A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.

Depolarization-induced calcium influx into rat cerebral cortex synaptosomes increased the phosphorylation of several synaptosomal proteins as examined by 32Pi incorporation. A phosphopeptide mapping technique involving NaDodSO4/ polyacrylamide gels has been used to show that phosphorylation of a Mr 87,000 substrate protein is stimulated by depolarizationinduced calcium influx. Phosphorylation of this Mr 87,000 substrate occurred in synaptosomal cytosol and was markedly stimulated by calcium/phosphatidylserine. Calmodulin inhibited this phosphorylation reaction. This substrate for calcium/phospholipid-dependent protein kinase is enriched in and appears to be specific to neurons.Calcium is recognized to play a key role in the regulation of neurotransmitter release from nerve endings (1). Increasing evidence indicates that some of the intracellular actions of calcium in neurons may be mediated through protein phosphorylation (see review in ref. 2). By using fractionated nerve endings (synaptosomes) from the brain, it was demonstrated that calcium influx induced by membrane depolarization resulted in increased phosphorylation of specific intraterminal proteins, the most prominent of which was protein I (3). Activation by calcium of the phosphorylation of protein I and of certain other synaptosomal proteins has been shown to be mediated by calmodulin-dependent protein kinases (4-6).Recently, a second species of calcium-dependent protein kinase has been described that requires phospholipid (7) rather than calmodulin for its activity. This enzyme occurs at a relatively high level in mammalian brain (8). We report here the occurrence of a Mr 87,000 phosphoprotein (termed the 87k protein) in the nerve terminals from rat cerebral cortex; the phosphorylation of this protein is regulated by Ca2' influx through activation of a Ca2+/phospholipid-dependent protein kinase.MATERIALS AND METHODS Materials. Bovine brain L-a-phosphatidyl-l-serine (98-99% pure) and diolein (99%) were purchased from Sigma. Calmodulin was purified from rabbit brain according to the procedure of Grand et al. (9). Standard protein I was purified from bovine brain by a modification (unpublished results) of the original procedure (10). The catalytic subunit of cAMP-dependent protein kinase was purified from bovine heart as described (11).Preparation of Synaptosomes. A crude mitochondrial fraction (P2) containing synaptosomes was prepared as described (3). For further subfractionation, the P2 pellet prepared from three rats was suspended in 5 ml of 0.32 M sucrose/5 mM Hepes, pH 7.4, at a protein concentration of 8-10 mg/ml. The suspension was layered on a step gradient consisting of the following sucrose solutions in 5 mM Hepes (pH 7.4): 2 ml, 1.5 M; 10 ml, 1.2 M; 6 ml, 1.0 M; 7 ml, 0.8 M. After centrifugation in a Beckman SW 25.1 rotor at 90,000 x g for 2 hr, 2.3-ml fractions were collected. Fractions enriched in myelin, synaptosomes, and mitochondria were identified by the use of appropriate enzyme markers (12,13). The fractions were diluted to ...

Under conditions that limit proteolytic degradation, the detergent-solubilized purified receptor protein from Torpedo californica exists in monomeric and dimeric forms. The purified receptor complex is composed of four different polypeptide subunits of apparent molecular weights 40 000, 50 000, 60 000, and 65 000. The individual polypeptides have been purified and their amino acid compositions have shown them to be relatively hydrophobic. In addition, the carbohydrate composition of the intact receptor complex and of the individual polypeptides has been determined. Amino acid analysis provided evidence for the occurrence of a component with chromatographic properties similar to those of phosphoserine. Treatment of receptor with CH3NH2 in base, a condition which provided quantitative modification of O-phosphoserine residues in beta-casein, completely eliminated the peak corresponding to phosphoserine following mild acid hydrolysis. We conclude that the receptor contains O-phosphoserine residues to the extent of approximately seven residues per molecule and these residues occur in all constituent polypeptides. Other forms of O-substituted serine and threonine were also shown to occur, most likely as glycosylated residues.

