共查询到20条相似文献,搜索用时 31 毫秒
1.
F Bonilla-Musoles F Raga NG Osborne J Blanes F Coelho 《Canadian Metallurgical Quarterly》1997,65(2):245-252
The combined effects of pressure and temperature on the activity of butyrylcholinesterase (BuChE) were investigated in the pressure range from 10(-3) to 5 kbar and temperature range from -10 degrees C to 70 degrees C. Inactivation of the enzyme showed a complex dependence on pressure and temperature. Under moderate pressures (1-3 kbar) at temperatures 40-65 degrees C BuChE was resistant to heat inactivation; under other conditions of pressure and temperature, the action of both parameters was synergistic and caused inactivation. Results allowed to construct a pressure-temperature kinetic phase diagram for the enzyme inactivation. The elliptic diagram for the irreversible transition active-->inactive BuChE as a function of both pressure and temperature has a positive angular coefficient. This indicates that pressure acts as a stabilizer of BuChE against heat denaturation. 相似文献
2.
GE Borgstahl HE Parge MJ Hickey MJ Johnson M Boissinot RA Hallewell JR Lepock DE Cabelli JA Tainer 《Canadian Metallurgical Quarterly》1996,35(14):4287-4297
Human manganese superoxide dismutase (MnSOD) is a homotetrameric enzyme which protects mitochondria against oxygen-mediated free radical damage. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical four-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites. The 2.5 A crystallographic structure of the naturally occurring polymorphic variant Ile58Thr MnSOD reveals that the helical hairpin mutation Thr58 causes two packing defects in each of the two four-helix bundles of the tetrameric interface. Similar mutations, expected to cause packing defects in the Cu,ZnSOD dimer interface, are associated with the degenerative disease amyotrophic lateral sclerosis. Ile58Thr MnSOD is primarily dimeric in solution and is significantly less thermostable than the normal enzyme, with decreases of 15 degrees C in the main melting temperature and 20 degrees C in the heat-inactivation temperature. Consequently, this mutant MnSOD is compromised at normal body temperatures: thermal inactivation, predicted from the decrease in thermal stability, occurs with a theoretical half-life of only 3.2 h at 37 degrees C (1.4 h at 41 degrees C), compared with 3.1 years for native MnSOD. This prediction is supported by direct measurements: incubation at 41.7 degrees C for 3 h has no effect on the activity of native MnSOD but completely inactivates mutant MnSOD. Rapid inactivation of Ile58Thr MnSOD at the elevated temperatures associated with fever and inflammation could provide an early advantage by killing infected cells, but also would increase superoxide-mediated oxidative damage and perhaps contribute to late-onset diseases. 相似文献
3.
J Saad-Nehme AL Bezerra LA Fornells JL Silva JR Meyer-Fernandes 《Canadian Metallurgical Quarterly》1997,52(7-8):459-465
A complete inactivation is observed after a 3 min pre-incubation at 70 degrees C with mitochondrial F0F1-ATPase complex depleted of the ATPase natural inhibitor protein (ammonium-Sephadex submitochondrial particles) and activated MgATP-submitochondrial particles (particles that after a 4 h-pre-incubation at 42 degrees C released the endogenous inhibitor protein). However, latent MgATP-submitochondrial particles (particles containing the inhibitor protein) pre-incubated under the same conditions are totally inactivated only after 15 min of pre-incubation. When ammonium-Sephadex particles are reconstituted with 20 micrograms/ml of purified ATPase inhibitor protein there is an increase of 15-fold in the half-time for thermal inactivation (t0.5), showing that the inhibitor protein protects the mitochondrial F0F1-ATPase complex against thermal inactivation. 相似文献
4.
Production and properties of glucose isomerase from a Co2+-sensitive Streptomyces species were studied. After 4 days of shaking cultivation at 30 degrees C and 200 rpm, a maximum of 1.1 enzyme units per ml of broth was obtained. Cell-free glucose isomerase, obtained from mycelia heat-treated in the presence of 0.5 mM Co2+, showed a 3.5-fold increase in specific activity over enzyme obtained from untreated mycelia. The optimum pH and temperature for the glucose isomerase were 7 to 8 and 80 degrees C, respectively. The Michaelis constant for fructose was 0.40 M. Mg2+ was found to enhance the glucose isomerase activity, whereas the effect of Co2+ on enzyme activity depended on the manner in which the enzyme was prepared. This glucose isomerase was quite heat stable, with a half-life of 120 h at 70 degrees C. 相似文献
5.
