Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

Ca2+-Dependent and Ca2+-Independent Protein Kinase C Changes in the Brains of Patients with Alzheimer's Disease

Abstract: We examined protein kinase C (PKC) activity in Ca²⁺-dependent PKC (Ca²⁺-dependent PKC activities) and Ca²⁺-independent PKC (Ca²⁺-independent PKC activities) assay conditions in brains from Alzheimer's disease (AD) patients and age-matched controls. In cytosolic and membranous fractions, Ca²⁺-dependent and Ca²⁺-independent PKC activities were significantly lower in AD brain than in control brain. In particular, reduction of Ca²⁺-independent PKC activity in the membranous fraction of AD brain was most enhanced when cardiolipin, the optimal stimulator of PKC-ε, was used in the assay; whereas Ca²⁺-independent PKC activity stimulated by phosphatidylinositol, the optimal stimulator of PKC-δ, was not significantly reduced in AD. Further studies on the protein levels of Ca²⁺-independent PKC-δ, PKC-ε, and PKC-ζ in AD brain revealed reduction of the PKC-ε level in both cytosolic and membranous fractions, although PKC-δ and PKC-ζ levels were not changed. These findings indicated that Ca²⁺-dependent and Ca²⁺-independent PKC are changed in AD, and that among Ca²⁺-independent PKC isozymes, the alteration of PKC-ε is a specific event in AD brain, suggesting its crucial role in AD pathophysiology.     

Ca2+-Independent Release of Glutamate During In Vitro Anoxia in Isolated Nerve Terminals

The effects of in vitro anoxia on the release of glutamate in isolated nerve terminals were studied. The extra-synaptosomal concentration of glutamate ([Glu]ext) under aerobic conditions was 2.3 microM and increased to 4.9 microM after 10 min of anoxia. However, when synaptosomes were incubated in the presence of lactate plus pyruvate instead of glucose, to prevent anaerobic glycolysis, anoxia induced an eightfold increase in the [Glu]ext. The accumulation of glutamate in the external medium during anoxia was Ca2+ independent and insensitive to a significant reduction of the Ca(2+)-dependent release of the amino acid. These results indicate that a Ca(2+)-independent efflux of cytoplasmic glutamate occurs during in vitro anoxia in isolated nerve terminals.     

Purification and Characterization of a Ca2+- and Calmodulin-Dependent Protein Kinase from Rat Brain

Abstract: A Ca²⁺- and calmodulin-dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800-fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S-300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited K _m values of 109 and 30 μM for ATP and chicken gizzard myosin light chain, respectively, and K _a values of 12 n M and 1.9 μM for brain calmodulin and Ca²⁺, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine-rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a Ca²⁺- and calmodulin-dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. Our findings indicate that there is a multifunctional Ca²⁺- and calmodulin-dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins.     

Ca2+-Independent, Ca2+-Dependent, and Carbachol- Mediated Arachidonic Acid Release from Rat Brain Cortex Membrane

Synaptoneurosomes obtained from the cortex of rat brain prelabeled with [14C]arachidonic acid [( 14C]AA) were used as a source of substrate and enzyme in studies on the regulation of AA release. A significant amount of AA is liberated in the presence of 2 mM EGTA, independently of Ca2+, primarily from phosphatidic acid and polyphosphoinositides (poly-PI). Quinacrine, an inhibitor of phospholipase A2 (PLA2), suppressed AA release by about 60% and neomycin, a putative inhibitor of phospholipase C (PLC), reduced AA release by about 30%. An additive effect was exhibited when both inhibitors were given together. Ca2+ activated AA release. The level of Ca2+ present in the synaptoneurosomal preparation (endogenous level) and 5 microM CaCl2 enhance AA liberation by approximately 25%, whereas 2 mM CaCl2 resulted in a 50% increase in AA release relative to EGTA. The source for Ca(2+)-dependent AA release is predominantly phosphatidylinositol (PI); however, a small pool may also be liberated from neutral lipids. Carbachol, an agonist of the cholinergic receptor, stimulated Ca(2+)-dependent AA release by about 17%. Bradykinin enhanced the effect of carbachol by about 10-15%. This agonist-mediated AA release occurs specifically from phosphoinositides (PI + poly-PI). Quinacrine almost completely suppresses calcium-and carbachol-mediated AA release. Neomycin inhibits this process by about 30% and totally suppresses the effect of bradykinin. Our results indicate that both phospholipases PLA2 and PLC with subsequent action of DAG lipase are responsible for Ca(2+)-independent AA release. Ca(2+)-dependent and carbachol-mediated AA liberation occurs mainly as the result of PLA2 action. A small pool of AA is probably also released by PLC, which seems to be exclusively responsible for the effect of bradykinin.     

