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Gizzard alpha-actinin binds 45Ca2+ as shown by the calcium overlay method. Flow dialysis measurements in 20 mM Hepes (pH 7.5) reveal 3.5 +/- 1.8 (S.D.) high affinity calcium binding sites per dimer, with Kd1 = 6.36 +/- 0.34 x 10(-6) M, and 87.3 +/- 7.2 sites with Kd2 = 1.66 +/- 0.44 x 10(-4) M. Chymotrypsin and thermolysin digestion yielded peptides of gizzard alpha-actinin which, if they included the putative EF-hands, bound 45Ca2+ in 10 mM imidazole-HCl (pH 7.4) or 60 mM KCl, 10 mM imidazole-HCl (pH 7.4). In addition, peptides which include a region of the molecule more than 27 kDa from the N-terminal also bind calcium. In contrast, when KCl in the binding buffer was increased to 120 mM, calcium binding was eliminated. Flow dialysis data revealed no high-affinity binding and 82.5 +/- 3.3 calcium binding sites with calculated affinities in the millimolar range. These are divalent cation binding sites, not Ca(2+)-specific sites, because they are eliminated by the addition of up to 5 mM Mg2+. Structural changes produced upon cation binding to alpha-actinin measured by circular dichroism, proteolysis and bisANS fluorescence are substantial when binding K+ with only small changes upon binding of Ca2+ or Mg2+ in the presence of 120 mM KCl. These results suggest that monovalent and divalent cations have different effects on different parts of the molecule with a complete elimination of 45Ca2+ binding to the EF-hands being produced by 120 mM KCl.

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An antibody recognizing all ankyrin isoforms was employed to localize ankyrin in the cerebellum by light and electron microscopy. White matter (myelinated fiber tracts) did not contain ankyrin. Granule cell bodies and axons contained ankyrin which extended into the parallel fibers and their synapses. Purkinje cells contained a nearly uniform plasma membrane undercoating of ankyrin in the cell body. However, in both of these cell types ankyrin was not detected in dendrites. Basket and stellate cells did not contain detectable ankyrin. Golgi neuroepithelial cell bodies and processes contained ankyrin while myelin and myelinated axons did not. These results indicate that the membrane skeletal protein ankyrin occurs in some, but not all, neuronal and glial cell types in the cerebellum. For the neurons in which it does occur its distribution is polarized, being limited to cell bodies and axons while not occurring in dendrites.

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Erythrocyte spectrin dimers and separated alpha- and beta-spectrin chains bound 45Ca2+ after electrophoresis on native or sodium dodecyl sulfate-polyacrylamide gels, blotting, and 45Ca2+ overlay. Flow dialysis and equilibrium dialysis revealed two binding components: high-affinity, Ca(2+)-specific sites with kd = 4 x 10(-7) M and n = 100 +/- 20 per dimer and a low-affinity (millimolar) divalent cation component. Whereas brain spectrin had only four high-affinity sites [Wallis, C. J., Wenegieme, E. F., & Babitch, J. A. (1992) J. Biol. Chem. 267, 4333-4337], erythrocyte spectrin had 25-fold more sites per dimer. In addition to possibly modifying spectrin interactions with calcium-dependent protease and actin, as suggested by previous work on the interaction of Ca2+ with brain spectrin, the approximately two high-affinity sites per repeating segment of erythrocyte spectrin appear to stabilize a folded conformation of repeat structures indicated by an entropy increase upon binding. These data support the hypothesis that divalent cation binding to erythrocyte spectrin has become specialized to stabilize the membrane skeletal network and the cell, making them flexible but resistant to shear under the stressful conditions of blood circulation.

