Presence Of Free Ca Research Articles

Chemical modification of contractile 3-nm-diameter filaments in Vorticella spasmoneme by diethyl-pyrocarbonate and its reversible renaturation by hydroxylamine

A peritrich ciliate possesses a zooid and a long stalk consisting of a bundle of 3-nm-diameter filaments. Glycerinated stalks can contract in the presence of free Ca 2+ and re-extend in the absence of free Ca 2+. In the present study, we demonstrated that histidine residue(s) played a critical role in spasmoneme contraction by using glycerinated stalk of Vorticella. Concentration-dependent inhibition of spasmoneme contraction was observed in the presence of reversible histidine-modifying reagent named diethyl-pyrocarbonate (DEPC). In addition, the contractility degree of DEPC-modified spasmoneme could be partially restored by hydroxylamine treatment. The 244 nm absorption of modified spasmoneme protein(s) increased with rising DEPC concentration and decreased following the addition of hydroxylamine treatment. Adding Ca 2+ before DEPC modification could prevent the spasmoneme contraction from inhibition of DEPC. Those results suggested that histidine residues were actively involved in spasmoneme contraction. Ca 2+-binding ability of spasmin was not inhibited by DEPC modification, which suggested that the essential histidine residues were not on the calcium-binding site of spasmin.

Purification and functional characterization of an 85-kDa gelsolin from the ascidians Microcosmus sulcatus and Phallusia mammilata

From the pharyngeal baskets of the ascidians Microcosmus sulcatus and Phallusia mammilata we have purified an 85-kDa protein that is characterized as a member of the gelsolin family. These proteins from both species show the same behaviour in functional assays. The ascidian gelsolin binds two actin monomers in a highly cooperative manner. This complex formation is Ca 2+-dependent, but not completely reversible, as on removal of Ca 2+ one actin monomer dissociates leaving a 1:1 complex between gelsolin and G-actin. The properties of F-actin severing and G-actin nucleation depend on the presence of free Ca 2+ in a micromolar range, with half maximum activation at about 3×10 −6 M. The protein becomes inactivated when Ca 2+ concentrations of 0.5 mM are exceeded. Fragmentation of F-actin by the ascidian gelsolin is comparably fast to that of vertebrate gelsolin. A steady state of actin fragmentation is reached within 2–4 s. Promotion of G-actin nucleation is also comparable to that of vertebrate gelsolin. Regarding functional aspects, the ascidian gelsolin is more closely related to vertebrate gelsolin than to an arthropod gelsolin from crayfish tail muscle.

Mechanochemical Synthesis of Tricalcium Aluminum Hydrate from Calcium Hydroxide-Boehmite Mixtures by Room Temperature Grinding.

Tricalcium aluminum hydrate (3CaO·Al2O3·6H2O：C3AH6) is synthesized mechanochemically from mixtures composed of calcium hydroxide (Ca(OH)2) and pseudo-boehmite (γ-AlO(OH)) powders by room temperature grinding using a planetary ball mill. Use of the boehmite sample with inferior crystallinity is more favorable for the mechanochemical synthesis rather than that with well crystalline one. The time required to form C3AH6 from the Ca(OH)2-γ-AlO(OH) mixture is much longer than that from the Ca(OH)2-gibbsite (Al(OH)3) one. Adsorbed water from air during grinding plays a significant role in the formation of C3AH6 from the former mixture. After water addition to Ca(OH)2-γ-AlO(OH) mixtures ground for various times, excess hydrated calcium aluminates such as C2AH8, C3AH8-12 and C2A0.5H6.5 are formed in the starting and the short time ground mixtures, while a few amount of these compounds is formed in these hydrated mixtures after prolonged grinding. Formation of these excess hydrated compounds, which belong to layered structural materials, is enhanced in the presence of free Ca, Al compounds and water.

Structure, function and regulation of Ca 2+-sensitive cytosolic phospholipase A2 (cPLA2)

The 85-kDa cytosolic PLA2 (cPLA2) is present in many cells and tissues and its unusual functional properties and catalytic mechanism are being elucidated. Notably, cPLA2 becomes catalytically active in the presence of free Ca 2+ concentrations as present in stimulated cells and preferentially cleaves arachidonic acid-containing phospholipids. A variety of agonists, growth factors and cytokines, as well as stressful stimuli activate cPLA2 to hydrolyze cellular phospholipids thereby liberating fatty acids and lysophospholipids and providing the precursor substrates for the biosynthesis of eicosanoids and platelet-activating factor. These products of cPLA2 contribute to inflammatory and degenerative disease states and cPLA2 is therefore an attractive target for the development of novel therapies.

NS 1619 activates BK Ca channel activity in rat cortical neurones

Single channel recordings of large conductance Ca 2+-activated K + (BK Ca) channels were made from neurones isolated from rat motor cortex. Application of levcromakalim, pinacidil or diazoxide had no effect on BK Ca channel activity in excised patches. In contrast, NS 1619 (1-(2′-hydroxy-5′-trifluoromethylphenyl)-5-trifluoromethyl-2(3 H)benzimidazalone) induced concentration-dependent activation of BK Ca channels with a calculated EC 50 of 32 μM. The NS 1619-induced activity was dependent on the presence of free Ca 2+ at the intracellular surface, but was not associated with a change in channel voltage sensitivity. Niflumic acid had no effect on BK Ca activity per se but prevented NS 1619-mediated activation.

