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1.
Enzymatic kinetic parameters of influenza A virus N1 neuraminidases (NA) chromatographically purified from several vaccine candidate strains were tested. With ionic strength held constant, Ca2+ or Mg2+ increased the initial rate of enzymatic activity. The 1934 and 1943 strains had statistically significant highest initial velocities, V(max)/K(m) and V(max). There were no significant differences among the influenza virus strains from 1947 to 1991. Measured K(m) for the 1943 strain (6.2 x 10(-5) M) was significantly higher than other strains (3.1-4.7 x 10(-5) M). V(max)/K(m) varied from 0.78 M(-1) s(-1) to 0.91 M(-1) s(-1) and V(max) varied from 3.0 s(-1) to 5.5 s(-1) before the addition of a divalent cation and increased approximately 2-fold for each of these kinetic parameters for each strain after the addition of exogenous Ca2+ or Mg2+. Dialysis reduced the initial velocity and immunogenicity of each strain with significant differences found among strains. Enzymatic activity and immunogenicity were partially restored by the addition of exogenous Ca2+. Nucleic acid sequence analysis could not predict these differences. Selection of vaccine strains must include analysis of antigenic changes, but also enzymatic studies and determination of the requirement of divalent cations to maintain immunogenicity and activity during production. 相似文献
2.
3.
Schertzer JD Green HJ Duhamel TA Tupling AR 《American journal of physiology. Endocrinology and metabolism》2003,284(3):E597-E610
Prolonged exercise followed by a brief period of reduced activity has been shown to result in an overshoot in maximal sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity [maximal velocity (V(max))] in rat locomoter muscles (Ferrington DA, Reijneveld JC, B?r PR, and Bigelow DJ. Biochim Biophys Acta 1279: 203-213, 1996). To investigate the functional significance and underlying mechanisms for the increase in V(max), we analyzed Ca(2+)-ATPase activity and Ca(2+) uptake in SR vesicles from the fast rat gastrocnemius muscles after prolonged running (RUN) and after prolonged running plus 45 min of low-intensity activity (RUN+) or no activity (REC45) and compared them with controls (Con). Although no differences were observed between RUN and Con, both V(max) and Ca(2+) uptake were higher (P < 0.05) by 43 and 63%, respectively, in RUN+ and by 35 and 34%, respectively, in REC45. The increase in V(max) was accompanied by increases (P < 0.05) in the phosphorylated enzyme intermediate measured by [gamma-(32)P]ATP. No differences between groups for each condition were found for the fluorescent probes FITC and (N-cyclohexyl-N(1)-dimethylamino-alpha-naphthyl)carbodiimide, competitive inhibitors of the nucleotide-binding and Ca(2+)-binding sites on the enzyme, respectively. Similarly, no differences for the Ca(2+)-ATPase were observed between groups in nitrotyrosine and phosphoserine residues, a measure of nitrosylation and phosphorylation states, respectively. Western blots indicated no changes in relative isoform content of sarcoendoplasmic reticulum (SERCA)1 and SERCA2a. It is concluded that the increase in V(max) of the Ca(2+)-ATPase observed in recovery is not the result of changes in enzyme nitroslyation or phosphorylation, changes in ATP and Ca(2+)-binding affinity, or changes in protein content of the Ca(2+)-ATPase. 相似文献
4.