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Brain spectrin alpha and beta chains bind 45Ca2+, as shown by the calcium overlay method. Flow dialysis measurements revealed eight high affinity binding sites/tetramer that comprise two binding components (determined by nonlinear regression analysis). The first component has one or two sites (kd = 2-30 x 10(-8) M), depending on the ionic strength of the binding buffer, with the remaining high affinity sites in the second component (kd = 1-3 x 10(-6) M). In addition, there is a variable, low affinity binding component (n = 100-400, kd = 1-2 x 10(-4) M). Magnesium inhibits calcium binding to the low affinity sites with a K1 = 1.21 mM. Proteolytic fragments from trypsin or chymotrypsin digests of brain spectrin bind 45Ca2+ if they include alpha domain IV, alpha domain III, or the amino-terminal half of the beta chain (but more than 25 kDa from the amino-terminal). These data suggest that calcium ions bind with high affinity to the putative EF-hands in alpha domain IV and to one site in the amino-terminal half of the beta chain that is associated with alpha domain IV in the native dimer. The localization is consistent with a direct calcium modulation of the spectrin-actin-protein 4.1 interaction. In addition, there appears to be one high affinity site near the hypersensitive region of alpha brain spectrin. All four proposed binding sites occur near probable calmodulin-binding or calcium-dependent protease cleavage sites.

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Glial fibrillary acidic protein (GFAP) is soluble in low ionic strength solutions but shows a strong tendency toward assembly with increasing ionic strength as revealed by electron microscopy and turbidity measurements. Increasing K+, Na+, and Li+ concentrations cause an increase followed by a decrease in GFAP turbidity with a maximum at 200 mM, but their effects are much weaker than effects of divalent cations at the same ionic strength. Ca2+, Mg2+, Mn2+, and Ba2+ promote assembly at millimolar concentrations, and 10 microM Cu2+ causes rapid aggregation. The critical concentration for GFAP assembly was 0.08 +/- 0.04 mg/mL in 2 mM Tris-HCl, 60 mM KCl, and 1 mM CaCl2, pH 6.8. The Mr 38,000 rod domain of GFAP obtained by limited chymotryptic digestion is more soluble in 100 mM imidazole hydrochloride buffer, pH 6.8, than the intact molecule, and removal of the end pieces greatly reduces the ability of GFAP to form filaments. BNPS-skatole (2-[(2-nitrophenyl)sulfenyl]-3-methyl-3-bromoindolenine) treatment releases a Mr 30,000 N-terminus and a Mr 20,000 C-terminus. The Mr 30,000 polypeptide shows a higher affinity than the Mr 20,000 fragment for intact GFAP. Arginine and lysine at low concentrations slightly accelerate GFAP assembly, but above 100 mM both amino acids inhibit assembly. ATP, GTP, CTP, and UTP do not show significant effects on GFAP assembly. Dephosphorylation by alkaline phosphatase slightly reduces the assembly ability of GFAP, but phosphatase-treated GFAP still is assembly competent.

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In our previous work [Yang, Z. W., & Babitch, J. A. (1988) Biochemistry (preceding paper in this issue)] divalent cations were found to be more effective promoters of astroglial filament formation than were monovalent cations. To determine if one or more divalent cation binding sites were the basis for this difference, glial fibrillary acidic protein (GFAP) was attached to nitrocellulose membranes and bathed in 1 microM 45CaCl2 in 60 mM KCl, 0.5 mM MgCl2, and 10 mM imidazole hydrochloride, pH 7.4. After removal of unbound 45Ca2+, GFAP was observed to bind calcium. Flow dialysis experiments showed that GFAP, dissolved in 2 mM Tris-HCl, pH 7.5, contained three classes of binding sites and 0.61 +/- 0.08 (SD), 1.7 +/- 0.4, and 4.6 +/- 0.2 sites per GFAP molecule with dissociation constants of 0.66 +/- 0.01 microM, 6.6 +/- 0.3 microM, and 44 +/- 1 microM, respectively. After addition of 0.5 mM MgSO4 to the flow dialysis solution, the high- and low-affinity sites were not observed while the remaining sites (1.95 +/- 0.15 per GFAP molecule) had a Kd = 2.16 +/- 0.25 microM. This showed that the high- and low-affinity sites are "Ca2+-Mg2+" sites while sites with intermediate affinity are calcium specific. To locate the calcium-binding regions, GFAP peptides were examined for calcium binding by calcium-45 autoradiography. The calcium-specific binding areas were localized in coil I. Computer-assisted analysis of the GFAP sequence revealed several EF-hand-like areas which could be the calcium binding sites. We conclude that divalent cations may play both structural and regulatory roles in astroglial intermediate filaments.