The TSH-Dependent Potassium Channel in a Cloned Rat Thyroid Cell Line

We found a TSH-dependent K +channel in the membrane of a rat thyroid cell line (FRTL-5 cell). This K + channel is activated by extracellular application of the thyroid stimulating hormone (TSH). Treatment of dibutyryladenosine cyclic monophosphate (Bt 2 cAMP) also activated the TSH-dependent K + channel. Intracellular application of protein kinase A activates this K + channel without the presence of free Ca 2+. These findings indicate that the thyroid K + channel is activated through the TSH-cAMP-protein kinase A system. This is the first report of a polypeptide hormone activated K + channel and it should be useful for examining the effects of this K + channel on thyroid cell functions.

Possible role during exocytosis of a ca 2+-activated channel in neurohypophysial granules

Ion channels from bovine neurohypophysial granules were incorporated into artificial lipid bilayers. The larger amplitude channel is permeable to cations and exhibits multiple conductances. The channel opens only in the presence of free Ca 2+, but is inhibited by relatively high Ca 2+ concentrations. Release of vasopressin from permeabilized neurohypophysial terminals also shows a similar biphasic dependence on Ca 2+. Release is selectively inhibited by low concentrations of the long-chain alcohol octanol, but not by high concentrations of ethanol, as is the neurosecretory granule Ca 2+-activated cation channel. Furthermore, Ca 2+-evoked release and channel activity are both inhibited by the long-chain tetraethylammonium analogs decamethonium and decyl-triethyl ammonium bromide. The close correlation between channel and release properties lead us to conclude that the Ca 2+-activated channel is involved in peptide secretion.

Antibody that recognizes conformations of calmodulin in the serum from patient with chronic active hepatitis

According to the effects of Ca++ on the reactivity, anti-calmodulin antibody in the sera of patients with autoimmune chronic active hepatitis was classified into three types, which were tentatively designated as type 1, 2 and 3. Type 1 antibody reacted with calmodulin only in the presence of free Ca++. Binding of type 2 antibody to calmodulin was inhibited by the presence of free Ca++. Type 3 antibody reacted with calmodulin regardless of the presence or absence of free Ca++. Thus the sera contained the populations of the antibody which recognized the different conformations of calmodulin molecule.

Studies of alkali-silica reaction. Part 3. Mechanisms by which NaCl and Ca(OH) 2 affect the reaction

Recently it has been observed that a concentrated solution of NaCl accelerates alkali-silica reaction and that the presence of free Ca(OH) 2 is a prerequisite for expansion to occur. This paper reports work done to understand the chemical processes involved. From the results of this investigation following mechanism has been proposed to explain the above observations. In the presence of free Ca(OH) 2, Na + ions from alkali salts and OH − ions from Ca(OH) 2 together with water molecules penetrate reactive grains. SiOSi bonds in reactive grains are broken by penetrating Na + and OH − ions thereby opening the grains for further penetration of materials. At the same time silica ions tend to diffuse out of the reactive grains. Expansion occurs when more materials enter the reactive grains than diffuse out.

Identification of calmodulin-sensitive and calmodulin-insensitive adenylate cyclase in rat kidney

Adenylate cyclase from rat kidney membranes solubilized with Lubrol-PX, was resolved into calmodulin-insensitive and calmodulin-sensitive forms using DEAE-Sephacel and calmodulin-Sepharose affinity chromatography. The major fraction, 90% of the activity recovered, did not bind to the calmodulin-sepharose in the presence of Ca 2+, and was insensitive to activation by calmodulin. The calmodulin-sensitive enzyme, approximately 10% of the recovered activity, bound to the affinity column and was eluted with buffer containing 2 m m EGTA. In the presence of free Ca 2+, calmodulin increased the specific activity of the calmodulin-sensitive adenylate cyclase from 15.2 to 60.4 pmol/mg protein −1 min −1. Maximum stimulation occurred at 0.035–0.076 m m Ca 2+. The apparent K a for calmodulin was 8 n m. The calmodulin-mediated increase in activity was inhibited by trifluoperazine, but not by its analog trifluoperazine-5-oxide. In contrast, trifluoperazine did not inhibit the calmodulin-insensitive activity. The GTP analog, guanyl-5′-yl imidodiphosphate, did not activate either fraction. Furthermore, activation by calmodulin did not require the presence of a guanyl nucleotide. The present finding of a calmodulin-sensitive form of adenylate cyclase in kidney raises the possibility that a calmodulin-mediated mechanism is involved in the formation of cAMP in this organ.

Possible mechanism of the inhibitory effect of ouabain on renin secretion from rat renal cortical slices.

