Citrate effects on the Ca2+-loading capacity of isolated rat liver mitochondria: interaction of citrate and ATP

The maximal amounts of Ca2+ being accumulated (delta Ca2+max) and H+ emitted (delta H+max) by Ca2+-loading mitochondria, with succinate (+rotenone) as respiratory substrate, were evaluated. delta Ca2+max was increased by providing either citrate or ATP to a Pi- and Mg2+-free medium. With citrate, delta H+max was only scarcely increased, so that the effect of the proton-carrying anion resulted essentially from an increase in the Ca2+/H+ ratio, i.e., from preservation of membrane potential. With ATP (+/- oligomycin), the Ca2+/H+ ratio was unaltered; i.e., the increase of delta Ca2+max was paralleled by a related increase in delta H+max. Mitochondria appeared to retain Ca at higher delta pH, i.e., at lower membrane potential, in the presence of ATP. With citrate and ATP together, both the Ca2+/H+ ratio and delta H+max were largely increased, and the product of these two terms, delta Ca2+max, was considerably enlarged. The effect of either citrate or ATP was markedly reinforced in the presence of the other anion. In addition to increasing the Ca2+/H+ ratio, citrate contributed to increasing delta H+max in the presence of ATP, i.e., apparently sensitized mitochondria to the action of ATP. A citrate-induced depression of Ca2+ cycling across the inner membrane, even though pronounced, did not account for the sensitization. Supraadditive effects of citrate and ATP persisted in the presence of MgCl2 and Pi, under conditions of massive Ca2+ loading, and may contribute to the high capacity of mitochondria, in situ, to retain calcium.     

A calcium-43 NMR study of calcium binding to an acidic proline-rich phosphoprotein from human saliva

The 43Ca NMR line width measured for Ca2+ bound to protein A, an acidic proline-rich salivary protein, is 1 order of magnitude narrower than has previously been observed for other proteins of similar molecular weight. The correlation times, quadrupole coupling constants, and chemical shifts estimated for Ca2+ ions bound to the intact protein (Mr approximately 10 000) and its 30 amino acid residue long acidic N-terminal TX peptide were indistinguishable within experimental error. These results--as well as the outcome of 1H NMR relaxation rate measurements--are indicative of extensive motions for the protein residues, which in turn give rise to a high degree of flexibility for the protein-bound Ca2+. Ca2+ titration and pH-dependent measurements on protein A, the TX peptide, and the dephosphorylated TX peptide established the importance of the two phosphoserine residues in the binding of Ca2+. Moreover, a comparison of the 43Ca NMR parameters with those obtained for other Ca2+-binding proteins suggests the presence of Ca2+-binding sites of similar symmetry in all these proteins. No evidence was found for a proposed interaction between the highly acidic N-terminal and the weakly basic C-terminal regions of protein A. In contrast, the high pH inflection that was observed in the pH titration curve for the intact protein was also found for the phospho and dephospho TX peptides, thus suggesting that basic moieties in the N-terminal region rather than those in the C-terminal region may be responsible for this observation.     

In vitro translation of polyadenylate-containing RNAs from dormant and germinating spores of the fungus Botryodiplodia theobromae

The oxaloacetate (OAA) decarboxylase (EC 4.1.1.3) activity of Acetobacter xylinum cells grown on glucose or glycerol is the same as that of cells grown on intermediates of the citrate cycle. The enzyme was purified 92-fold from extracts, and its molecular weight was determined to be 100,000 by gel filtration. Initial velocity studies revealed marked positive cooperativity for OAA (Hill coefficient [n(H)] = 1.8; S(0.5) = 21 mM). The affinity of the enzyme for OAA was markedly increased upon addition of nicotinamide adenine dinucleotide (NAD), NAD phosphate (NADP), and some other pyridine nucleotides. S(0.5(OAA)) decreased to 1 mM but n(H) and V(max) were unchanged. Saturation kinetics for the pyridine nucleotides were hyperbolic, and a half-maximal effect was obtained with 8 muM NAD and 30 muM NADP. The enzyme also catalyzed the exchange of (14)CO(2) into OAA but not the net carboxylation of pyruvate. Exchange activity, too, exhibited sigmoidal kinetics for OAA and was strongly stimulated by NAD at low substrate concentrations. The enzyme was inhibited by acetate competitively with respect to OAA. The K(I) for acetate (12 mM) was well within the physiological range of this compound inside the cell. The regulatory properties of the decarboxylase with respect to OAA cooperativity, NAD activation, and acetate inhibition were retained in situ within permeabilized cells. These properties seem to provide for a control mechanism which could insure the maintenance of OAA and the citrate cycle during growth of cells on glucose and, conversely, the required supply of pyruvate during growth on intermediates of the citrate cycle.     