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Using flow dialysis, we found two classes of calcium-binding sites on tubulin: high-affinity binding sites (1.56 +/- 0.38 per tubulin dimer) with a dissociation constant of (4.86 +/- 0.12).10(-6) M and low-affinity binding sites (5.82 +/- 0.50 per tubulin dimer) with a dissociation constant of (6.4 +/- 0.4).10(-5) M. In the presence of 6.10(-5) M MgSO4, we found 0.64 +/- 0.18 calcium-binding sites per tubulin dimer with a dissociation constant of (4.7 +/- 0.5).10(-6) M and 1.2 +/- 0.2 sites per dimer with a dissociation constant of (3.5 +/- 0.4).10(-5) M. Under controlled conditions, trypsin and chymotrypsin selectively cleaved alpha- and beta-subunits, respectively, forming major fragments of 35 kDa and 20 kDa from the alpha-subunit, and major fragments of 31 kDa and 22 kDa from the beta-subunit. The high-affinity calcium-binding sites were detected in the carboxyl-terminal region of each tubulin subunit. Computer analysis of the subunit amino-acid sequences suggested possible locations of the putative calcium-binding sites.

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Amino acid sequences near the carboxy terminal end of the Electrophorus electricus electric organ and rat brain sodium channel polypeptides were discovered to be putative EF hand calcium binding sites. This conclusion was made using the following criteria: the Tufty-Kretsinger and Szebenyi-Moffat EF hand tests, a computer generated analysis, the revised guidelines of Chou & Fasman, and sequence comparisons with other published EF hand calcium binding regions. These results suggest that the sodium channel may be a calcium binding protein.

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Chicken brain postsynaptic density (PSD) polypeptides, obtained by treating synaptosomes with 0.5% Triton X-100 and then further purified on a sucrose gradient, are demonstrated to contain four basic proteins of 76K (pI greater than 9.2), 58K (pI 8.1-8.8, heterogeneous), 40K (pI 9.0), and 24K (pI 8.9). Nonequilibrium pH gradient-sodium dodecyl sulfate two-dimensional gels further reveal six more basic proteins with pI values higher than 9.2: 76K, 52K, 47K, 45K, 36K, and 34K. These basic proteins are a major part of the total chicken PSD polypeptides appearing on the gels. Some of these basic proteins (58K, 52K, 47K, 36K, 24K, and two at 76K) are distinguishable from those of brain mitochondria, the major contaminant. The 40K and 34K proteins may be common mitochondrial polypeptides. The 45K protein is probably a mitochondrial contaminant. A number of proteins including 76K (synapsin I-like protein) and 58K, along with some other minor ones, can be phosphorylated by endogenous protein kinase(s) in the presence of Ca2+, Mg2+, and [gamma-32P]ATP. No PSD basic proteins bind Ca2+.

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Chick brain synaptosomes were fractionated by affinity chromatography on concanavalin A-Sepharose. Three subfractions were obtained. One, designated UBF, was not bound to the affinity adsorbent and represented 36% of the total synaptosomal protein treated with the beads. A second fraction, designated BF1, adhered to concanavalin A-Sepharose exclusively through its carbohydrate recognition site. The third fraction, called BF2, bound to the beads through hydrophobic interactions and represented about 20% of the total synaptosomal protein. About 20% of the total synaptosomal protein was found to be retarded on three ligand-less gels, with potential for only hydrophobic interactions. This binding can be reversed, however, by ethylene glycol, a result indicating hydrophobic binding sites on the synaptosomes. Enzyme marker studies and electron microscopy showed differences between UBF, BF1, and BF2, mainly with respect to mitochondrial contamination. Binding studies with [3H]-Con A show the absence of Con A-specific carbohydrates from the surface of UBF or BF2. As expected strong and specific binding between [3H]-Con A and [3H] BF1 was observed. These findings are discussed in relation to a model for the interior working of the synaptosomes.

