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1.
In addition to the (Na ++K +)ATPase another P-ATPase, the ouabain-insensitive Na +-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg 2+, MgATP and furosemide on the Na +-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na +-ATPase at pH 7.0 are: (a) K0.5 for Na +=8.9±2.2 mM; (b) K0.5 for MgATP=1.8±0.4 mM; (c) two sites for free Mg 2+: one stimulatory ( K0.5=0.20±0.06 mM) and other inhibitory ( I0.5=1.1±0.4 mM); and (d) I0.5 for furosemide=1.1±0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na + ( K0.5=19.5±0.4) and MgATP ( K0.5=3.4±0.3 mM) but increases the apparent affinity for furosemide (0.18±0.02 mM) and Mg 2+ (0.05±0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na + ( K0.5=18.7±1.5 mM) and furosemide ( I0.5=3.04±0.57 mM) but does not change the apparent affinity to MgATP and Mg 2+. The data presented in this paper indicate that the modulation of the Na +-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg 2+ and MgATP complex may play an important role in the Na +-ATPase physiological regulatory mechanisms. 相似文献
2.
Chaetomium thermophilum CT2 produced endocellulases at 50 °C, when grown on 2% microcrystalline cellulose, 1% soluble starch, and 0.4% yeast extract medium. A major endocellulase component was purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography and gel filtration on Sephacryl S-100. The molecular weight of the enzyme was estimated to be 67.8 kDa and the enzyme was found to be a glycoprotein containing 18.9% carbohydrate. The Km of the purified enzyme for carboxymethyl cellulose, sodium salt (CMC), was 4.6 mg ml −1. The enzyme displayed highest activity towards CMC and significantly lower activities towards phosphoric acid swollen cellulose and filter paper. The activity was enhanced in the presence of Na +, K + and Ca 2+ but inhibited by Hg 2+, Zn 2+, Ag +, Mn 2+, Ba 2+, Fe 2+, Cu 2+, Mg 2+ and NH 4+. Optimum activity was at 60 °C and pH 4.0. The enzyme was stable over 60 min incubation at 60 °C and half-life at 70, 80 and 90 °C was approximately 45, 24 and 7 min, respectively. 相似文献
3.
Recently, we demonstrated that angiotensin-(1–7) (Ang-(1–7)) stimulates the Na +-ATPase activity through a losartan-sensitive angiotensin receptor, whereas bradykinin inhibits the enzyme activity through the B 2 receptor [Regul. Pept. 91 (2000) 45; Pharmacol. Rev. 32 (1980) 1]. In the present paper, the effect of bradykinin (BK) on Ang-(1–7)-stimulated Na +-ATPase activity was evaluated. Preincubation of Na +-ATPase with 10 −9 M Ang-(1–7) increases enzyme activity from 7.9±0.9 to 14.1±1.5 nmol Pi mg −1 min −1, corresponding to an increase of 79% ( p<0.05). This effect is reverted by bradykinin in a dose-dependent manner (10 −14–10 −8 M), reaching maximal inhibitory effect at 10 −9 M. Des-Arg 9 bradykinin (DABK), an agonist of B 1 receptor, at the concentrations of 10 −9–10 −7 M, does not mimic the BK inhibitory effect, and des-Arg 9-[Leu 8]-BK (DALBK), a B 1 receptor antagonist, at the concentrations of 10 −10–10 −7 M, does not prevent the inhibitory effect of BK on Ang-(1–7)-stimulated enzyme. On the other hand, HOE 140, an antagonist of B 2 receptor, abolishes the inhibitory effect of BK on the Ang-(1–7)-stimulated enzyme in a dose-dependent manner, reaching maximal effect at 10 −7 M. Taken together, these data indicate that stimulation of B 2 receptors by BK can counteract the stimulatory effect of Ang-(1–7) on the proximal tubule Na +-ATPase activity. 相似文献
4.
The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg DCW−1 (dry cell weight). The kinetic parameters, Km and Vmax, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min −1 mg DCW−1, respectively. The measured apparent activation energy, 25.54 kJ mol −1, indicated a partial control by mass transport, more likely through the cell wall. UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg
ml−1) indicated: lower diffusional control (Ea=37.73 kJ mol−1); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg
ml−1 of resting cells, with a maximum volumetric productivity of 0.5 g l−1 h−1. 相似文献
5.