Nuclear magnetic resonance study on rabbit skeletal troponin C: calcium-induced conformational change

Rabbit skeletal muscle troponin C (TnC) was investigated by means of 1H NMR in the presence of dithiothreitol that prevents dimerization of the protein. Two-dimensional (2D) 1H NMR spectra were observed in order to assign resonances to specific amino acids. One-dimensional 1H NMR spectra were observed as a function of Ca2+ concentration. The Ca2+-induced spectral change is categorized into two types: type 1 corresponds to the conformational change of the C-terminal-half domain (Ca2+ high-affinity sites) and type 2 to that of the N-terminal-half domain (Ca2+ low-affinity sites). From the 2D NMR spectra and Ca2+ titration data, it was suggested that (1) amide protons of Gly-108, Ile-110, Gly-144, and Ile-146 are hydrogen-bonded when the C-terminal-half domain binds 2 mol of Ca2+ and (2) hydrogen bonds of Gly-108, Ile-110, Gly-144, and Ile-146 are destroyed or weakened when the C-terminal-half domain releases 2 mol of Ca2+. Nuclear Overhauser enhancement difference spectra as well as the Ca2+ titration data suggested that a hydrophobic cluster is formed in the C-terminal-half domain when the C-terminal-half domain binds 2 mol of Ca2+. A hydrophobic cluster exists in the N-terminal-half domain without regard to Ca2+ binding to the N-terminal-half domain. The spectra of Tyr-10 showed both types of spectral change during the Ca2+ titration. The results suggested that Tyr-10 of apo-TnC interacts with the C-terminal-half domain.     

A link between citrate and proton release by proteoid roots of white lupin (Lupinus albus L.) grown under phosphorus-deficient conditions?

White lupin (Lupinus albus L.) is able to acclimate to phosphorus deficiency by forming proteoid roots that release a large amount of citric acid, resulting in the mobilization of sparingly soluble soil phosphate in the rhizosphere. The mechanisms responsible for the release of organic acids have not been fully elucidated. In this study, we focused on the link between citrate and malate release and the release of H+ and other inorganic ions by proteoid roots of white lupin. The release of citrate was closely correlated with the release of H+, K+, Na+ and Mg2+, but not with that of Ca2+. The stoichiometric relationships between citrate release and the release of H+, K+, Na+ and Mg2+ were 1 : 1.3, 1 : 2.1, 1 : 1.5 and 1 : 0.47, respectively. Similar correlations were found between exudation of malate and cations. During 30 min incubation, fusicoccin addition stimulated H+ and malate release, but not citrate release. A concomitant stimulation of H+, malate and citrate release was measured after 60 min incubation. Vanadate inhibited the release of H+ and malate, but not that of citrate. Anthracene-9-carboxylic acid, an anion channel blocker, caused a concomitant decrease in release of citrate, malate and H+. We conclude that for export of citrate across the plasma membrane of proteoid root cells, H+ release is not strictly related to citrate release. Other cations such as K+ and Na+ can also serve as counterions for citrate release. In contrast, malate release shows a strong H+ release dependency.     

Relationship between phosphatidylinositol synthesis and recovery of 5-hydroxytryptamine-responsive Ca2+ flux in blowfly salivary glands.

Each salivary gland contains about 135 pmol of phosphatidylinositol. In glands prelabelled by incubation for 1 h with [32P]Pi or [3H]inositol there was a subsequent breakdown of 80% of the labelled phosphatidylinositol over a 2 h incubation period with 10 micrometer-5-hydroxytryptamine. However, there was no detectable decrease either in total phosphatidylinositol based on phosphorus analysis by chemical estimation or in the radioactivity of [32P]phosphatidylinositol in salivary glands of flies raised from the larval stage on diets containing[32P]Pi and whose phospholipids were uniformly labelled. These results suggest that the pool of phosphatidylinositol involved with Ca2+ gating is a small fraction of the total phosphatidylinositol content. Furthermore it is this small compartment that is preferentially radioactively labelled during short-term incubations with radioactively labelled precursors. In salivary glands incubated for 2 h with 10 micrometer-5-hydroxytryptamine there was a marked decrease in the flux of 45Ca2+ across the gland. After removal of the hormone, incubation of salivary glands for 1 h in the presence of 2mM-inositol, but not choline or ethanolamine, resulted in a recovery of hormone-responsive 45Ca2+ flux. Quantitative studies revealed that less than 9 pmol of phosphatidylinositol must be formed to fully restoret he 5-hydroxytryptamine-responsive 45Ca2+ flux.     