It is shown, that for correct definition of "basal" Ca(2+)-independent Mg(2+)-dependent ATPase ac-activity (10-13 mmol Pi/hour on 1 mg of protein) in a fraction of uterus smooth muscle cell plasma membranes is necessary to use in medium without calcium of an incubation not only EGTA and digitonin--of the factor of infringement in activity by this subcellular structure, but inhibitors of others Mg(2+)-dependent ATP-hydrolyse enzymatic systems localized as in plasma membrane (Na+, K(+)-ATPase) and in others subcellular frames, first of all, in mitochondria (Mg(2+)-ATPase) and endoplasmic reticulum (transport Ca2+, Mg(2+)-ATPase). In the case of a sacolemal fraction of a smooth muscle the contribution of others Mg(2+)-dependent ATP-hydrolyse systems in a common enzymatic hydrolysis ATP, which unconnected to functioning "basal" Ca(2+)-independent Mg(2+)-dependent ATPase, is very appreciable and achieves 35%. The researches, carried out in the frameworks of definition of initial velocity of enzymatic reaction, have enabled to define its some properties--cationic and anionic specificity, and also sensitivity to action of some inhibitors. It has appeared, that the "basal" Ca(2+)-independent Mg(2+)-dependent ATP-hydrolyse reaction is nonspecific rather both in relation to cations of divalent metals Me2+, and cations of monovalent metals and anions, which were utilized for support of ionic strength. The cations La--antagonist of cations Ca--practically did not influence enzymatic activity. The non-specific inhibitors transport of ATPases--p-chloromercuribenzoate, o-vanadate and eosine Y with a various degree of efficiency inhibited "basal" Ca(2+)-independent Mg(2+)-dependent ATP-hydrolyse reaction. On the basis of the analysis of the own and literary data the conclusion is made that "basal" Ca(2+)-independent Mg(2+)-dependent ATPase of a smooth muscle cell plasma membrane is considerably less sensitive to action of nonspecific inhibitors of the Ca(2+)-transporting systems, than these systems. 相似文献
5.
Green HJ Burnett M Duhamel TA D'Arsigny C O'Donnell DE Webb KA Ouyang J 《American journal of physiology. Cell physiology》2008,295(2):C350-C357
The objective of this study was to investigate the hypothesis that alterations in sarcoplasmic reticulum (SR) Ca(2+)-cycling properties would occur in skeletal muscle in patients with moderate to severe chronic obstructive pulmonary disease (COPD). To investigate this hypothesis, tissue samples were obtained from the vastus lateralis of 8 patients with COPD [age 65.6 +/- 3.2 yr; forced expiratory volume in 1 s (FEV(1))/forced vital capacity (FVC) = 44 +/- 2%; mean +/- SE] and 10 healthy age-matched controls (CON, age 67.5 +/- 2.5 yr; FEV(1)/FVC = 77 +/- 2%), and homogenates were analyzed for a wide range of SR properties. Compared with CON, COPD displayed (in mumol.g protein(-1).min(-1)) a 16% lower maximal Ca(2+)-ATPase activity [maximal velocity (V(max)), 158 +/- 10 vs. 133 +/- 7, P < 0.05] and a 17% lower Ca(2+) uptake (4.65 +/- 0.039 vs. 3.85 +/- 0.26, P < 0.05) that occurred in the absence of differences in Ca(2+) release. The lower V(max) in COPD was also accompanied by an 11% lower (P < 0.05) Ca(2+) sensitivity, as measured by the Hill coefficient (defined as the relationship between Ca(2+)-ATPase activity and free cytosolic Ca(2+) concentration for 10-90% V(max)). For the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms, SERCA1a was 16% higher (P < 0.05) and SERCA2a was 14% lower (P < 0.05) in COPD. It is concluded that moderate to severe COPD results in abnormalities in SR Ca(2+)-ATPase properties that cannot be explained by changes in the SERCA isoform phenotypes. The reduced catalytic properties of SERCA in COPD suggest a disturbance in Ca(2+) cycling, possibly resulting in impairment in Ca(2+)-mediated mechanical function and/or second messenger regulated processes. 相似文献
6.
Characterization of cadmium uptake in Lactobacillus plantarum and isolation of cadmium and manganese uptake mutants. 总被引:1,自引:0,他引:1
Two different Cd(2+) uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn(2+) uptake system which also takes up Cd(2+) and is induced by Mn(2+) starvation. The calculated K(m) and V(max) are 0.26 microM and 3.6 micromol g of dry cell(-1) min(-1), respectively. Unlike Mn(2+) uptake, which is facilitated by citrate and related tricarboxylic acids, Cd(2+) uptake is weakly inhibited by citrate. Cd(2+) and Mn(2+) are competitive inhibitors of each other, and the affinity of the system for Cd(2+) is higher than that for Mn(2+). The other Cd(2+) uptake system is expressed in Mn(2+)-sufficient cells, and no K(m) can be calculated for it because uptake is nonsaturable. Mn(2+) does not compete for transport through this system, nor does any other tested cation, i.e., Zn(2+), Cu(2+), Co(2+), Mg(2+), Ca(2+), Fe(2+), or Ni(2+). Both systems require energy, since uncouplers completely inhibit their activities. Two Mn(2+)-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn(2+) for growth as the parental strain. Mn(2+) starvation-induced Cd(2+) uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn(2+) or Cd(2+) accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn(2+) and Cd(2+) uptake system. 相似文献
7.