Ca2+ -Dependent Release from Rat Brain of Cannabinoid Receptor Binding Activity

As a result of the identification, pharmacological characterization, and localization of the cannabinoid receptor in the CNS, the existence of an endogenous ligand for this receptor can be hypothesized. Following the premise that such a substance could have the properties of a neuromodulator being stored in intracellular vesicles, we tested the ability of increased intracellular Ca2+ levels to stimulate release. We demonstrate here that the Ca2+ ionophore A23187 can induce release of cannabinoid-like binding activity in the presence but not in the absence of Ca2+. The effect of A23187 was maximal at 1.2 microM, consistent with vesicular release. It was necessary to increase the concentration of extracellular free Ca2+ to greater than 60 nM to evoke release. The released cannabinoid-like binding activity displaced [3H]CP-55940 binding to cannabinoid receptors in rat synaptosomal membranes in a concentration-dependent manner. This is the first report of a substance present endogenously in brain that can be released in a Ca(2+)-dependent manner and that binds to the cannabinoid receptor.     

Allosteric Activation of Brain Mitochondrial Ca2+ Uptake by Spermine and ty Ca2+: Brain Regional Differences

Analysis of the initial rates of 45Ca2+ uptake by rat brain mitochondria in Ca2+-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid buffers indicated that nontelencephalic mitochondria exhibited both a much less pronounced stimulatory effect of spermine and significantly more hyperbolic kinetics of Ca2+ uptake than telencephalic mitochondria. Nontelencephalic mitochondria were also markedly less susceptible to a Ca2+-induced hysteretic allosteric activation of the Ca2+ uniporter. A new Ca2+ loading procedure, which strikingly illustrates differences in mitochondrial Ca2+ buffering characteristics, is also described. In this procedure, low concentrations of Ca2+ (1, 2, or 5 microM) were repetitively added to mitochondria every 30 s while changes in free Ca2+ concentration were recorded. Spermine induced a marked attenuation of the rise in free Ca2+ level under these conditions. Steady-state rates of Ca2+ uptake were determined by a quantitative analysis of the buffering of repetitive Ca2+ additions, and, again, brain regional differences were qualitatively similar to those observed in the initial rate kinetics; Ca2+ uptake by nontelencephalic mitochondria in the steady state was markedly less responsive to stimulation by spermine and appeared to have a more hyperbolic dependence on Ca2+ in the absence of spermine. These results also suggest that there is a lag time in the activation of the uniporter by Ca2+, in addition to the hysteresis that has previously been observed in the deactivation of the uniporter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local Anesthetics Inhibit the Ca2+,Mg2+-ATPase Activity of Rat Brain Synaptosomes

Many biochemical effects of local anesthetics are expressed in Ca2+-dependent processes [Volpi M., Sha'afi R.I., Epstein P.M., Andrenyak P.M., and Feinstein M.B. (1981) Proc. Natl. Acad. Sci. USA 78, 795-799]. In this communication we report that local anesthetics (dibucaine, tetracaine, lidocaine, and procaine and the analogue quinacrine) inhibit the Ca2+-dependent and the Mg2+-dependent ATPase activity of rat brain synaptosomes and of membrane vesicles derived from them by osmotic shock. This inhibition is induced by concentrations of these drugs close to their pharmacological doses, and a good correlation between K0.5 of inhibition and their relative anesthetic potency is found. The Ca2+-dependent ATPase is more selectively inhibited at lower drug concentrations. The physiological relevance of these findings is discussed briefly.     

