticulocyte-rich blood, two different protein kinases, a low and a high Ka enzyme, were tentatively differentiated by kinetic data; the apparent activation constant for the high Ka enzyme (approximately less than 5 x 10(-8) M) was in the concentration range of the binding constants determined on cytoplasmic preparations. The activity of the high Ka protein kinase was several fold higher in reticulocyte-rich than in reticulocyte-poor cytoplasmic fractions, while the activity of the low Ka enzyme was obviously independent of the reticulocyte content. From the results obtained, it is concluded that in premature rat erythrocytes, membrane protein(s) may serve as protein substrates for cAMP-dependent protein kinase(s) located in the cytoplasm. This assumption was supported by experiments with intact erythrocytes (prelabelled with inorganic 32P-phosphate) from reticulocyte-rich blood: isoprenaline, theophylline, and also dibutyryl-cAMP significantly increased phosphorylation of membrane protein of these cells. From the results presented (and others previously reported) it becomes evident that only premature rat erythrocytes, i.e. reticulocytes, are equipped with a beta-adrenergic receptor-effector system consisting of a beta-adrenergically stimulated adenyl cyclase and cAMP-dependent protein kinase(s). Obviously, the adrenergic receptor system and also part of the effector system is lost during the process of red cell maturation.