The interactions of adenylates with allosteric citrate synthase.

Evidence is presented that a number of derivatives of adenylic acid may bind to the allosteric NADH binding site of Escherichia coli citrate synthase. This evidence includes the facts that all the adenylates inhibit NADH binding in a competitive manner and that those which have been tested protect an enzyme sulfhydryl group from reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) in the same way that NADH does. However, whereas NADH is a potent inhibitor of citrate synthase, most of the adenylates are activators. The best activator, ADP-ribose, increases the affinity of the enzyme for the substrate, acetyl-CoA, and saturates the enzyme in a sigmoid manner. A fluorescence technique, involving the displacement of 8-anilino-1-naphthalenesulfonate from its complex with citrate synthase, is used to obtain saturation curves for several nucleotides under nonassay conditions. It is found that acetyl-coenzyme A, coenzyme A, and ADP-ribose all bind to the enzyme cooperatively, and that the binding of each becomes tighter in the presence of KCl, the activator, and oxaloacetic acid (OAA), the second substrate. Another inhibitor, alpha-ketoglutarate, can complete with OAA in the absence of KCl but not in its presence. The nature of the allosteric site of citrate synthase, and the modes of action of several activators and inhibitors, are discussed in the light of this evidence.     

The extracellular zinc-sensing receptor mediates intercellular communication by inducing ATP release

Taste and salivary secretion disorders have been linked to zinc deficiency, indeed zinc is found in secretory granules in the salivary gland. The signaling role for the zinc release in this tissue, however, is poorly understood. Here, we address the signaling pathways and physiological role of the zinc-sensing receptor, ZnR, in the ductal salivary gland cell line, HSY. Exposure of these cells to zinc triggered intracellular Ca2+ release from thapsigargin-sensitive stores. The G alpha q inhibitor, YM-254890 (1 microM), eliminated the Zn2+-dependent Ca2+ response, demonstrating that ZnR is a G alpha q-coupled receptor. Dose-response curves yielded an apparent K0.5 of 36 microM and a Hill coefficient of 7 in the absence of extracellular Ca2+, and K0.5 of 55 microM with a Hill coefficient of 3 in its presence. This indicates that although Zn2+ is essential for ZnR activation, Ca2+ may affect the receptor co-operativity. The homologous desensitization pattern of ZnR was characterized by pre-exposure of cells to Zn2+ at concentrations found to activate the receptor. Re-exposure of cells to Zn2+ elicited an attenuated Zn2+-dependent Ca2+ response for at least 3 h, indicating that the ZnR is strongly desensitized by Zn2+. Finally, we studied the paracrine affects of ZnR using a co-culture consisting of the HSY cells and vascular smooth muscle cells (VSMCs). While no Zn2+-dependent Ca2+ release was observed in VSMC alone, application of Zn2+ to the co-culture induced a Ca2+ rise in both HSY cells and VSMC. This Ca2+ rise was inhibited by the ATP scavenger, apyrase. Taken together, our results demonstrate that ZnR activity is monitored in salivary cells and is modulated by extracellular Ca2+. We further show that ZnR enhances secretion of ATP, thereby linking zinc to key signaling pathways involved in modification of salivary secretions by the ductal cells.     

Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae

Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulate Ca2+ ion in the presence of ATP, not in the presence of ADP or adenyl-5'-yl imidodiphosphate. Calcium transport showed saturation kinetics with a Km value of 0.1 mM and optimal pH of 6.4. Ca2+ ion incorporated in the vesicles was exchangeable and released completely by a protonophore uncoupler, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847), or calcium-specific ionophore, A23187. The transport required Mg2+ ion but was inhibited by Cu2+ or Zn2+ ions, inhibitors of H+-ATPase of the vacuolar membrane. The transport activity was sensitive to the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked Ca2+ uptake completely, but valinomycin stimulated it 1.35-fold. These results indicate that an electrochemical potential difference of protons is a driving force for this Ca2+ transport. The ATP-dependent formation of the deltapH in the vesicles and its partial dissipation by CaCl2 were demonstrated by fluorescence quenching of quinacrine. This Ca2+ uptake by vacuolar membrane vesicles is suggested to be catalyzed by a Ca2+/H+ antiport system.     