Identification and Purification of Calcium-Independent Phospholipase A2 from Bovine Brain Cytosol

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a Novel Phospholipase A2 Activity in Human Brain

Abstract: Phospholipases A₂ (PLA₂) are a family of enzymes that catalyze the removal of fatty acid residues from phosphoglycerides. The enzyme is postulated to be involved in several human brain disorders, although little is known regarding the status of PLA₂ activity in human CNS. We therefore have characterized some aspects of the PLA₂ activity present in the temporal cortex of human brain. More PLA₂ activity was found in the membrane (particulate) fraction than in the cytosolic fraction. The enzyme could be solubilized from particulate material using 1 M potassium chloride, and was capable of hydrolyzing choline phosphoglyceride (CPG) and ethanolamine phosphoglyceride (EPG), with a preference (approximately eightfold) for EPG over CPG. When the solubilized particulate enzyme was subjected to gel filtration chromatography, PLA₂ activity eluted in a high molecular mass fraction (∼180 kDa). PLA₂ activity was weakly stimulated by dithiothreitol, strongly stimulated by millimolar concentrations of calcium ions, and inhibited by brief heat treatment at 57°C, bromophenacyl bromide, the arachidonic acid derivative AACOCF₃, γ-linolenoyl amide, and N -methyl γ-linolenoyl amide. Thus, whereas the human brain enzyme(s) characterized in our study displays some of the characteristics of previously characterized PLA₂s, it differs in several key features.     

Characterization of Exchange Inhibitory Peptide Effects on Na+/Ca2+ Exchange in Rat and Human Brain Plasma Membrane Vesicles

Abstract: The inhibitory effects of Na⁺/Ca²⁺ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na⁺/Ca²⁺ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na⁺-dependent Ca²⁺ uptake in both rat brain [IC₅₀ = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC₅₀ = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ～80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na⁺-dependent Ca²⁺ release, and the inhibitory effect was enhanced by increasing the extravesicular Na⁺ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na⁺. When XIP was added at steady state (5 min after the initiation of intravesicular Na⁺-dependent Ca²⁺ uptake), it was found that the intravesicular Ca²⁺ content declined with time. Analysis of unidirectional fluxes for Ca²⁺ at steady state showed that 50 µM XIP inhibited Ca²⁺ influx and efflux ～85 and 70%, respectively. This result suggested that XIP inhibited both Na⁺/Ca²⁺ exchange and Ca²⁺/Ca²⁺ exchange but had no effect on the passive release pathway for Ca²⁺. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na⁺ or other monovalent cations, e.g., K⁺, is required for XIP to have its inhibitory effect on Ca²⁺ transport.     

Dibutyryl-Cyclic GMP Stimulation of Ca2+-ATPase Activity in Rat Brain Synaptic Membranes

The effects of dibutyryl cyclic AMP (db-cAMP) and dibutyryl cyclic GMP (db-cGMP) were tested on Ca2+-ATPase, Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities in lysed synaptosomes prepared from whole rat brains (minus cerebellum). At concentrations from 0.1 to 2.0 mM, db-cGMP produced a selective, concentration-dependent increase in Ca2+-ATPase activity. Both db-cGMP and db-cAMP slightly reduced Mg2+-ATPase activity, whereas neither compound had concentration-dependent effects on (Ca2+ + Mg2+)-ATPase activity. These findings suggest that the Mg2+-independent, Ca2+-ATPase activity in rat brain is regulated by a cyclic GMP-dependent process. Further, the data provide evidence that the Ca2+-ATPase activity in lysed synaptosomal membranes represents an enzyme that is distinguishable from both the Mg2+ -and (Ca2+ + Mg2+)-ATPase.     

