Stimulation of brain adenylate cyclase activity by the undecapeptide substance P and its modulation by the calcium ion

Synthetic substance P stimulated adenylate cyclase activity in particulate preparations from rat and human brain.The concentration of substance P for half maximal stimulation in rat brain was 1.8 · 10⁻⁷ M.The stimulatory effect of substance P on the rat brain adenylate cyclase activity was 88% compared with 48% by noradrenalin, 163% by prostaglandin E₁ and 184% by prostaglandin E₂.Both the basal and substance P-stimulated adenylate cyclase activity in rat brain were inhibited by concentration of Ca²⁺ above 10⁻⁶ M.The chelating agent ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid at a concentration of 0.1 mM reduced the basal adenylate cyclase activity by 64% and eliminated the substance P-stimulated activity.The inhibition by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid was completely reversed by increasing concentrations of Ca²⁺.     

Radioiodination of brain tubulin with Bolton-Hunter reagent

Radio-iodination of tubulin can be achieved by Bolton-Hunter reagent both in the absence and presence of microtubule associated proteins. Specific radioactivities as high as 400 C_i/mmole tubulin dimer can be obtained, i.e. an average of 0.2 molecule of reagent is bound per molecule of tubulin. About 80 % of the [¹²⁵I]- labelled tubulin keeps its ability to assemble in microtubules and polymerizes with the same critical concentration as the native tubulin, which makes the method adequate for preparing tracer tubulin useful for in vivo and in vitro studies. Both α and β subunits are labelled, 60 % of the radiolabel being bound to the β subunit.     

Specific binding of the calcium-dependent regulator protein to brain membranes from the guinea pig

The interaction of a calcium-dependent regulator protein (CDR) of brain adenylate cyclase (EC 4.6.1.1) with synaptic membranes from guinea pig brain was examined using ¹²⁵I-CDR as a tracer molecule. ¹²⁵I-CDR binding was reversible, saturable, and temperature sensitive. The same Ca²⁺ and Mg²⁺ dependence was observed for ¹²⁵I-CDR binding and for brain adenylate cyclase activation by CDR.     

Detection of an initial burst of Ca2+ translocation in sarcoplasmic reticulum.

Rapid quench methods were used to determine Ca²⁺ uptake, ATPase phosphorylation and Pi production in the transient state of Sarcoplasmic Reticulum. It was found that within 20 milliseconds of the addition of ATP maximal levels of phosphorylated enzyme intermediate are reached and an initial burst of Ca²⁺ uptake is completed. This burst, kinetically distinct from the following transport activity, is related to the phosphorylated intermediate with a molar ratio of two.     

Effects of calcium ions and quinolinic acid on rat kidney mitochondrial kynurenine aminotransferase

At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl₂, apparently because Ca⁺⁺ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg⁺⁺, Mn⁺⁺, Na⁺, K⁺, and NH₄⁺ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca⁺⁺ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca⁺⁺ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. $\text{in vivo}$ depends on the ability of that agent to chelate Ca⁺⁺.     

Increase in polymerized liver tubulin during stimulation of hepatic plasma protein secretion in the rat

Involvement of hepatic microtubules in plasma protein secretion by the liver was investigated by stimulating protein secretion in rat liver and then measuring the different forms of tubulin. Total and free tubulin were estimated in liver supernatants by the [³H] colchicine-binding assay. Polymerized tubulin, assumed to reflect the presence of microtubules, was calculated from the difference between total and free tubulin. To enhance liver plasma protein secretion, an acute inflammatory reaction was induced in one group of rats and a nephrotic syndrome in another. In both cases, total liver tubulin increased significantly compared to normal animals, but free tubulin was unchanged. Accordingly, polymerized tubulin rose by 50% during the inflammatory reaction and by 90% during the nephrotic syndrome. These results support the hypothesis that hepatic microtubules are involved in plasma protein secretion by the liver and also suggest that enhanced secretion requires additional microtubules.     

Hormonal stimulation of cAMP-dependent protein kinase in rat pancreas

The phosphorylation of myelin (basic protein) purified from rabbit brain was markedly stimulated by exogenously added calmodulin in the presence of calcium and inhibited by W-7(N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), a calmodulin interacting agent, in a dose-dependent fashion. However, exogenously added myelin basic protein free from protein kinase activity could not serve as a substrate of this calmodulin dependent protein kinase, suggesting that this kinase catalyzes the phosphorylation of the enzyme-substrate complex. These results suggest that a calmodulin-dependent protein kinase complex with the substrate (basic protein) is located in the myelin membrane of the central nervous system.     