We examined the mechanism through which leptin increases Na +, K +-ATPase activity in the rat kidney. Leptin was infused under anaesthesia into the abdominal aorta proximally to the renal arteries and then Na +, K +-ATPase activity was measured in the renal cortex and medulla. Leptin (1 μg/kg min) increased Na +, K +-ATPase activity after 3 h of infusion, which was accompanied by the increase in urinary H 2O 2 excretion and phosphorylation level of extracellular signal regulated kinase (ERK). The effect of leptin on ERK and Na +, K +-ATPase was abolished by catalase, specific inhibitors of epidermal growth factor (EGF) receptor, AG1478 and PD158780, as well as by ERK inhibitor, PD98059, and was mimicked by both exogenous H 2O 2 and EGF. The effect of leptin was also prevented by the inhibitor of Src tyrosine kinase, PP2. Leptin and H 2O 2 increased Src phosphorylation at Tyr 418. We conclude that leptin-induced stimulation of renal Na +, K +-ATPase involves H 2O 2 generation, Src kinase, transactivation of the EGF receptor, and stimulation of ERK. 相似文献
6.
The H +-ATPase from chloroplasts, CF 0F 1, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid—base transition ΔpH (pH in = 5.0, pH out = 8.5) and a K +/valinomycin diffusion potential, Δφ (K +in = 0.6 mM, K +out = 60 mM). A rate of 250 s −1 was observed with the reduced enzyme (85 s −1 in the absence of Δφ). A rate of 50 s −1 was observed with the oxidized enzyme under the same conditions (15 s −1 in the absence of Δφ). The reconstituted enzyme contained 2 ATP bound per CF 0F 1 and 1 ADP bound per CF 0F 1. Upon energisation the enzyme was activated and 0.9 ADP per CF 0F 1, was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 10 5 M −1·s −1 under energized and de-energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de-energized conditions. 相似文献
7.
A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg 2+ ions. Ca 2+, dithiothreitol, and Mn 2+ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave Vmax and Km values of 154 U mg −1 and 1.3 mg ml −1. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date. 相似文献
8.
1. 1. (Mg2+ + Ca2+) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg2+ + Ca2+) ATPase seen during development was greatest in the synaptic membrane preparations. 2. 2. At 37°C both Na+ or K+ at concentrations higher than 30 mM inhibited the microsomal Mg2+ ATPase, but the (Mg2+ + Ca2+) ATPase was stimulated by both Na+ and K+. Synaptic membrane Mg2+ ATPase was inhibited by concentrations higher than 100 mM K+; Na+ however stimulated this enzyme at all concentrations. Much of this Na+ stimulated activity was ouabain sensitive. Synaptic membrane (Mg2+ + Ca2+) ATPase was stimulated by Na+ or K+, this stimulation follows approximate saturation kinetics with an apparent Km of 18.8 mM Na+ or K+. 3. 3. Arrhenius plots of microsomal (Mg2+ + Ca2+) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole−1 above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg2+ + Ca2+) ATPase from both immature and adult brain.
Author Keywords: Brain; ATPase; temperature; development; synaptic membranes 相似文献
9.
Reconstituted Na +,K +-ATPase from either pig kidney or shark rectal glands was phosphorylated by cAMP dependent protein kinase, PKA. The stoichiometry was 0.9 mole P i/mole -subunit in the pig kidney enzyme and 0.2 mol P i/mol -subunit in the shark enzyme. In shark Na +,K +-ATPase PKA phosphorylation increased the maximum hydrolytic activity for cytoplasmic Na + activation and extracellular K + activation without affecting the apparent Km values. In contrast, no significant functional effect after PKA phosphorylation was observed in pig kidney Na +,K +-ATPase. 相似文献
10.
Isolated rat hepatocytes posses a saturable glucocorticoid uptake system with high affinity ( Kd value = 2.8 ± 0.7 × 10 −8 M; 318,000 ± 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30–40% if the cells are incubated simultaneously with [ 3H]corticosterone and either SH-reagents ( N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na +/K +-ATPase-inhibitors, ouabain and quercetine. These Na +/K +-ATPase-blockers exert half-maximal inhibition at 3 × 10 −7 and 3 × 10 −6 M, respectively. A slight increase in K + concentration and a corresponding decrease in Na + in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5- and 5β-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake. 相似文献
11.