Kinetic Characterization of Ca2+ Transport in Synaptic Membranes

Lysed synaptosomal membranes were prepared from brain cortices of HA/ICR Swiss mice, and the ATP-stimulated Ca2+ uptake, Ca2+-stimulated Mg2+-dependent ATPase activity, and the Ca2+-stimulated acyl phosphorylation of these membranes were studied. The Km values for free calcium concentrations ([Ca2+]f) for these processes were 0.50 microM, 0.40 microM, and 0.31 microM, respectively. Two kinetically distinct binding sites for ATP were observed for the ATP-stimulated Ca2+ uptake and the Ca2+-stimulated Mg2+-ATPase activity. The high-affinity Km values for ATP for these two processes were 16.3 microM and 28 microM, respectively. These results indicate that the processes studied operate in similar physiological concentration ranges for the substrates [Ca2+]f and ATP under identical assay conditions and, further, that these processes may be functionally coupled in the membrane.     

Adenosine A1 Agonists and the Ca2+ Channel Agonist Bay K 8644 Produce a Synergistic Stimulation of the GTPase Activity of Go in Rat Frontal Cortical Membranes

Abstract: The identity and role of G proteins in coupling adenosine receptors to effectors have been studied to a limited degree. We have identified the G proteins whose GTPase activity is stimulated by adenosine receptor agonists in neuronal membranes. (R)-Phenylisopropyladenosine, 2-chloroadenosine, and N-ethylcarboxamideadenosine produced a concentration-dependent stimulation of GTPase. At 10^?5M, the increase above basal GTPase in frontal cortex was 25 ± 4, 20 ± 3, and 8 ± 1%, respectively, and in the cerebellum 55 ± 2, 41 ± 4, and 22 ± 2%, respectively. The effects of (R)-phenylisopropyladenosine and 2-chloroadenosine were inhibited by (1) A₁ antagonists (76–96% reduction), (2) pretreatment with pertussis toxin (90–100% reduction), and (3) antibodies raised against the α-subunit of G_i and G_o (55–57% reduction by each), suggesting that A₁ receptors interact equally with G_i and G_o. (R)-Phenylisopropyladenosine increased the binding of a nonhydrolyzable analogue of GTP to membranes in a pertussis toxin-sensitive manner, indicative of activation of G_i or G_o. Previously, (±)-Bay K 8644 enhanced GTP hydrolysis by G_o but not G_i. Now we report a profound synergistic stimulation of GTPase in the presence of (R)-phenylisopropyladenosine and (±)-Bay K 8644 (10^?7 to 10^?5M). (±)-Bay K 8644 had no effect on nucleotide exchange and, thus, cannot activate G_o. It appears that a positive cooperative stimulation of G_o occurs when it is first activated by A₁ receptors and subsequently interacts with the L-type Ca²⁺ channel.     

Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes

Abstract: The effects of K⁺ depolarization and of stimulation by veratridine on apparent cytosolic free Ca²⁺ ([Ca²⁺]_cyt) and net Ca²⁺ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca²⁺]_cyt was determined with fura-2, and Ca²⁺ accumulation was measured with tracer ⁴⁵Ca. [Ca²⁺]_cyt was ～ 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K⁺]₀, was increased from the normal 5 mM to 30 or 50 mM, ⁴⁵Ca uptake and [Ca²⁺]_cyt both increased within 1 s. Both increases were directly related to [Ca²⁺]₀ for [Ca²⁺]₀= 0.02–1.2 mM; however, the increase in ⁴⁵Ca uptake greatly exceeded the increase in [Ca²⁺]_cyt. With small Ca²⁺ loads ≤100 μmol/L of cell water, equivalent to the Ca²⁺ entry during a train of ≤60 impulses), the ⁴⁵Ca uptake exceeded the increase in [Ca²⁺]_cyt by a factor of nearly 1,000. This indicates that ～99.9% of the entering Ca²⁺ was buffered and/or sequestered within ～ 1 s. With larger Ca²⁺ loads, a larger fraction of the entering Ca²⁺ was buffered; ～99.97% of the load was buffered with loads of 250–425 μmol/L of cell water. The ratio between the total Ca²⁺ entry and the increase in [Ca²⁺]_cyt, the “calcium buffer ratio”β, was therefore ～ 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca²⁺]_cyt, which ranged between ～7,300:1 and 12,800:1. When the synaptosomes were activated with 10 μM veratridine ([Ca²⁺]₀= 0.2–0.6 mM), ⁴⁵Ca influx and [Ca²⁺]_cyt increased progressively for ～10 s (β= 2,700:13,050:1) and then leveled off. Application of 10 μM tetrodotoxin before the introduction of veratridine prevented the increases in ⁴⁵Ca influx and [Ca²⁺]_cyt. Application of 10 μM tetrodotoxin after 5–10 s of exposure to veratridine caused both the [Ca²⁺]_cyt and the veratridine-stimulated ⁴⁵Ca within the terminals to decline, thereby demonstrating that the Ca²⁺ loading is reversible in the presence of extracellular Ca²⁺. These data show that synaptosomes are capable of buffering and metabolizing Ca²⁺ in a manner expected for intact neurons.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Calmodulin Stimulation of Ca2+-Dependent ATP Hydrolysis and ATP-Dependent Ca2+ Transport in Synaptic Membranes