Chlorpromazine inhibits the calcium-mediated effects of S-100 protein(s) on assembled brain microtubule proteins, but not those on microtubule protein assembly

We have examined the S-100-chlorpromazine interplay at the level of brain microtubule proteins in vitro. The results indicate that in the presence of 0.12 M KCl and 10 microM free Ca2+ the inhibitory effect of S-100 on microtubule assembly is additive to that of chlorpromazine, but S-100 fails to potentiate the disassembling effect of 0.1 mM Ca2+ if added to assembled microtubule proteins after chlorpromazine and Ca2+, probably because of inhibition of S-100 by the phenothiazine. Chlorpromazine does not compete with S-100 for binding to purified tubulin.     

Quantitation and characterization of the microtubule associated MAP2 in porcine tissues and its isolation from porcine (PK15) and human (HeLa) cell lines

A radioimmunoassay developed for the microtubule associated protein MAP₂ shows that this protein, or related polypeptides are present in all the porcine tissues studied. Nervous tissues (brain, 11 μg MAP₂/mg protein; cerebellum, 9.7 μg MAP₂/mg protein) contain much higher levels of MAP₂ than non-nervous tissues (kidney, 104 ng MAP₂/mg protein; lung 89 ng MAP₂/mg protein; spleen 66 ng MAP₂/mg protein; thyroid 21 ng MAP₂/mg protein; liver 9.7 ng MAP₂/mg protein). A heat resistant protein doublet of 300,000 with the ability to promote microtubule polymerization has been purified from pig kidney cells by affinity chromatography using MAP₂ antibodies. Using a similar purification method a protein of 200,000 daltons has been isolated from Hela cells.     

Crotoxin effects on Torpedo californica cholinergic excitable vesicles and the role of its phospholipase A activity

The phospholipolytic neurotoxin from $\text{Crotalus}\text{durissus}\text{terrificus}$, crotoxin, is able to produce a dose- and time-dependent block of carbachol-stimulated ²²Na efflux from pre-loaded $\text{Torpedo}\text{californica}$ excitable vesicles. The blocking activity is dependent on calcium and is abolished by chemical modification with p-bromophenacyl bromide. The isolated basic subunit, crotoxin B, produces an identical block, whereas the isolated acidic subunit, crotoxin A, has no detectable effect. Neither crotoxin nor crotoxin B antagonizes the binding of $\text{[}{}^{125}\text{I]-α-bungarotoxin}$ to purified acetylcholine receptor, although, at high concentrations, they antagonize its binding to acetylcholine receptor-rich membrane fragments. Certain phospholipase A₂ enzymes and the fatty acid products of their digestion can mimic the crotoxin action. It is therefore suggested that, although considered a pre-synaptic neurotoxin, crotoxin can have $\text{in}\text{vitro}$ post-synaptic effects, possibly mediated by its endogeneous phospholipase A₂ activity.     

Study of the mitochondrial phosphate carrier in the course of calcium phosphate accumulation: A requirement for Mg2+ and ADP of its sensitivity to thiol reagents

1. The accumulation of calcium phosphate driven by succinate oxidation is ADP-dependent. In its absence the accumulation stops after a short incubation time and the oxygen uptake is permanently stimulated. This uncoupled oxygen uptake is insensitive to the inhibitors of phosphate transport, like mersalyl and N-ethylmaleimide. When ADP plus Mg²⁺ are added to the medium, or when ADP is added in the initial presence of magnesium, the inhibitory action of the thiol reagents on oxygen uptake is re-established. ADP alone or Mg²⁺ alone are without any effect.2. Phosphate/phosphate exchange has been studied, in the absence of ADP, when calcium phosphate accumulation had stopped and oxygen uptake is uncoupled. Under these conditions the exchange process becomes insensitive to thiol reagents. Sensitivity is recovered solely in the presence of ADP plus Mg²⁺.3. When mitochondrial swelling is studied according to the method of Chappell, it also appears that the phosphate carrier loses it sensitivity to mersalyl in the absence of ADP, which confirms the data obtained with phosphate/phosphate exchange experiments. When ADP plus Mg²⁺ are added (or present), together with mersalyl, the action of the thiol inhibitor is recovered. ADP and magnesium are inactive separately. EGTA plus Mg²⁺ (but not EGTA plus ADP) may substitute for ADP plus Mg²⁺ in this process.4. A possible interaction between the magnesium binding site and the phosphate carrier is considered and discussed.     