The role of Na + in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na +-motive respiratory chain of V. alginolyticus. Na + loaded cells incubated in a K + or Li + medium fail to synthesize ATP in response to lactate addition. The addition of Na + at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na + level. Neither lactate oxidation nor Δω generation coupled with this oxidation is increased by external Na + in the Na +-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse ΔP Na, i.e., [Na +] in > [Na +] out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K +-loaded cells incubated in a high Na + medium, i.e., when Δp Na of the proper direction ([Na +] in < [Na +] out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K + medium (Δp Na is low) is decreased by CCCP even without monensin. Artificial formation of Δp Na by adding 0.25 M NaCl to the K +-loaded cells (Na + pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na + pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na + instead of H + as the coupling ion. 相似文献
12.
Na +-dependent uptake of excitatory neurotransmitter glutamate in astrocytes increases cell energy demands primarily due to the elevated ATP consumption by glutamine synthetase and Na +, K +-ATPase. The major pool of GLAST/EAAT1, the only glutamate transporter subtype expressed by human fetal astrocytes in undifferentiated cultures, was restricted to the cytoplasmic compartment. Elevated glutamate concentrations (up to 50 μM) stimulated both glutamate uptake and Na +, K +-ATPase activity and concomitantly increased cell surface expression of GLAST and FXYD2/γ subunit of Na +, K +-ATPase. Intracellular accumulation of glutamate or its metabolites per se was not responsible for these changes since metabolically inert transport substrate, d-aspartate, exerted the same effect. Nanomolar concentrations of TFB-TBOA, a novel nontransportable inhibitor of glutamate carriers, almost completely reversed the action of glutamate or d-aspartate. In the same conditions (i.e. block of glutamate transport) monensin, a potent Na + ionophore, had no significant effect neither on the activation of Na +, K +-ATPase nor on the cell surface expression of γ subunit or GLAST. In order to elucidate the roles of γ subunit in the glutamate uptake-dependent trafficking events or the activation of the astroglial sodium pump, in some cultures γ subunit/FXYD2 was effectively knocked down using siRNA silencing. Unlike the blocking effect of TFB-TBOA, the down-regulation of γ subunit had no effect neither on the trafficking nor activity of GLAST. However, the loss of γ subunit effectively abolished the glutamate uptake-dependent activation of Na +, K +-ATPase. Following withdrawal of siRNA from cultures, the expression levels of γ subunit and the sensitivity of Na +, K +-ATPase to glutamate/aspartate uptake have been concurrently restored. Thus, the activity of GLAST directs FXYD2 protein/γ subunit to the cell surface, that, in turn, leads to the activation of the astroglial sodium pump, presumably due to the modulatory effect of γ subunit on the kinetic parameters of catalytic subunit(s) of Na +, K +-ATPase. 相似文献
13.
To quantitatively understand intracellular Na + and Cl − homeostasis as well as roles of Na +/K + pump and cystic fibrosis transmembrane conductance regulator Cl − channel ( ICFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na +, K +, Ca 2+ and Cl −), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca 2+ transient and cell shortening, and the decreases in both Cl − concentration and cell volume. These results are consistent with experimental data. Increasing the density of ICFTR shortened APD and augmented the peak amplitudes of the L-type Ca 2+ current ( ICaL) and the Ca 2+ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of ICaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na + during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na + affinity of Na +/K + pump by protein kinase A. However it was predicted that Na + increases at higher beating rate because of larger Na + influx through forward Na +/Ca 2+ exchange. It was demonstrated that dynamic changes in Na + and Cl − fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes. 相似文献
14.