The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex. 相似文献
8.
A peroxidase (PD-cP; 0.47 mg/100 g leaves) was purified from autumn leaves of Phytolacca dioica L. and characterized. PD-cP was obtained by acid precipitation followed by gel-filtration and cation exchange chromatography. Amino acid composition and N-terminal sequence of PD-cP up to residue 15 were similar to that of Spinacia oleracea (N-terminal pairwise comparison showing four amino acid differences). PD-cP showed a molecular mass of approx. 36 kDa by SDS-PAGE, pH and temperature optima at 3.0 and 50.0°C, respectively and seasonal variation. The Michaelis-Menten constant (K(M)) for H(2)O(2) was 5.27 mM, and the velocity maximum (V(max)) 1.31 nmol min(-1), while the enzyme turnover was 0.148 s(-1). Finally, the presence of Ca(2+) and Mg(2+) enhanced the PD-cP activity, with Mg(2+) 1.4-fold more effective than Ca(2+) 相似文献
9.
Yolanda Gutiérrez-Martín Francisco Javier Martín-Romero Fernando Henao Carlos Gutiérrez-Merino 《Free radical biology & medicine》2002,32(1):46-55
A sustained increase of intracellular free [Ca(2+)] ([Ca(2+)](i)) has been shown to be an early event of neuronal cell death induced by peroxynitrite (ONOO(-)). In this paper, chronic exposure to ONOO(-) has been simulated by treatment of rat brain synaptosomes or plasma membrane vesicles with repetitive pulses of ONOO(-) during at most 50 min, which efficiently produced nitrotyrosine formation in several membrane proteins (including the Ca(2+)-ATPase). The plasma membrane Ca(2+)-ATPase activity at near-physiological conditions (pH 7, submicromolar Ca(2+), and millimolar Mg(2+)-ATP concentrations), which plays a major role in the control of synaptic [Ca(2+)](i), can be more than 75% inhibited by a sustained exposure to micromolar ONOO(-) (e.g., to 100 pulses of 10 microM ONOO(-)). This inhibition is irreversible and mostly due to a decreased V(max), and to the 2-fold increase of the K(0.5) for Ca(2+) stimulation and about 5-fold increase of the K(M) for Mg(2+)-ATP. [Ca(2+)](i) increases to >400 nM when synaptosomes are subjected to this treatment. Reduced glutathione can afford only partial protection against the inhibition produced by micromolar ONOO(-) pulses. Therefore, inhibition of the plasma membrane Ca(2+)-pump activity during chronic exposure to ONOO(-) may account by itself for a large and sustained increase of intracellular [Ca(2+)](i) in synaptic nerve terminals. 相似文献
10.
T A Duhamel R D Stewart A R Tupling J Ouyang H J Green 《Journal of applied physiology》2007,103(4):1212-1220
The study investigated the hypothesis that three consecutive days of prolonged cycle exercise would result in a sustained reduction in the Ca(2+)-cycling properties of the vastus lateralis in the absence of changes in the sarcoplasmic (endoplasmic) reticulum Ca(2+)-ATPase (SERCA) protein. Tissue samples were obtained at preexercise (Pre) and postexercise (Post) on day 1 (E1) and day 3 (E3) and during recovery day 1 (R1), day 2 (R2), and day 3 (R3) in 12 active but untrained volunteers (age 19.2 +/- 0.27 yr; mean +/- SE) and analyzed for changes (nmol.mg protein(-1).min(-1)) in maximal Ca(2+)-ATPase activity (V(max)), Ca(2+) uptake and Ca(2+) release (phase 1 and phase 2), and SERCA isoform expression (SERCA1a and SERCA2a). At E1, reductions (P < 0.05) from Pre to Post in V(max) (150 +/- 7 vs. 121 +/- 7), Ca(2+) uptake (7.79 +/- 0.28 vs. 5.71 +/- 0.33), and both phases of Ca(2+) release (phase 1, 20.3 +/- 1.3 vs. 15.2 +/- 1.1; phase 2, 7.70 +/- 0.60 vs. 4.99 +/- 0.48) were found. In contrast to V(max), which recovered at Pre E3 and then remained stable at Post E3 and throughout recovery, Ca(2+) uptake remained depressed (P < 0.05) at E3 Pre and Post and at R1 as did phase 2 of Ca(2+) release. Exercise resulted in an increase (P < 0.05) in SERCA1a (14% at R2) but not SERCA2a. It is concluded that rapidly adapting mechanisms protect V(max) following the onset of regular exercise but not Ca(2+) uptake and Ca(2+) release. 相似文献
11.