Fission yeast homolog of neuronal calcium sensor-1 (Ncs1p) regulates sporulation and confers calcium tolerance

The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 microm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Delta) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl(2)). Analysis of Ncs1p mutants lacking myristoylation (Ncs1p(G2A)) or deficient in Ca2+ binding (Ncs1p(E84Q/E120Q/E168Q)) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Delta, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1Delta was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+-intolerance of ncs1Delta was not affected by cAMP or recoverin, suggesting that the two ncs1Delta phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p.     

Characterization and Ca2+ Requirement of Histamine-Induced Catecholamine Secretion in Cultured Bovine Chromaffin Cells

The stimulation of cultured bovine chromaffin cells with histamine induced a continuous catecholamine secretion (EC50 = 3 x 10(-7) M) via the H1 receptor, in addition to an initial catecholamine burst due to a nonspecific stimulatory effect at higher doses (greater than or equal to 10(-4) M). The continuous secretion showed little desensitization and lasted for more than 1 h. In fura-2-loaded cells, the stimulation with histamine evoked a transient rise of intracellular free Ca2+ concentration ([Ca2+]i) which lasted only for a few minutes and was followed by a sustained [Ca2+]i rise which continued for more than 20 min. The addition of an activator for the L-type voltage-sensitive Ca2+ channel, i.e., Bay K 8644 (1 microM), facilitated the sustained [Ca2+]i rise, as well as the secretion, whereas the addition of relatively high concentrations of Ca(2+)-channel blockers (10 microM) suppressed the sustained [Ca2+]i rise and part of the secretion. Removal of extracellular Ca2+ completely abolished continuous secretion and sustained [Ca2+]i rise. When the external Ca2+ level was elevated, both sustained [Ca2+]i rise and continuous secretion were enhanced in a similar Ca(2+)-dependent manner, showing saturation with around 1-3 mM Ca2+. This Ca2+ dependence was clearly different from that observed with high K+ and nicotine, which is mediated by the L-type Ca2+ channel, in which the responses showed little or no saturation when the Ca2+ level was increased. The results indicate that stimulation with histamine induces a continuous secretion via the H1 receptor, in addition to a transient and nonspecific secretion at higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium transport into the vacuole of oat roots. Characterization of H+/Ca2+ exchange activity

Calcium (Ca2+) is sequestered into vacuoles of oat root cells through a H+/Ca2+ antiport system that is driven by the proton-motive force of the tonoplast H+-translocating ATPase. The antiport has been characterized directly by imposing a pH gradient in tonoplast-enriched vesicles. The pH gradient was imposed by diluting K+-loaded vesicles into a K+-free medium. Nigericin induced a K+/H+ exchange resulting in a pH gradient of 2 (acid inside). The pH gradient was capable of driving 45Ca2+ accumulation. Ca2+ uptake was tightly coupled to H+ loss as increasing Ca2+ levels progressively dissipated the steady state pH gradient. Ca2+ uptake displayed saturation kinetics with a Km(app) for Ca2+ of 10 microM. The relative affinity of the antiporter for transport of divalent cations was Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. La3+ or Mn2+ blocked Ca2+ uptake possibly by occupying the Ca2+-binding site. Ruthenium red (I50 = 40 microM) and N,N'-dicyclohexylcarbodiimide (I50 = 3 microM) specifically inhibited the H+/Ca2+ antiporter. When driven by pH jumps, the H+/Ca2+ exchange generated a membrane potential, interior positive, as shown by [14C]SCN accumulation. Furthermore, Ca2+ uptake was stimulated by an imposed negative membrane potential. The results support a simple model of one Ca2+ taken up per H+ lost. The exchange transport can be reversed, as a Ca2+ gradient (Ca2+in greater than Ca2+out) was effective in forming a pH gradient (acid inside). We suggest that the H+/Ca2+ exchange normally transports Ca2+ into the vacuole; however, under certain conditions, Ca2+ may be released into the cytoplasm via this antiporter.     