Cordyceps militaris mycelium produced mainly Cu, Zn containing superoxide dismutase (Cu, Zn-SOD). Cu, Zn-SOD activity was detectable in the culture filtrates, and intracellular Cu, Zn-SOD activity as a proportion protein was highest in early log phase culture. The effects of Cu 2+, Zn 2+, Mn 2+ and Fe 2+ on enzyme biosynthesis were studied. The Cu, Zn-SOD was isolated and purified to homogeneity from C. militaris mycelium and partially characterized. The purification was performed through four steps: (NH 4) 2SO 4 precipitation, DEAE-sepharose™ fast flow anion-exchange chromatography, CM-650 cation-exchange chromatography, and Sephadex G-100 gel filtration chromatography. The purified enzyme had a molecular weight of 35070 ± 400 Da and consisted of two equal-sized subunits each having a Cu and Zn element. Isoelectric point value of 7.0 was obtained for the purified enzyme. The N-terminal amino acid sequence of the purified enzyme was determined for 12 amino acid residues and the sequences was compared with other Cu, Zn-SODs. The optimum pH of the purified enzyme was obtained to be 8.2–8.8. The purified enzyme remained stable at pH 5.8–9.8, 25 °C and up to 50 °C at pH 7.8 for 1.5 h incubation. The purified enzyme was sensitive to H 2O 2, KCN. 2.5 mM NaN 3, PMSF, Triton X-100, β-mercaptoethanol and DTT showed no significant inhibition effect on the purified enzyme within 5 h incubation period. 相似文献
15.
Steady-state current-voltage relationships (SSCVRs) of the plasma membrane of human T-lymphocytes were studied at the physiological temperature of 37°C by using the whole-cell patch-clamp technique. SSCVRs displayed a characteristic N-like shape with a negative resistance region (NRR) in a voltage range of −45 to −35 mV. The majority of cells assayed revealed SSCVR patterns crossing the V-axis at three points (in mV): V1 = −55 to −45, V2 = −40 to −35, V3 = −30 to −10. SSCVRs of T-cells activated by phytohaemagglutinin (48–96 h) also displayed NRR, but crossed the V-axis at one point only ( V1 = −55 to −60 mV). It implies the possibility of two stable levels of membrane potential ( V1 and V3) for the resting T-cells, but only one ( V1) for activated T-cells. These data thus account for the triggering property of T-cell membrane potential previously reported. The NRR can be explained on the basis of the Hodgkin-Huxley type n4j model of K + channel kinetics. According to the model the possibility for a membrane to have on or two stable levels of membrane potential depends on the ratio of selective K + conductance to non-selective leaky conductance ( Gk/ Gleak). The steady-state level of K + conductance in resting T-lymphocytes proved to be sensitive to Ca 2+. Buffering Ca 2+ ions from either external or internal solution resulted in an appreciable increase in K + conductance. The possibility for membrane potential have two stable levels of membrane potential in connection with the Ca 2+ dependence of K + conductance was supposed to be important for Ca 2+-signalling during T-cell activation. 相似文献
16.
The effect of extracellular Na + ([Na +] e) removal on agonist-induced granule secretion in platelets in relation to [ph] i and [Ca 2+] i changes was investigated. Substitution of [Na +] e with choline + of K + resulted in a significant enhancement of 5HT secretion induced by thrombin, collagen, U46619 and the protein kinase C activators, PMA and diC 8. Increases in [Ca 2+] i induced by thrombin and U46619 were slightly inhibited or unaffected in these buffers, but [pH] i increases induced by thrombin, U46619, PMA and diC 8 were abolished and a drop in [pH] i (0.05–0.1 units below resting) was observed. Although preincubation with potassium acetate produced a big drop in [pH] i and greatly increased secretion with all the agonists, particularly in the absence of [Na +] e, clear evidence that [pH] i rises due to Na +/H + exchange are inhibitory to secretion was obtained only with thrombin. Thus, (i) NH 4Cl, which restored the increase in [pH] i in the absence of [Na +] e reduced the potentiated secretory response to thrombin, (ii) no increase in thrombin-induced secretion was observed when Na + was replaced with Li +, which allowed a normal increase in [pH] i and (iii) ethyl isopropyl amiloride (EIPA) abolished the [pH] i rise and potentiated thrombin-induced secretion. With collagen and U46619, the results suggest that removal of [Na +] e per se rather than inhibition of Na +/H + exchange results in enhanced secretion. It is concluded that [Na +] e per se and [pH] i elevations via Na +/H + exchange both have important inhibitory roles in the control of platelet granule secretion. 相似文献
17.
