Stimulatory Effect Of Calcium Research Articles

Investigation of calcium-dependent activity and conformational dynamics of zebra fish 12-lipoxygenase

BackgroundA 12-lipoxygenase in zebra fish (zf12-LOX) was found to be required for normal embryonic development and LOXs are of great interest for targeted drug designing. In this study, we investigate the structural-functional aspects of zf12-LOX in response to calcium. MethodsA soluble version of zf12-LOX was created by mutagenesis. Based on multiple sequence alignment, we mutated the putative calcium-responsive amino acids in N-PLAT domain of soluble zf12-LOX. Using a series of biophysical methods, we ascertained the oligomeric state, stability, structural integrity and conformational changes of zf12-LOX in response to calcium. We also compared the biophysical properties of soluble zf12-LOX with the mutant in the absence and presence of calcium. ResultsHere we provide a detailed characterization of soluble zf12-LOX and the mutant. Both proteins exist as compact monomers in solution, however the enzyme activity of soluble zf12-LOX is significantly increased in presence of calcium. We find that the stimulatory effect of calcium on zf12-LOX is related to a change in protein structure as observed by SAXS, adopting an open-state. In contrast, enzyme with a mutated calcium regulatory site has reduced activity-response to calcium and restricted large re-modeling, suggesting that it retains a closed-state in response to calcium. Taken together, our study suggests that Ca2+-dependent regulation is associated with different domain conformation(s) that might change the accessibility to substrate-binding site in response to calcium. General significanceThe study can be broadly implicated in better understanding the mode(s) of action of LOXs, and the enzymes regulated by calcium in general.

Regulation of ATP production: dependence on calcium concentration and respiratory state.

Nanomolar free calcium enhances oxidative phosphorylation. However, the effects over a broad concentration range, at different respiratory states, or on specific energy substrates are less clear. We examined the action of varying [Ca2+] over respiratory states ranging 4 to 3 on skeletal muscle mitochondrial respiration, potential, ATP production, and H2O2 production using ADP recycling to clamp external [ADP]. Calcium at 450 nM enhanced respiration in mitochondria energized by the complex I substrates, glutamate/malate (but not succinate), at [ADP] of 4-256 µM, but more substantially at intermediate respiratory states and not at all at state 4. Using varied [Ca2+], we found that the stimulatory effects on respiration and ATP production were most prominent at nanomolar concentrations, but inhibitory at 10 µM or higher. ATP production decreased more than respiration at 10 µM calcium. However, potential continued to increase up to 10 µM; suggesting a calcium-induced inability to utilize potential for phosphorylation independent of opening of the mitochondrial permeability transition pore (MTP). This effect of 10 µM calcium was confirmed by direct determination of ATP production over a range of potential created by differing substrate concentrations. Consistent with past reports, nanomolar [Ca2+] had a stimulatory effect on utilization of potential for phosphorylation. Increasing [Ca2+] was positively and continuously associated with H2O2 production. In summary, the stimulatory effect of calcium on mitochondrial function is substrate dependent and most prominent over intermediate respiratory states. Calcium stimulates or inhibits utilization of potential for phosphorylation dependent on concentration with inhibition at higher concentration independent of MTP opening.

Stimulatory effect of calcium on metabolism and its sensitivity to pH in kidney mitochondria.

The relationship between mitochondrial matrix free Ca2+ concentration ([Ca2+]m) and pH was evaluated by incubating isolated rat kidney mitochondria with different extramitochondrial Ca2+ concentrations ([Ca2+]e) at medium pH (pHe) 7.0 and 7.4. [Ca2+]m was monitored using the fluorescent signal from mitochondria loaded with the Ca2+ indicator fura 2. The changes in [Ca2+]m were compared with alpha-ketoglutarate dehydrogenase (alpha-KGDH) flux, measured as O2 consumption (nmol.min-1.mg protein-1) from 185 microM alpha-ketoglutarate (alpha-KG). The apparent dissociation constant of the matrix fluorescent probe for Ca2+ was determined in each experiment and was 323 +/- 45 nM (n = 14). When mitochondria were exposed to [Ca2+]e below 160 nM, [Ca2+]m was greater at pHe 7.0 than at pHe 7.4. However, above 160 nM [Ca2+]e, [Ca2+]m plateaued at pHe 7.0 but rose progressively at pHe 7.4. Increasing [Ca2+]m by consecutive additions of Ca2+ to the medium had a significantly more pronounced acceleratory effect on alpha-KG oxidation at pHe 7.0 than at pHe 7.4. Kinetic analysis of alpha-KGDH revealed a 45% decrease in the Michaelis constant (Km) for alpha-KG at pHe 7.0, but the Km was unchanged at pHe 7.4 with elevation of [Ca2+]m from 32 to 751 nM. Maximal velocity (Vmax) increased significantly at both pHe values. Half-maximal alpha-KG oxidation occurred at [Ca2+]m of 76 +/- 11 nM and 105 +/- 31 nM at pHe 7.0 and 7.4, respectively. These studies demonstrate a direct, pH-sensitive correlation between [Ca2+]e and [Ca2+]m; [Ca2+]m changed over a range that may regulate alpha-KGDH flux in intact kidney mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Difference in the property of the nerve calmodulin-dependent protein kinase between kdr-type, pyrethroid-resistant and susceptible strains of Musca domestica and Blattella germanica