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At least 19 calcium-binding proteins were detected in avian brain subfraction using 45Ca2+ binding to proteins immobilized in polyacrylamide gels. Half of the 45Ca2+ binding proteins were observed in presynaptic cytoplasm. Two-dimensional gel electrophoresis of this material revealed at least 14 45Ca2+ binding polypeptides besides calmodulin. These proteins may be important in brain and nerve terminal function.

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We refined the method of Schibeci and Martonosi (1980a) to enhance detection of calcium-binding proteins in polyacrylamide gels using 45Ca2+. Our efforts have produced a method which is shorter, has 40-fold greater sensitivity over the previous method, and will detect 'EF hand'-containing calcium-binding proteins in polyacrylamide gels below the 0.5 microgram level. In addition, this method will detect at least one example from every described class of calcium-binding protein, including lectins and gamma-carboxyglutamic acid containing calcium-binding proteins. The method should be useful for detecting calcium-binding proteins which may trigger neurotransmitter release.

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Chick brain membranes were fractionated by affinity chromatography on AH-Sepharose 6MB linked to p-sulfamylbenzoic acid, an inhibitor of carbonic anhydrase. Two major fractions were obtained. One, designated unbound fraction, representing 42% of the total membrane protein, eluted freely from the affinity adsorbent. A second fraction, designated specifically bound fraction, contained 36% of the total membrane proteins. In addition, 11% of the total membranes bound non-specifically and could be eluted only by the use of shearing forces. Various lines of evidence indicated that the sulfonamide binding site of membrane-bound carbonic anhydrase, in addition to the carbonic anhydrase inhibitor immobilized to AH-Sepharose 6MB, was responsible for the observed specific binding. The specifically bound fraction was highly enriched in carbonic anhydrase while the unbound fraction was completely devoid of this enzyme activity.

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When erythrocytes are incubated with 32Pi, incorporation of label into phosphoproteins is a gradual process, increasing for at least 2 hours. Membrane phospholipids also are labeled. Exogenous protein kinase substrates are unlabeled in these incubations. This suggests that labeling by 32Pi occurs into polypeptides inside the erythrocytes. When erythrocytes are incubated with [gamma-32P]ATP and active protein kinase, membrane polypeptides are not labeled. Only exogenously added protein kinase substrates and the regulatory subunit of protein kinase (and its contaminants) are labeled. This suggests that labeling from [gamma-32P]ATP and active protein kinase occurs in the compartment outside the erythrocytes. Apyrase (EC 3.6.1.5) eliminates such labeling, demonstrating that it was occurring in the compartment external to the erythrocytes. However, in incubations of cells with 32Pi, apyrase has no effect on the incorporation into membrane polypeptides and phospholipids, demonstrating that this labeling occurs on the inside of the membrane. Thus, additions of apyrase to intact particles incubated with protein kinase substrates and 32Pi provides a method for identifying internally exposed polypeptides in the plasma membranes of a variety of systems.

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A search for the presence of troponin in brain reveals that troponin is below 0.00037% of total bovine brain soluble protein. Troponin levels were examined using G-actin-linked Sepharose affinity chromatography and 45Ca binding. The chromatographic and 45Ca binding experiments revealed the presence of several actin and calcium-binding proteins, none of which corresponded to any troponin subunit. In addition, troponin was not found in any chick brain subfraction analyzed, and the level of troponin in chick nerve ending cytoplasm enriched for troponin was less than 0.023%. Considering that substantial amounts of myosin and actin occur in brain, these findings indicate that troponin is not likely to be a regulator of putative brain actomyosin interactions. The significance of these results and their relation to proposed models for neurotransmitter release is discussed.