Novel fold and carbohydrate specificity of the potent anti-HIV cyanobacterial lectin from Oscillatoria agardhii

Oscillatoria agardhii agglutinin (OAA) is a recently discovered cyanobacterial lectin that exhibits potent anti-HIV activity. Up to now, only its primary structure and carbohydrate binding data have been available. To elucidate the structural basis for the antiviral mechanism of OAA, we determined the structure of this lectin by x-ray crystallography at 1.2 Å resolution and mapped the specific carbohydrate recognition sites of OAA by NMR spectroscopy. The overall architecture of OAA comprises 10 β-strands that fold into a single, compact, β-barrel-like domain, creating a unique topology compared with all known protein structures in the Protein Data Bank. OAA sugar binding was tested against Man-9 and various disaccharide components of Man-9. Two symmetric carbohydrate-binding sites were located on the protein, and a preference for Manα(1–6)Man-linked sugars was found. Altogether, our structural results explain the antiviral activity OAA and add to the growing body of knowledge about antiviral lectins.     

Ca2+-induced conformational changes in cardiac troponin C as measured by N-(1-pyrene)maleimide fluorescence

Bovine cardiac troponin C was modified by N-(1-pyrene)maleimide at Cys-35 and Cys-84; the Ca2+-induced conformational changes were followed by measuring pyrene fluorescence. In isolated troponin C, the saturation of Ca2+, Mg2+-sites leads to a simultaneous increase in the pyrene monomer as well as to a decrease in the pyrene excimer fluorescence, whereas the saturation of Ca2+-specific sites results in a slight decrease in the fluorescence of pyrene monomer. Troponin T does not influence the dependence of pyrene-troponin C fluorescence on Ca2+ concentration. Within the equimolar complex of troponin C and troponin I, the saturation of Ca2+, Mg2+-sites has no effect on pyrene fluorescence, whereas the saturation of Ca2+-specific sites leads to a simultaneous decrease of both pyrene monomer and pyrene excimer fluorescence. It is supposed that troponin I diminishes the conformational changes in troponin C that are induced by the saturation of Ca2+, Mg2+-sites and enhances the conformational changes induced by the saturation of Ca2+-specific sites of troponin C.     

Ion-binding to phospholipids. Interaction of calcium and lanthanide ions with phosphatidylcholine (lecithin).

Surface chemical and nuclear magnetic resonance (NMR) techniques have been used to study the interaction of Ca2+ and lanthanides with lecithins. With both methods positive reactions were detected at metal concentrations greater than 0.1 mM. 1H and 31P high-resolution NMR spectra obtained with single bilayer vesicles of lecithin were invariant up to Ca2+ concentrations of 0.1 M indicating that there is only a loose association between Ca2+ and the phospholipid. The weak interaction between Ca2+ and lecithin is confirmed by both surface chemical and NMR techniques showing that the packing of egg lecithin molecules present in bilayers does not change up to Ca2+ concentrations of about 0.1 M. The packing was also independent of pH between 1--10. Contradictory results have been reported in the literature concerning the question of Ca2+ binding to lecithins. The conflicting results are shown to have arisen from differences in the experimental conditions and differences in the sensitivity of the physical methods used by various authors to study Ca2+ -lecithin interactions. An estimate of the strength of binding and molecular details of the interaction were derived using paramagnetic lanthanides as isomorphous replacements for Ca2+. From the changes in chemical shifts induced in the presence of lanthanides an apparent binding constant KA approximately 30 l/mol was calculated at lanthanide concentrations greater than 10 mM. Using surface chemical methods it was shown that this KA is up to 10 times larger than that for Ca2+ binding. The complete assignment of the 1H NMR spectrum of lecithin, including the resonances from the relatively immobilized glycerol group, was determined to derive molecular details of the cation-lecithin interaction. From spin-lattice relaxation-time measurements and line broadening in the presence of GdCl3 it is concluded that the cations are bound to the phosphate group and that this is the only binding site. The absolute proton shifts induced by paramagnetic lanthanides depended on the nature of the ion, but the shift ratios standardised to the shift of the O3POCH2 (choline) signal were invariant throughout the lanthanide series indicating that the shifts are purely pseudocontact. In contrast the 31P shifts were found to contain significant contact contributions. These findings are consistent with a weak interaction and with the phosphate group being the binding site. The absolute shifts but not the shift ratios depended on the anion present indicating that the cation binding may be accompanied by binding of anions. Contrary to negatively charged phospholipids the interaction of lanthanides with lecithins was enhanced as the ionic strength was increased by adding NaCl. This was explained in terms of steric hindrance due to the extended conformation of the lecithin polar group.     

Phosphoinositide synthesis and Ca2+ gating in blowfly salivary glands exposed to 5-hydroxytryptamine.