Histidine decarboxylase from Lactobacillus 30a is rapidly and irreversibly inactivated upon incubation with L-histidine methyl ester. The rate of inactivation is first-order with respect to remaining active enzyme and exhibits saturation kinetics with a kinact of 1.2 mM and an apparent first-order rate constant of 0.346 min-1 at pH 4.8 and 25 degrees C. On complete inactivation, 3 mol of [14C]histidine (from L-[14C]histidine methyl ester) and 2 mol of 14C (from L-histidine [14C]methyl ester) are bound in nondialyzable form per mol (190 000 g) of protein inactivated with a corresponding loss of three of the five DTNB-titratable--SH groups that are essential for activity of the native enzyme. Imidazole propionate, a competitive inhibitor of the enzyme, protects against inactivation, loss of --SH groups, and incorporation of radioactivity from both the histidine and the methyl ester moieties of the labeled inhibitor, and kinetic evidence indicates that imidazole propionate and histidine methyl ester compete for binding at the active site of histidine decarboxylase in a mutually exclusive manner. Treatment of the labeled protein with either alkali or hydroxylamine results in the quantitative release of radioactivity. These data suggest that inactivation of histidine decarboxylase by L-histidine methyl ester results from two different modes of interaction between the inhibitor and the active site of histidine decarboxylase; the major interaction involves an essential -SH group. 相似文献
6.
Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding. 相似文献
7.
The arginine-specific reagents phenylglyoxal and butane-2,3-dione irreversibly inactivate the Tritrichomonas foetus hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) and Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The inactivation of the tritrichomonal enzyme by phenylglyoxal follows time-dependent and concentration-dependent pseudo-first-order kinetics. Complete protection against inactivation is afforded by the addition of 25 microM GMP, whereas 5-phosphoribosyl-1-diphosphate (PRibPP) at 50-250 microM can only slow down the inactivation, without being protective. Digestion of [7-(14)C]phenylglyoxal-modified enzyme with trypsin and separation of the peptides by reverse-phase HPLC shows that only one radioactive peak is greatly diminished by incubation with 25 microM GMP or 1 mM PRibPP. Mass-spectral analysis identifies Arg155 as the target site of two molecules of phenylglyoxal that is protected by the substrates. This amino acid residue is positioned next to Tyr156, which is a highly conserved aromatic residue among all the purine phosphoribosyltransferases (PRT) and is always found stacked on top of the purine substrate. This may explain why phenylglyoxal labeling of Arg155 inactivates the enzyme and why GMP can protect Arg155 more effectively than PRibPP. Among the purine PRT in our possession, only schistosomal HGPRT, the only other enzyme that contains an arginine residue at the corresponding location (Arg187), was susceptible to phenylglyoxal and butane-2,3-dione. The presence of Lys185-Phe186 and Ser179-Trp180 at the corresponding locations in human HGPRT and Giardia lamblia GPRT, respectively, may explain their resistance to phenylglyoxal. Thus, Arg155 in T. foetus HGXPRT and Arg187 in S. mansoni HGPRT will be attractive targets for future studies. 相似文献
8.
Polyguanylic acid (poly(G)) was synthesized from GDP in a yield of 60-75% by Thermus thermophilus polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) at 70 degrees C, pH 8.5 in the presence of Mg2+. The yield was dependent on the ratio of GDP to Mg2+, but was independent of the concentrations of enzyme or substrate. The maximal rate of GDP polymerization was obtained when the ratio of GDP to Mg2+ was 3:1. However, by prolonged incubation, the higher initial ratio of over 4:1 was preferred because of the rapid consumption of GDP in the reaction mixture. Poly(G) prepared by 1 h incubation was heterogeneous in size from 5 S to over 23 S, but by prolonged incubation of 19 h the size of product converged to 9 S as judged by sucrose density gradient centrifugation. Its chain length was determined by terminal nucleoside analysis to be 200 nucleotides long. 相似文献
9.