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Chick synaptosomes were found to remain intact following fixation in the presence of 0.2 M sucrose or 0.3 M sucrose. Increasing the sucrose concentration up to 0.4 M significantly decreased the number of intact synaptosomes; however, typical intrasynaptosomal organelles such as synaptic vesicles and mitochondria were visible in all preparations. This work suggests possibly important species differences in synaptosomal structure between chick and rat synaptosomes.

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Protein I has been identified and compared in membranes prepared from chick and rat forebrain. Based upon five criteria known to characterize protein I, namely, (1) its ability to serve as a substrate for both the cyclic AMP-dependent protein kinase and (2) the Ca2+- dependent, calmodulin-requiring protein kinase, (3) its ability to be extracted from membranes at low pH, (4) its characteristic pattern of digestion by collagenase, and (5) its existence as a basic protein, we have determined that although protein I of rat brain consists of the usual doublet polypeptides Ia and Ib, only a single chick forebrain polypeptide is detectable which possesses protein I-like properties.

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The protein kinase activities endogenous to synaptic membranes prepared by an identical procedure from avian (chick) and mammalian (rat) brains were compared. Both species showed similar responses towards both protein kinase effector molecules cyclic adenosine monophosphate and Ca2+. Kapp for cyclic adenosine monophosphate-dependent protein kinase activity occurred at 0.4-0.8 microM cAMP and Kapp for Ca2+-dependent, calmodulin-requiring protein kinase activity occurred at 1-2 microM Ca2+ (free ion concentration) both in the absence or presence of calmodulin added to the reaction mixture. This suggests that endogenous calmodulin in these membranes was able to modulate the Ca2+-dependent, calmodulin requiring protein kinase activity. After EGTA-treatment of the membranes to remove endogenous Ca2+ and calmodulin, no significant response towards Ca2+ on the phosphorylation of the membrane polypeptides was measured unless exogenous calmodulin was added after which the Kapp for Ca2+ was increased to 15 microM Ca2+ (free ion concentration). There was a difference in the maximal levels of kinase activity in these membranes with chick membranes containing 57% less cyclic adenosine monophosphate-dependent protein kinase activity, but 65% more Ca2+-dependent, calmodulin-requiring protein kinase activity than the rat membranes. Similar results were determined when either low (5 microM) or high (5.8 microM) concentrations of adenosine 5'-triphosphate were added to the reaction mixtures. Besides certain species differences in the molecular weights of the resulting phosphoproteins, we observed several major differences with respect to the absence or presence of some of the phosphoproteins. Chick synaptic membranes may lack the cyclic adenosine monophosphate-requiring, microtubule-associated phosphoprotein, MAP2, one of the 2 neurospecific, cyclic adenosine monophosphate-requiring and Ca2+, calmodulin-requiring phosphoproteins (Protein Ib, although Protein Ia apparently is present), and the Ca2+-requiring, calmodulin-independent, ACTH-sensitive phosphoprotein, B50. The phenothiazines, trifluoperazine, fluphenazine and chlorpromazine were found to inhibit the Ca2+-dependent, calmodulin-requiring protein kinase activities of both the chick and rat synaptic membranes. This inhibition appeared to be specific for calmodulin because at the same concentrations the phenothiazine analogue, chlorpromazine-sulfoxide, had no effect on this activity. Also found to inhibit Ca2+-dependent calmodulin-requiring protein kinase activity were dibucaine and adrenocorticotropin. These data suggest that rat forebrain synaptic plasma membranes are activated by cyclic adenosine monophosphate

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Mild detergent extraction of chick brain synaptic plasma membranes followed by gel electrophoresis suggests that synaptic plasma membrane tubulin is an integral component. Although some of the synaptic plasma membrane tubulin might be aggregates, that possibility is not supported by the observation that tubulin aggregates that are added to synaptosomes before synaptic subfractionation do not partition with synaptic plasma membranes during membrane isolation.

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