Moreno-Enriquez A Evangelista-Martinez Z Gonzalez-Mondragon EG Calderon-Flores A Arreguin R Perez-Rueda E Huerta-Saquero A 《Journal of microbiology and biotechnology》2012,22(3):292-300
We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters K(m), V(max), and k(cat) for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at 50 degrees C, but the optimal temperature of activity was 37 degrees C. It also showed maximal and optimal activities at pH 9.0. The values of K(m), V(max), k(cat), and k(cat)/K(m) were 8.9 +/- 0.967 × 10?3 M, 128 +/- 2.8 U/mg protein, 106 +/- 2 s?1, and 1.2 +/- 0.105 × 10? M?1s?1, respectively. The L-asparaginase activity was reduced in the presence of Mn2?, Zn2?, Ca2?, and Mg2? metal ions for about 52% to 31%. In addition, we found that NH??, L-Asp, D-Asn, and beta-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobialtype asparaginase II family. 相似文献
12.
The action of sodium nitroprusside, nitrite-anions and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (Ca(2+)-independent) enzymatic activity in myometrium sarcolemma fraction is investigated. It is established, that 0.1 mM sodium nitroprusside and 10(-8)-10(-5) M nitrite-anions essentially reduce Ca2+, Mg(2+)-ATPase activity whereas Mg(2+)-ATPase proved to be absolutely resistant to them. At rather high concentration of nitrite-anions (0.1 mM) appreciable stimulation of Ca2+, Mg(2+)-ATPase was observed. Hydrogen peroxide (10(-8)-10(-4)), depending on the concentration suppressed both enzymes activity. However, Ca2+, Mg(2+)-ATPase proved to be more sensitive to the action of H2O2 (seeming K(i) = 0.42 +/- 0.1 microM), than Mg(2+)-ATPase (seeming K(i) = 3.1 +/- 0.9 microM). At presence of 1 mM ditiothreitole (a reducer of SH groups of the membrane surface) action of investigated substances considerably decreased. Reagents on carboxic- (dicyclogexilcarbodiimid) and amino- groups of the membrane (trinitrobenzolsulfonic acid) inhibited both Ca2+, Mg(2+)-ATPase, and Mg(2+)-ATPase activity in membrane fractions. In the presence of noted reagents sodium nitroprusside and nitrite-anions action was not almost shown. Hence, nitrogen oxide, nitrite-anions and hydrogen peroxide suppress Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (only hydrogen peroxide) activity in the plasmatic membrane of myometrium cells, and this action can be connected with direct updating of superficial chemical groups of the membrane. 相似文献
13.
The enzymatic properties of an ecto-5'-nucleotidase were described in Trichomonas vaginalis. The enzyme hydrolyzes nucleoside monophosphates in optimum pH values of 7.5 and 6.5 for the 30236 strain and for the 30238 strain, respectively. Mg(2+) and Ca(2+) were activators of AMP hydrolysis in both strains. The apparent K(m) (Michaelis constant) values for Mg(2+)-AMP were 111.4+/-28.1 microM (mean+/-SD, n=3) for 30236 strain and 420.2+/-35.7 microM (mean+/-SD, n=3) for 30238 strain. The ecto-5'-nucleotidase activity was insensitive to levamisole and tetramisole, inhibitors of alkaline phosphatases, whereas alpha,beta-methylene-ADP inhibited the enzymatic activity of both strains. Our results showed that the AMP hydrolysis presents differences in some kinetic parameters between the two strains investigated. An analysis of the enzymatic chain involved in the ATP hydrolysis to adenosine will contribute to understanding the biochemical aspects of the parasite and the mechanisms related to host-parasite interactions. 相似文献
14.
