Studies on the Orientation of Cyclic AMP-Dependent Protein Kinase in Human Erythrocyte Membranes

Abstract

The topographic location of cyclic AMPdependent protein kinase has been studied in preparations of permeable and sealed membranes derived from human erythrocytes. Both the catalytic and cyclic AMP-binding components of protein kinase are localized on the inner, cytoplasmic, surface of the plasma membrane.3':5'-Cyclic AMP has been implicated in the regulation of a variety of membrane-associated functions including: contact inhibition (1, 2), hormone secretion (3), cellular permeability (4), and synaptic transmission (5, 6). It is possible that membrane-associated, cyclic AMP-dependent protein kinases could alter the structure and function of cell membranes by catalyzing the phosphorylation of specific polypeptides in plasma membranes (7-13).Plasma membranes derived from human erythrocytes contain greater than 70% of the total protein kinase activity found in these cells (14), and catalyze the cyclic AMPcontrolled phosphorylation of two endogenous polypeptide chains (9-11). These observations suggest that erythrocyte membranes provide an appropriate system for investigations on protein kinase-mediated alterations in the properties of membranes.Since the protein components of the erythrocyte membrane are asymmetrically positioned with respect to the cytoplasmic and exterior membrane surfaces (15), the biological role of cyclic AMP in membranes may be uniquely determined by the topographic distribution of the enzymes involved in the metabolism and action of the cyclic nucleotide. In the present experiments the spatial orientations of the catalytic and cyclic AMP-binding components of protein kinase were studied in permeable ghosts, sealed rightside-out ghosts, and sealed inside-out vesicles prepared according to the methods of Steck and Kant (16,17) and Bodemann and Passow (18). The disposition of protein kinase in the membrane was determined by comparing its accessibility in the various membrane preparations to the accessibility of enzymes known to be associated with either the cytoplasmic or the exterior surfaces of the membrane (15-17). (EC 1.6.4.3) by the procedure of Wallach and Kamat (22). Assays of enzymic and cyclic AMP-binding activities in salt-sealed ghosts were performed in saline buffered with 10 mM Tris -HCl, pH 7.4, to prevent lysis. Substitution of buffered saline for the standard buffers had no effect on the enzymic and binding activities in any of the membrane preparations. Inside-out vesicles and Mg2+-sealed ghosts were not lysed by incubation in the standard assay buffers. MATERIALS AND METHODS RESULTSThe data on the accessibility of cyclic AMP-dependent protein kinase and three reference enzymes are presented in Table 1. Acetylcholinesterase, a marker for the exterior surface of the erythrocyte membrane (23), is completely accessible in permeable ghosts and sealed, rightside-out membranes, but is relatively inaccessible in sealed, inside-out membranes. Conversely, glyceraldehyde-3-phosphate dehydrogenase, an indicator of the inner surface of the membrane (24), is completely available i...

The Organization of Proteins in the Human Red Blood Cell Membrane

“…The enzyme appears to reside exclusively at the cytoplasmic side of the membrane (106), a further argument for the presence of the phosphorylated proteins at that surface. Whether the cyclic AMP binding site, which has also been ascribed to the inner surface of the erythrocyte membrane (106,139), is the same as the regulatory site of the protein kinase is uncertain, since the cyclic AMP concentration required for half-maximal saturation of the binding site (3 nM) is 10 100 times lower than that required for half-maximal stimulation of phosphorylation (135,136,139). Photoaffinity labeling was recently used to demonstrate that the 50,000-dalton polypeptide whose phosphorylation is most stimulated by cyclic AMP probably corresponds to this cyclic AMP binding site (140).…”

The band 3 protein of the human red cell membrane: A review

“…Labeling is not stimulated by cyclic AMP (adenosine monophosphate) in vitro. The phosphate appears to be introduced only from the cytoplasmic surface [77] . Drickamer [50, 511 has found that phosphorylation occurs primarily within 10,000 daltons of the cytoplasmic N terminus of band 3, but that some 32Pi can also be detected in fragments ascribed to the center of the polypeptide chain.…”

Substrate heterogeneity of component a of the human erythrocyte membrane

“…This behavior has recently been demonstrated with respect t o the major sialoglycoprotein (17). Component a migrates as a wide band at an apparent molecular (18). Carbohydrates have been repeatedly demonstrated on the outer surface available to the external environment of the erythrocyte (19)(20)(21).…”