The effects of calcium, clamodulin and 1R-deltamethrin on protein phosphorylation processes were investigated using lysed synaptosomal preparations isolated from the nervous system of the suceptible and kdr-resistant German cockroaches and houseflies. By comparing interstrain differences in phosphorylation activities of isolated, lysed synaptic membranes, it was concluded that the stimulatory effect of calcium alone on protein phosphorylation was the same in the preparations from the susceptible and the kdr-resistant strains of the German cockroach. However, calmodulin, added in the presence of Ca 2+, significantly increased the level of phosphorylation of the two putative subunits of calcium-calmodulin-dependent protein kinase (CCPK) from the susceptible strains, but its effect on the same enzyme in the kdr-resistant strains of the German cockroach and housefly was much less. 1R-deltamethrin at 10 −8 M inhibited both the total protein phosphorylation and the phosphorylation on the two subunits of CCPK in the susceptible and kdr-resistant strains of the German cockroach. Depolarization induced in intact synaptomomes by veratridine or “high K +” in the presence of 1R-deltamethrin at 10 −10 M and 10 −6 M had the effect of significantly increasing the total level of endogenous protein phosphorylation in the susceptible strain, but the increase was not significant in the kdr-resistant strain of the German cockroach. An identical experimental approach on kdr and the susceptible strain of the housefly produced essentially the same results, indicating that the CCPK enzyme from the kdr-type resistant insects behave in a different manner from the susceptible counterparts in terms of their responsiveness to calmodulin.

Calcium influx similarly stimulates the release of chorionic gonadotrophin and placental lactogen from human placental explants

Summary The releases of human chorionic gonadotrophin (hCG) and of human placental lactogen (hPL) by explants from normal term placentae were both stimulated by calcium, barium, ionomycin, and forskolin. The stimulatory effect of calcium could be thwarted by cobalt, a calcium influx blocker. These results are consistent with the view that an increased calcium inflow provokes the secretion of both hCG and hPL from human placental explants.

The effect of ions, ion channel blockers, and ionophores on uptake of vitellogenin into cockroach follicles

Since calcium plays an important role in vitellogenin binding and uptake in Nauphoeta cinerea and because calcium channels have been described in follicles of this species, we investigated the effect of various ions, ionophores, and ion channel blockers on vitellogenin uptake in vitro. Calcium significantly stimulated vitellogenin uptake; this effect could be substituted best by barium and less well by strontium and magnesium. The stimulatory effect of calcium, and to a certain extent also that of barium, was dependent on the vitellogenin concentration, whereas the effect of strontium and magnesium was not. In the presence of calcium, vitellogenin uptake was inhibited by barium, strontium, and magnesium as well as by the transition elements nickel, cobalt, and zinc, but not by manganese which had a stimulatory effect. Valinomycin, verapamil, tetraethylammonium, and atropin reduced vitellogenin uptake, while amiloride and ouabain were ineffective. Our results indicate that calcium inward (and possibly potassium outward) fluxes play an important role in vitellogenin uptake.

Inhibition of Reproduction of Phytophthora by the Calmodulin-interacting Compounds Trifluoperazine and Ophiobolin A

SUMMARY: Sporangium production in Phytophthora palmivora was inhibited by trifluoperazine (TFP) and ophiobolin A. TFP was inhibitory to a similar extent to cultures both in light and in darkness, although the stimulatory effect of calcium and the inhibitory effect of EGTA were much greater in the dark. Sporangium and oospore production in P. cactorum were inhibited by TFP. Thus development of both asexual and sexual reproductive structures involves a calcium/calmodulin interaction.

Effects of an acute calcium load on plasma ACTH, cortisol, aldosterone and renin activity in man.

Plasma adrenocorticotrophin (ACTH), cortisol and aldosterone increased during and after iv administration of calcium gluconate in 4 normal subjects, one patient with hypoparathyroidism and one patient with hypothyroidism. On the other hand, there was a decrease in plasma renin activity but only in the normal subjects. Plasma ACTH and cortisol responses to calcium were abolished whereas plasma aldosterone response persisted in 2 normal subjects pre-treated with dexamethasone. The results observed after calcium administration were compared to those observed after infusion of the solvent only in 6 normal subjects and 4 thyroidectomized patients who were studied twice at 3 day intervals. Plasma ACTH, cortisol and aldosterone were higher when calcium was administered. Plasma renin activity was not statistically different whether or not calcium had been injected in the subjects studied twice. These results demonstrate a direct effect of calcium on ACTH and aldosterone secretion which is not mediated by calcitonin and parathyroid hormone. The stimulatory effect of calcium on cortisol secretion depends on the increase in plasma ACTH.