In this paper, a novel and unique ginsenoside Rg(1)-hydrolyzing β-D-glucosidase from Penicillium sclerotiorum was isolated, characterized, and generally described. The β-glucosidase is an ~180 kDa glycoprotein with pI 6.5, and consists of four identical subunits of ~40 kDa. The β-glucosidase was active in a narrow pH range (4-5) and at relatively high temperature (60-70°C). The optimal activity against p-nitrophenyl-β-D-glucopyranoside (pNPG) was as follows: pH 4.5 and temperature 65°C. Under these conditions, the K(m) of the enzyme was 0.715 mM with a V(max) of 0.243 mmol nitrophenol/min mg. Metal ions such as Ba(2+), K(+), Fe(3+), and Co(2+) significantly promoted the enzymatic activity, while Ca(2+), Mg(2+), and Ag(+) inhibited its activity. Of the tested substrates, only ginsenoside Rg(1) could be specifically hydrolyzed by the β-glucosidase at the C6-glucoside to form the rare ginsenoside F(1). These properties were novel and different from those of other previously described glycosidases. 相似文献
15.
The permeability transition pore as a Ca(2+) release channel: new answers to an old question 总被引:1,自引:0,他引:1
Mitochondria possess a sophisticated array of Ca(2+) transport systems reflecting their key role in physiological Ca(2+) homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca(2+) uptake, the ruthenium red (RR)-sensitive mitochondrial Ca(2+) uniporter (MCU); and one main mechanism for Ca(2+) release, the RR-insensitive 3Na(+)-Ca(2+) antiporter. An additional mechanism for Ca(2+) release is provided by a Na(+) and RR-insensitive release mechanism, the putative 3H(+)-Ca(2+) antiporter. A potential kinetic imbalance is present, however, because the V(max) of the MCU is of the order of 1400nmol Ca(2+)mg(-1) proteinmin(-1) while the combined V(max) of the efflux pathways is about 20nmol Ca(2+)mg(-1) proteinmin(-1). This arrangement exposes mitochondria to the hazards of Ca(2+) overload when the rate of Ca(2+) uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca(2+)]. In this short review we discuss the hypothesis that transient opening of the Ca(2+)-dependent permeability transition pore may provide mitocondria with a fast Ca(2+) release channel preventing Ca(2+) overload. We also address the relevance of a mitochondrial Ca(2+) release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals. 相似文献
16.
The C-terminal segment of the loop between transmembrane helices 2 and 3 (A(L) region) of the plasma membrane Ca(2+) pump (PMCA) is not conserved in other P-ATPases. Part of this region, just upstream from the third transmembrane domain, has been associated with activation of the PMCA by acidic lipids. cDNAs coding for mutants of the Ca(2+) pump isoform h4xb with deletions in the A(L) region were constructed, and the proteins were successfully expressed in either COS or Chinese hamster ovary cells. Mutants with deletions in the segment 296-349 had full Ca(2+) transport activity, but deletions involving the segment of amino acids 350-356 were inactive suggesting that these residues are required for a functional PMCA. In the absence of calmodulin the V(max) of mutant d296-349 was similar to that of the recombinant wild type pump, but its K(0.5) for Ca(2+) was about 5-fold lower. The addition of calmodulin increased the V(max) and the apparent Ca(2+) affinity of both the wild type and d296-349 enzymes indicating that the activating effects of calmodulin were not affected by the deletion. At low concentrations of Ca(2+) and in the presence of saturating amounts of calmodulin, the addition of phosphatidic acid increased about 2-fold the activity of the recombinant wild type pump. In contrast, under these conditions phosphatidic acid did not significantly change the activity of mutant d296-349. Taken together these results suggest that (a) deletion of residues 296-349 recreates a form of PMCA similar to that resulting from the binding of acidic lipids at the A(L) region; (b) the A(L) region acts as an acidic lipid-binding inhibitory domain capable of adjusting the Ca(2+) affinity of the PMCA to the lipid composition of the membrane; and (c) the function of the A(L) region is independent of the autoinhibition by the C-terminal calmodulin-binding region. 相似文献
17.