1. The effects of ouabain on renin secretion by rat renal cortical slices were studied. 2. Renin secretion was inhibited by 10(-3) M-ouabain in the presence of free Ca (10(-4) to 2.6 x 10(-3) M). Inhibition was blocked at Ca less than 10(-8) M. 3. The effect of free Ca on ouabain-inhibition was shown to be independent of the presence of EGTA, completely reversible, and unrelated to passive leakage of renin from non-viable cells, as assessed by simultaneous release of lactate dehydrogenase activity (LDH). 4. It is proposed that, as a result of inhibition of Na, K-ATPase by ouabain, (a) intracellular Na increases in the renin-secreting juxtaglomerular cells, (b) intracellular Ca increases, via an Na-Ca exchange mechanism, and (c) that Ca accumulation, in some unknown manner, inhibits renin secretion.

The role of Ca(OH) 2 in the breakdown of Portland cement concrete due to alkali-silica reaction

Both field observations and subsequent laboratory experiments showed that the presence of free Ca(OH) 2 is a necessary condition for the breakdown of a Portland cement concrete structure due to the destructive alkali-silica reaction. The complete removal of free Ca(OH) 2 either by leaching with a concentrated solution of CaCl 2 or by reacting with a pozzolana suppresses alkali-silica reaction even when excess alkali metal salt ,including hydroxide ,is present in the structure. Attention has been drawn to certain practical implications of the above results.

Catalytic and regulatory properties of two forms of bovine heart cyclic nucleotide phosphodiesterase

The soluble supernatant fraction of bovine heart homogenates may be fractionated on a DEAE cellulose column into two cyclic nucleotide phosphodiesterases (EC 3.1.4.-): P I and P II phosphodiesterases, in the order of emergence from the column. In the presence of free Ca 2+, the P I enzyme may be activated several fold by the protein activator which was discovered by Cheung ((1971) J. Biol. Chem. 246, 2859–2869). The P II enzyme is refractory to this activator, and is not inhibited by the Ca 2+ chelating agent, ethylene glycol bis ( β- aminoethyl ether)-N,N′- tetraacetate (EGTA). The activated activity of P I phosphodiesterase may be further stimulated by imidazole or NH 4 +, and inhibited by high concentrations of Mg 2+. These reagents have no significant effect on either the P II enzyme or the basal activity of P I phosphodiesterase. Although both forms of phosphodiesterase can hydrolyze either cyclic AMP or cyclic GMP, they exhibit different relative affinities towards these two cyclic nucleotides. The P I enzyme appears to have much higher affinities toward cyclic GMP than cyclic AMP. K m values for cyclic AMP and cyclic GMP are respectively 1.7 and 0.33 mM for the non-activated P I phosphodiesterase; and 0.2 and 0.007 mM for the activated enzyme. Each cyclic nucleotide acts as a competitive inhibitor for the other with K i values similar to the respective K m values. In contrast with P I phosphodiesterase, P II phosphodiesterase exhibits similar affinity toward cyclic AMP and cyclic GMP. The apparent K m values of cyclic AMP and cyclic GMP for the P II enzyme are approx. 0.05 and 0.03 mM, respectively. The kinetic plot with respect to cyclic GMP shows positive cooperativity. Each cyclic nucleotide acts as a non-competitive inhibitor for the other nucleotide. These kinetic properties of P I and P II phosphodiesterase of bovine heart are very similar to those of rat liver cyclic GMP and high K m cyclic AMP phosphodiesterases, respectively (Russel, Terasaki and Appleman, (1973) J. Biol. Chem. 248, 1334).

Effects of tropomyosin, troponin and Ca 2+ on the interaction between F-actin and heavy meromyosin

1. In the absence of ATP, H-meromyosin (heavy meromyosin) bound with the F-actin-tropomyosin-troponin complex up to the molar ratio of H-meromyosin to actin of 1:1, independently of the concentration of Ca 2+. 2. In the presence of free Ca 2+ above about 1 μM, with an increasing amount of H-meromyosin bound to a fixed amount of the F-actin-tropomyosin-troponin complex, the degree of flow birefringence decreased and the extinction angle increased. The minimum value of the birefringence and the maximum value of the extinction angle were found at the molar ratio of H-meromyosin to actin of 1:2. A further increase of bound H-meromyosin to actin restored both the degree of birefringence and the extinction angle to nearly the same level as the F-actin-tropomyosin-troponin complex only. In the absence of free Ca 2+, the birefringence did not change with the binding of H-meromyosin. 3. This sensitivity of birefringence to the concentration of Ca 2+ appeared only in the presence of tropomyosin and troponin. At a fixed ratio of H-meromyosin, actin and tropomyosin, the birefringence in the absence of Ca 2+ increased with increasing amount of added troponin up to the weight ratio of troponin to actin of 1:6; whereas the birefringence in the presence of Ca 2+ did not change. 4. At a fixed ratio of H-meromyosin to actin, the birefringence changed with increasing amount of tropomyosin added up to the weight ratio of tropomyosin to actin of 1:6; above this ratio, the birefringence was constant. 5. Subfragment S-1, prepared by the chymotryptic digestion of myosin, bound to F-actin, but the birefringence did not change even in the presence of tropomyosin and troponin.