Stimulatory effect of calcium on photoactivation of the water oxidation system in dark-grown spruce chloroplasts

The water-oxidation system in chloroplasts from Picea abies leaves grown without light was activated and stabilized upon exposure of the isolated chloroplasts to weak light. The rate of electron transport from diphenylcarbazide to 2,6-dichloroindophenol in the photosystem II remained constant. Ca2+ added to the chloroplast suspension was incorporated into thylakoid membranes during illumination and strongly stimulated the photoactivation of latent water-oxidation system but not electron transport in photosystem II. It is concluded that the site which requires Ca2+ and is activated by light is the water-oxidation system.

The stimulatory effect of calcium on Na,K-ATPase of nervous tissue

The stimulatory effect of calcium on Na,K-ATPase of nervous tissue

Characterisation of somatostatin release from the pancreas

The effect of calcium on somatostatin secretion was investigated in the isolated, perfused canine pancreas preparation and compared with those of acetylcholine, glucose, isoproterenol and arginine. Calcium (5 mmol/l) stimulated somatostatin release in a typical biphasic response pattern being about 5 times as potent as acetylcholine (1 mumol/l), arginine (5 mmol/l), and isoproterenol (2 ng/ml) while the release of insulin and glucagon in response to calcium and the other secretagogues were of the same magnitude. Somatostatin release increased progressively when perfusate calcium was increased step-wise from 0 through 1.25 and 2.5 to 5.0 mmol/l. Calcium stimulated the secretion of somatostatin in the absence of glucose. The stimulatory effect of calcium was, however, modulated by the glucose concentration being about twice as large at 200 mg/100 ml as at 25 mg/100 ml glucose in the perfusion medium.

Gastrointestinal effect of calcitonin: Inhibition of gastrin secretion

The stimulatory effect of calcium on gastric acid secretion is well-known (6, 4), but the mechanism unknown. Gastrin secretion may be involved since calcium infusions increase the concentration of gastrin in serum (7).

The stimulatory effect of calcium on the synthesis of cartilage proteoglycan

Calcium stimulates the uptake of [ 35 S] SO 4 into cartilage at physiological concentration for ionized calcium. This effect can be blocked by puromycin, indicating calcium stimulates the synthesis of proteoglycan. It is proposed that this effect is mediated by alteration of the negative charge density of the polyanionic proteoglycan.

The effect of calcium on labeling of nucleic acids with radioactive precursors in heart tissue slices

Heart tissue slices were incubated in the presence of various radioactive precursors of pyrimidine nucleotides. Labeling of RNA of the tissue slices was greater with [ 3H]uridine, [ 3H]UMP, and [ 3H]cytidine than with [ 3H]uracil or [ 3H]orotic acid. The labeling of RNA was linear for at least 4 h, and all components of RNA (28 S, 19 S and 4 S) were stimulated with an increase in calcium concentration in the medium from 10 −5 to 10 −3 m. Contrarily, the labeling of DNA with [ 3H]thymidine was not influenced by changes in calcium in the medium. Under conditions where RNA labeling with [ 3H]uridine was stimulated with an increase in calcium, there was no change in radio-activity in the acid soluble pool. Chromatography of the acid soluble pool showed a shift in distribution of radioactivity toward nucleotides and away from uracil when calcium concentration in the medium was increased. The stimulatory effect of calcium on RNA labeling was not replaced with other divalent cations. The data suggest that calcium may influence RNA metabolism in heart tissue, possibly through an effect on intermediate pyrimidine metabolism.

Stimulatory effects of calcium on protein synthesis in adrenal (and thyroidal) cell-free systems as related to trophic hormone action.

Calcium directly stimulates incorporation of [H3]-leucine into protein during incubations of adrenal 15,000 × g-supernatant fluid which contains microsomes and soluble cell fraction. Effects of calcium are observed at concentrations as low as 10-7 M, and the peak effect usually occurs at 10-5 M. Since ATP, GTP, and magnesium are present in excess, the effect of calcium is not related to these factors. Calcium does not stimulate formation of the amino acyl- transfer-RNA complex, but does enhance the transfer of amino acid from the amino acyl- transfer-RNA complex to protein. The stimulatory effect of calcium is also demonstrable when purified polysomes are used in place of microsomes in the incubations, but does not appear to be due to changes in the ribosome-polysome fraction. The effect of calcium is apparent only if the soluble cell fraction is derived from control adrenals. With soluble cell fraction from ACTH-stimulated adrenals (wherein transfer enzyme activity is already enhanced), there is little o...

Stimulation of Mitosis in Bone Marrow and Thymus of Normal and Irradiated Rats by Divalent Cations and Parathyroid Extract

Intraperitoneal injections of calcium and magnesium chlorides stimulated the entry of cells into mitosis in both bone marrow and thymus tissue of normal and irradiated rats, and thus reduced the mitotic delay induced by radiation exposure. Strontium and sodium chlorides were without effect. The stimulatory effect of calcium was evident in bone marrow even after 1000 R of whole-body irradiation. A subcutaneous injection of parathyroid extract was similar to calcium in stimulating mitosis in the bone marrow of irradiated rats, but the extract had no effect on mitosis in the thymus gland.