The effects of Mg(2+) and K(+) ions on the self-splicing inhibition of the td (thymidylate synthase gene) intron RNA by spectinomycin were investigated. The maximum splicing activity occurred at 20 mM KCl. The K(m) and V(max) values for GTP in the presence of 5 mM Mg(2+) are 2.25 microM and 0.55 min(-1), whereas those for GTP both in the presence of 5 mM Mg(2+) and 5 mM K(+) are 1.23 microM and 0. 46 min(-1), respectively. Spectinomycin at 10 mM concentration inhibited the splicing by about 10%, but at 20 mM concentration, the splicing rate was inhibited by about 63%. The splicing inhibition by the low concentration of spectinomycin was overcome markedly as the concentration of Mg(2+) ion was raised. At 30 mM spectinomycin, however, the splicing inhibition was not significantly affected by increasing the concentration of Mg(2+). A similar activation of the splicing rate was observed as the concentration of K(+) ion was increased. The concentration of K(+) ion required for the normal recovery of the splicing was much higher than that of Mg(2+) ion. Unlike Mg(2+) ion, 30 mM K(+) ion effectively alleviated the splicing inhibition by spectinomycin at its high concentration. The results indicate that K(+) and Mg(2+) ions may show mechanistically different interactions with spectinomycin in the self-splicing reaction of the td intron RNA. 相似文献
18.
Anandamide, an endogenous cannabinoid receptor ligand, was rapidly metabolized by Tetrahymena pyriformis in vivo. Metabolic products were mainly phospholipids as well as neutral lipids, including small amounts of free arachidonic acid. Anandamide amidohydrolase activity was detected in the culture medium by the release of [3H]arachidonic acid from [3H]anandamide, in a time- and concentration-dependent manner. Kinetic experiments demonstrated that the released enzyme had an apparent K(m) of 3.7 microM and V(max) 278 pmol/min/mg protein. Amidohydrolase activity was maximal at pH 9-10, was abolished by phenylmethylsulfonyl fluoride and was Ca(2+)- and Mg(2+)-independent. Thus, T. pyriformis is capable of hydrolyzing anandamide in vivo and releasing amidohydrolase activity. 相似文献
19.
Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site. 相似文献
20.
Braga CA Pinto JR Valente AP Silva JL Sorenson MM Foguel D Suarez MC 《The international journal of biochemistry & cell biology》2006,38(1):110-122
The C-domain of troponin C, the Ca(2+)-binding subunit of the troponin complex, has two high-affinity sites for Ca(2+) that also bind Mg(2+) (Ca(2+)/Mg(2+) sites), whereas the N-domain has two low-affinity sites for Ca(2+). Two more sites that bind Mg(2+) with very low affinity (K(a)<10(3)M(-1)) have been detected by several laboratories but have not been localized or studied in any detail. Here we investigated the effects of Ca(2+) and Mg(2+) binding to isolated C-domain, focusing primarily on low-affinity sites. Since TnC has no Trp residues, we utilized a mutant with Phe 154 replaced by Trp (F154W/C-domain). As expected from previous reports, the changes in Trp fluorescence revealed different conformations induced by the addition of Ca(2+) or Mg(2+) (Ca(2+)/Mg(2+) sites). Exposure of hydrophobic surfaces of F154W/C-domain was monitored using the fluorescence intensity of bis-anilino naphthalene sulfonic acid. Unlike the changes reported by Trp, the increments in bis-ANS fluorescence were much greater (4.2-fold) when Ca(2+)+Mg(2+) were both present or when Ca(2+) was present at high concentration. Bis-ANS fluorescence increased as a function of [Ca(2+)] in two well-defined steps: one at low [Ca(2+)], consistent with the Ca(2+)/Mg(2+) sites (K(a) approximately 1.5 x 10(6)M(-1)), and one of much lower affinity (K(a) approximately 52.3M(-1)). Controls were performed to rule out artifacts due to aggregation, high ionic strength and formation of the bis-ANS-TnC complex itself. With a low concentration of Ca(2+) (0.6mM) to occupy the Ca(2+)/Mg(2+) sites, a large increase in bis-ANS binding also occurred as Mg(2+) occupied a class of low-affinity sites (K(a) approximately 59 M(-1)). In skinned fibers, a high concentration of Mg(2+) (10-44 mM) caused TnC to dissociate from the thin filament. These data provide new evidence for a class of weak binding sites for divalent cations. They are located in the C-domain, lead to exposure of a large hydrophobic surface, and destabilize the binding of TnC to the regulatory complex even when sites III and IV are occupied. 相似文献