Changes in plasmalemma K +Mg 2+-ATPase dephosphorylating activity and H + transport were examined in freezing-tolerant and non-tolerant genotypes of the perennial grass species Festuca pratensis Huds. Enzyme activity and ΔμH + were measured in plasmalemma fractions isolated from basal nodes and roots. Three types of experiments were undertaken: (i) a field experiment, utilizing the seasonal growth and cessation cycle of a perennial plant; (ii) a cold acclimation experiment in hydroponics; and (iii) an instant freezing test. A specific fluctuation in K +Mg 2+-ATPase activity was found throughout the seasonal growth of the plants (i). The K +Mg 2+-ATPase activity peaks for both the basal node and the root plasmalemma were determined early in the spring before the renewal of growth. The lowest activity values in roots occurred at the time approaching flowering, and in basal nodes at the transition into the growth cessation. The K +Mg 2+-ATPase activity was approximately 50% lower in the basal node plasmalemma of freezing-tolerant plants than of non-tolerant ones, when assessed at the optimal growth stage in hydroponics. In hydroponics (ii) and in the freezing test (iii), temperature stress was followed by a more pronounced change in the level of K +Mg 2+-ATPase activity than in that of H + transport, and this change was more clearly differentiated in the basal node plasmalemma of contrasting genotypes than in the roots. Stress response was manifested differently in freezing-tolerant and non-tolerant plants at cold acclimation (4–2 °C) and at freezing (−8 °C) temperatures. Proton transport regulation via coupled changes in the hydrolysed ATP/transported proton ratio, as an attribute of freezing-tolerant plants, is discussed. 相似文献
18.
The role of the heme propionate groups in determining the electron transfer and electrostatic properties of myoglobin have been studied by thermodynamic, kinetic, and spectroscopic studies of horse heart myoglobin in which the heme propionate groups are esterified. Spectroelectrochemical analysis has established that the Em,7 of dimethylester heme-substituted Mb (DME-Mb) ( Em,7 = 100.2(2) mV vs. NHE (Normal Hydrogen Electrode) (25 °C) is increased 40 mV relative to that of the native protein with ΔH° = −12.9(2) kcal/mol and Δ S° = −51.0(8) cal/mol/deg (pH 7.0, μ = 0.1 M (phosphate)). The second order rate constant for reduction of DME-metMb by Fe(EDTA) 2− is increased > 400-fold relative to that for reduction of native metMb to a value of 1.34(2) × 10 3 M −1 s −1 with Δ S‡ = −13(1) cal/mol/deg and Δ H‡ = 9.2(3) (pH 7.0, μ = 0.1 M (phosphate)). Analysis of the pH dependences of the reduction potential and rate constant for reduction by Fe(EDTA) 2− demonstrates that heme propionate esterification introduces significant changes into the electrostatic interactions in myoglobin. These changes are also manifested by differences in the pH dependences of the 1H NMR spectra of native and DME-metMb that reveal shifts in p Ka values for specific His residues as the result of heme propionate esterification. In sum, the current results establish that heme propionate esterification not only affects the electron transfer properties of myoglobin but also influences the titration behavior of specific His residues. 相似文献
19.
The effects of N-ethylmaleimide (NEM) on mouse platelet serotonin (5-HT) and 86Rb + uptake were studied. The 5-HT transport system showed a biphasic response to increasing concentrations of NEM, with low concentrations (25–50 μM) stimulating and high concentrations (200–400 μM) inhibiting 5-HT transport. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked NEM-induced stimulation of 5-HT transport. The kinetics of 5-HT uptake indicated that NEM (50 μM) markedly increased the maximal rate of 5-HT transport ( Vmax control = 28.4±1.4 pmol/10 8 platelets/4 min vs Vmax NEM = 64.5±9.5 pmol/10 8 platelets/4 min but had no significant effect on the Km value. Platelet Na + K + ATPase activity was determined by measuring 86Rb + uptake. Platelet 86Rb + uptake showed a biphasic response to NEM, with low concentrations (25–100 μM) significantly stimulating and high concentrations (400 μM) inhibiting uptake. These changes in platelet 86Rb + uptake paralleled the biphasic changes in 5-HT transport. In the presence of fluoxetine, 5-HT transport was markedly inhibited but no change in the ability of NEM to stimulate 86Rb + uptake was observed. These data suggest that low concentrations of NEM activate plasma membrane Na + K + ATPase which results in a marked stimulation of platelet 5-HT transport. 相似文献
20.
Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75–80 °C and its half life was 30 min at 70 °C. The Km and Vmax were calculated to be, respectively, 0.33 mM and 188 μM min −1 mg −1 with p-nitrophenyl palmitate ( pNPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well. 相似文献
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