Interaction of tyrosine hydroxylase with tubulin

Bovine adrenal medulla tyrosine hydroxylase associates with microtubules during tubulin assembly. Limited proteolytic digestion of tyrosine hydroxylase does not affect the enzymatic activity but prevents its association with tubulin. A possible interpretation is that an ionic interaction occurs between microtubules and a negatively charged region of the enzyme which is removed by the protease treatment. Tyrosine hydroxylase is able to induce purified tubulin assembly as do the microtubule associated proteins; however, the association induced by tyrosine hydroxylase corresponds to the formation of aggregates or organized structures different from microtubules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and electron microscopy of proteins obtained from bovine adrenal medulla show the presence of tubulin in this tissue.     

Release of calcium from membranes and its relation to phagocytotic metabolic changes: A fluorescence study on leukocytes loaded with chlortetracycline

A fluorescent probe chlortetracycline was used to monitor the mobilization of intracellular divalent cations of leukocytes. When the chlortetracycline-loaded cells were stimulated with cytochalasin D or $\text{E. coli}$, a fluorescence change ascribable to the release of calcium from the intracellular hydrophobic environment was observed. The dose-response curve of the fluorescence change and that of the superoxide release of the cells were very similar. An intracellular calcium antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate inhibited both metabolic and fluorescence changes in parallel. A supposition that an intracellular mobilization of calcium ions is stimulating the metabolic change was supported.     

Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

Effects of beta-bungarotoxin on mitochondrial respiration are caused by associated phospholipase A activity.

Mitochondria prepared from tissue that had been incubated with β-bungarotoxin exhibited abnormal respiration. The respiratory rate in the presence of substrate only was apparently normal, but it did not increase upon the addition of ADP. This effect could also be obtained by treatment with $\text{V. russelli}$ phospholipase A or oleate. Treatment with lesser amounts of these agents caused the mitochondria to become uncoupled.     

A study of tubulin dimer conformation by near-UV circular dichroism

The near-UV circular dichroism properties of tubulin dimer have been measured for different preparative methods. Tubulin dimer was obtained from assembly compenent microtubule protein by gel filtration, or phosphocellulose ion-exchange chromatography in the presence of magnesium. Tubulin dimer prepared by the protocol of Weisenberg R.C. and Timasheff, S.N. (1970) Biochemistry 9, 4110–4116, was found to be markedly different due to some apparently irreversible change in conformation. We conclude that the removal of microtubule-associated proteins by phosphocellulose ion-exchange chromatography in the presence of magnesium can be performed without affecting the conformation of native tubulin dimer as judged by near-UV circular dichroism.     

Slow transition of phosphoenzyme from ADP-sensitive to ADP-insensitive forms in solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum: evidence for retarded dissociation of Ca2+ from the phosphoenzyme.

Solubilized Ca²⁺, Mg²⁺-ATPase of sarcoplasmic reticulum was phosphorylated with ATP without added MgCl₂. The phosphoenzyme formed was ADP-sensitive. Ca²⁺ in the medium was chelated after phosphorylation. This induced a slow transition of the phosphoenzyme from ADP-sensitive to ADP-insensitive forms. The ADP-sensitivity was restored by subsequent addition of CaCl₂. These results showed that the transition was caused by dissociation of Ca²⁺ bound to the phosphoenzyme. Further observations indicated that, when Ca²⁺ in the medium was chelated, Ca²⁺ bound to the phosphoenzyme was dissociated much more slowly than Ca²⁺ bound to the dephosphoenzyme. This suggests a possible formation of the occluded form of the Ca²⁺-binding site in the phosphoenzyme.     

Polyamine-stimulated phosphorylation of proteins from corn (Zea mays L.) coleoptiles

The effect of polyamines (spermine, spermidine and putrescine) on in vitro phosphorylation of proteins from corn coleoptiles was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine promoted the phosphorylation of several membrane and soluble proteins and most of the proteins phosphorylated were different from those phosphorylated in the presence of calcium. Spermidine promoted the phosphorylation to a lesser extent and putrescine had very little stimulatory effect. Spermine-promoted phosphorylation of soluble proteins was dependent upon the presence of Mg2+ and was discernible at 100 microM spermine concentration.