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.     

Purification and Characterization of Posterior Pituitary Calmodulin and Its Activation of Neurosecretosome Ca2++ Mg2+-ATPase Activities

Abstract: Calmodulin was isolated as an electrophoretically homogeneous protein from bovine posterior pituitary glands. The yield indicated that this gland is a particularly rich source. Purified bovine posterior pituitary calmodulin and bovine brain calmodulin had identical electrophoretic mobilities on 10% and 12% polyacrylamide gels. The protein was further identified by molecular weight determination and by amino acid analysis which showed that it contained trimethyllysine, one residue per molecule. Bovine posterior pituitary calmodulin was found to activate a preparation of calmodulin-deficient phosphodiesterase from bovine heart. In addition, pituitary calmodulin stimulated Ca2⁺+ Mg²⁺-ATPase activity associated with a purified nerve ending plasma membrane fraction. This dependence could only be demonstrated after successive washing of the membranes with EGTA buffers, a procedure designed to remove endogenous calmodulin.     

Purification of a Synaptic Membrane Na+/Ca2+ Antiporter and Immunoextraction with Antibodies to a 36-kDa Protein

The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na+/Ca2+ exchange activity were examined and a series of chromatographic procedures were used for the isolation of a protein involved in this transport activity. The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of 20% (vol/vol) glycerol led to optimal solubilization, and soybean phospholipids in low-pH medium were found to produce optimal reconstitution of activity after dialysis to remove the detergent. Sequential chromatography steps involving the use of gel filtration on Sephacryl S-400 HR, ion exchange on diethylaminoethyl-Sephacel, and metal chelate chromatography on tris-(carboxymethyl)ethylenediamine loaded with LaCl3 led to the isolation of a fraction highly enriched in both Na+/Ca2+ exchange activity and two protein bands identified by denaturing electrophoresis. The estimated molecular masses of the two proteins were 50 and 36 kDa. Development of polyclonal antibodies to the 36-kDa protein permitted immunoextraction of greater than 95% of the antiporter activity from solubilized synaptic plasma membranes. These antibodies cross-reacted with the electroeluted 50-kDa protein on enzyme-linked immunosorbent assays, suggesting a close relationship between the two proteins. These results indicate that the 36-kDa protein is at least a component of the brain membrane Na+/Ca2+ antiporter.     

Activation of Brain Guanylate Cyclase by Phospholipase A2

Various pure snake venom phospholipases A2 were used for studying their effect on guanylate cyclase activity. All the phospholipases A2 tested were found to activate guanylate cyclase from a rat brain homogenate. It was shown that particulate guanylate cyclase was especially affected. Intact glial cells incubated in presence of phospholipase A2 showed also an increased guanylate cyclase activity, demonstrating that the phospholipase effect, observed in disrupted cells, occurs also at the cellular level. These results suggest that in intact cells membrane-bound phospholipase A2 activity could be involved in the modulation of the cellular cyclic GMP content.