Calmodulin was previously found to inhibit the Ca2 /phospholipid-dependent phosphorylation of an endogenous substrate, called the 87-kilodalton protein, in a crude extract prepared from rat brain synaptosomal cytosol. We investigated the mechanism of this inhibition, using Ca2+/phospholipid-dependent protein kinase and the 87-kilodalton protein, both of which had been purified to homogeneity from bovine brain. Rabbit brain calmodulin and some other Ca2+-binding proteins inhibited the phosphorylation of the 87-kilodalton protein by this kinase in the purified system. Calmodulin also inhibited the Ca2+/phospholipid-dependent phosphorylation of HI histone, synapsin I, and the 6 subunit of the acetylcholine receptor, with use of purified components. These results suggest that calmodulin may be a physiological regulator of Ca2+/phospholipid-dependent protein kinase.Intracellular calcium is recognized to play a key role in the regulation of numerous physiological processes (e.g., exocytosis and muscle contraction). Many of the intracellular actions of calcium appear to be mediated by calcium-dependent protein phosphorylation (1,2). Two classes of calciumdependent protein kinases have been identifed-namely, Ca2+/calmodulin-dependent protein kinases (2) and Ca2+/ phospholipid-dependent protein kinase (also known as protein kinase C) (3). Ca2+/phospholipid-dependent protein kinase has been purified to homogeneity from rat brain (4) and pig spleen (5) and has been characterized in detail. This kinase has been suggested to play a physiological role in the regulation of hormone and neurotransmitter release. For example, serotonin release from platelets may be mediated through the Ca2+/phospholipid-dependent phosphorylation of an endogenous 40-kilodalton (kDa) substrate (6, 7).A physiological role for Ca2+/phospholipid-dependent protein kinase in neurons has been suggested by evidence that depolarization-induced calcium influx into intact synaptosomes increased the phosphorylation of an 87-kDa protein (87kDa) through activation of this kinase (8). The Ca2+/ phospholipid-dependent stimulation of 87kDa phosphorylation, observed in a calmodulin-depleted preparation of synaptosomal cytosol, was inhibited by the addition of exogenous calmodulin (8). We report here the demonstration of this calmodulin inhibition, using Ca2+/phospholipid-dependent protein kinase and 87kDa that had been purified to homogeneity. MATERIALS AND METHODSMaterials. Fresh bovine brains were transported on ice to the laboratory from a local slaughterhouse. Rat brain cytosol was depleted of calmodulin as described for rat brain synaptosomal cytosol (8). Calmodulin was purified from rabbit brain by the procedure of Grand et al. (9). Human erythrocyte calmodulin, bovine brain S-100 protein, and rat muscle parvalbumin were purchased from Calbiochem-Behring. Rabbit skeletal muscle troponin C was a gift from P. Leavis (Boston Biomedical Research Institute). S-100 protein, parvalbumin, and troponin C were found to be free of calmodulin contamination by the...

Brief freezing as a means of transiently permeabilizing synaptosomes was explored. Rat brain synaptosomes frozen and thawed in the presence of 5% dimethyl sulfoxide, a cryoprotectant, were shown to release, in a calcium‐dependent manner, previously accumulated [3H]norepinephrine and [14C]acetylcholine in response to elevated [K+]o. In addition, synaptosomes subjected to freeze/thaw were shown to retain their ability to exhibit resting protein phosphorylation, as well as stimulated protein phosphorylation occurring in response to calcium influx. Brief freezing of synaptosomes in the presence of [γ‐32P]ATP and either the catalytic subunit of cyclic AMP‐dependent protein kinase or calcium/calmodulin‐dependent protein kinase II rendered the synaptosomal interior accessible to these agents, as reflected by the phosphorylation of substrate proteins, such as synapsin I, which reside within the nerve terminal. Inclusion of inhibitors of these protein kinases during freeze/thaw blocked synaptosomal protein phosphorylation, indicating that the inhibitors were also introduced. After freezing, the synaptosomes resealed rapidly and spontaneously, as shown by the inability of any of the agents to elicit an effect on phosphorylation when added at the end of the freezing period. The permeabilization procedure should contribute to an understanding of the functional roles of phosphoproteins, and of their associated protein kinases and protein phosphatases, in nerve terminals.