Blowfly salivary glands, previously exposed to 10 microM-5-hydroxytryptamine for 30 min, demonstrated a rapid compensatory resynthesis of [3H]inositol-labelled phosphatidylinositol 4,5-bisphosphate when allowed to recover in medium containing 3-5 microM-inositol. Phosphatidylinositol 4,5-bisphosphate comprised 70% of the total [3H]-phosphoinositide, and there was a corresponding decrease in the formation of [3H]-phosphatidylinositol. Subsequent addition of 5-hydroxytryptamine produced an equivalent breakdown of the newly synthesized phosphoinositides but little 45Ca2+ gating. Increasing the inositol concentration in the medium to 300 microM produced a 14-fold stimulation of phosphatidylinositol synthesis but only a 5-fold increase in phosphatidylinositol 4,5-bisphosphate synthesis. Increasing the inositol concentration in the medium from 3 microM to 300 microM resulted in a progressively greater recovery of the 45Ca2+-gating response. At 300 microM-inositol there was an 85% recovery of 45Ca2+-gating response. These results indicate that conversion of phosphatidylinositol into phosphatidylinositol 4,5-bisphosphate occurs in blowfly salivary glands and is secondary to an initial breakdown of the phosphoinositides. Recovery of Ca2+ gating is dependent on the restoration of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate to appropriate concentrations.     

Phosphatidylinositol-hydrolysing enzymes in blowfly salivary glands.

The salivary glands of adult blowflies (Calliphora erythrocephala) contain enzymes that hydrolyse phosphatidylinositol, predominantly by a Ca2+-independent deacylation, though a Ca2+-dependent phosphodiesterase (phospholipase C) activity could be detected. The deacylating enzymes could also hydrolyse phosphatidylcholine and phosphatidylethanolamine, and were secreted in the saliva. Homogenization of salivary glands prelabelled with [3H]inositol resulted in a rapid deacylation of the endogenous 3H-labelled phosphatidylinositol; this hydrolysis was unaffected by addition of 5-hydroxytryptamine to the homogenate.     

Substrate polarization in enzyme catalysis: QM/MM analysis of the effect of oxaloacetate polarization on acetyl-CoA enolization in citrate synthase

Citrate synthase is an archetypal carbon-carbon bond forming enzyme. It promotes the conversion of oxaloacetate (OAA) to citrate by catalyzing the deprotonation (enolization) of acetyl-CoA, followed by nucleophilic attack of the enolate form of this substrate on OAA to form a citryl-CoA intermediate and subsequent hydrolysis. OAA is strongly bound to the active site and its alpha-carbonyl group is polarized. This polarization has been demonstrated spectroscopically, [(Kurz et al., Biochemistry 1985;24:452-457; Kurz and Drysdale, Biochemistry 1987;26:2623-2627)] and has been suggested to be an important catalytic strategy. Substrate polarization is believed to be important in many enzymes. The first step, formation of the acetyl-CoA enolate intermediate, is thought to be rate-limiting in the mesophilic (pig/chicken) enzyme. We have examined the effects of substrate polarization on this key step using quantum mechanical/molecular mechanical (QM/MM) methods. Free energy profiles have been calculated by AM1/CHARMM27 umbrella sampling molecular dynamics (MD) simulations, together with potential energy profiles. To study the influence of OAA polarization, profiles were calculated with different polarization of the OAA alpha-carbonyl group. The results indicate that OAA polarization influences catalysis only marginally but has a larger effect on intermediate stabilization. Different levels of treatment of OAA are compared (MM or QM), and its polarization in the protein and in water analyzed at the B3LYP/6-31+G(d)/CHARMM27 level. Analysis of stabilization by individual residues shows that the enzyme mainly stabilizes the enolate intermediate (not the transition state) through electrostatic (including hydrogen bond) interactions: these contribute much more than polarization of OAA.     

菠萝叶片PEP羧激酶与底物结合时构象的变化

菠萝叶片PEP羧激酶与底物OAA和ATP及配基Mn~(2+)等结合时引起紫外差示吸收光谱峰位和方向上的变化。OAA与酶结合诱导产生的差示吸收光谱在268—280nm处有一个宽负峰。ATP与酶结合出现两个差示负峰(242.5和273.5nm)。双底物OAA和ATP同时与酶结合,光谱上呈现252nm和268nm两个峰。Mn~(2+)不论与ATP或与ATP+OAA一起与酶反应时,皆使原来的峰位漂移,且正负方向逆转。酶蛋白在323nm有最大的荧光发射。OAA引起荧光发射强度增大,ATP及ATP+Mn~(2+)则减弱荧光发射。Mn~(2+)与OAA及ATP的复合效应导致荧光强度进一步减弱。