It was previously demonstrated that while lysogenic development of bacteriophage lambda in Escherichia coli proceeds normally at low temperature (20-25 degrees C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the pE promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage lambda cIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37 degrees C, while no phage progeny are observed at 20 degrees C. Contrary to previous reports, it is possible to demonstrate that pE promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20 degrees C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage lambda is neither inhibited at 20 degrees C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between lambda phage and lambda plasmid DNA at low temperature. 相似文献
10.
The chitinolytic activity of Verticillium cfr. lecanii A3, a strain isolated from continental Antarctica, was compared to those of two selected strains of Trichoderma harzianum. After 72 h of incubation at 25 degrees C in media containing chitin as the sole carbon source, all strains showed the same enzyme activity (ca. 230 mU/ml); at 15 degrees C, the levels of enzyme activity of the three strains were similar to those obtained at 25 degrees C. At 5 degrees C, in contrast, the activity of V. lecanii was ca. 4 times higher than those of both strains of T. harzianum (203 and 57 mU/ml, respectively; incubation time 144 h). The chitinase of V. lecanii, purified by preparative isoelectric focusing and ion-exchange chromatography, was shown to be a glycoprotein with apparent molecular weight of 45 kDa and isoelectric point of 4.9. The enzyme was active over a broad range of temperatures (5-60 degrees C): at 5 degrees C, its relative activity was still 50% of that recorded at 40 degrees C (optimal temperature). V. lecanii and its purified chitinase showed clear inhibitory effects on the growth of some test moulds such as Mucor plumbeus, Cladosporium cladosporioides, Aspergillus versicolor and Penicillium verrucosum: observations under the light and scanning electron microscopes revealed that growth inhibition was accompanied by mycelial damage and cell lysis. 相似文献
11.
T Wartmann A Krüger K Adler BM Duc I Kunze G Kunze 《Canadian Metallurgical Quarterly》1995,68(3):215-223
Arxula adeninivorans Ls3 is described as an ascomycetous, arthroconidial, anamorphic, xerotolerant yeast, which was selected from wood hydrolysates in Siberia. By using minimal salt medium or yeast-extract-peptone-medium with glucose or maltose as carbon source it was shown that this yeast is able to grow at up to 48 degrees C. Increasing temperatures induce changes in morphology from the yeast phase to mycelia depending on an altered programme of gene expression. This dimorphism is an environmentally conditioned (reversible) event and the mycelia can be induced at a cultivation temperature of 45 degrees C. Depending on the morphology of strain Ls3 (yeast phase or mycelia) the secretion behaviour as well as the spectrum of polypeptides accumulated in the culture medium changed. The activities of the accumulated extracellular enzymes glucoamylase and invertase were 2 to 3 times higher in cultures grown at 45 degrees C than in those grown at 30 degrees C. While the level of the glucoamylase protein secreted from mycelia between 45 and 70 hours did not change, biochemical activity decreased after a cultivation time of 43 hours. It was shown that this effect depended on both the catabolic repression of the glucoamylase by glucose and the thermal inactivation of this enzyme in media without or with low concentrations of starch or maltose. 相似文献
12.
The kinetics of inactivation of cytochrome P450 2B1, the major phenobarbital inducible rat hepatic P450, by N-benzyl-1-aminobenzotriazole (BBT) were characterized. Purified, reconstituted P450 2B1 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylase activity was inhibited by BBT in a mechanism-based manner. The loss of O-deethylase activity followed pseudo-first-order kinetics and was NADPH and BBT dependent. After a 5 min incubation, greater than 90% of the 2B1 activity was lost, whereas more than 70% of the ability of the reduced enzyme to bind CO was maintained. Inclusion of 10 mM glutathione in the inactivation reaction lowered the rate of inactivation (k(inactivation)) and increased the partition ratio without significantly affecting the inactivator concentration required for half-maximal inactivation (K(I)). The maximal rate constant for inactivation at 23 degrees C was 0.24 min(-1) without and 0.15 min(-1) with glutathione. The apparent K(I) was 2 microM in both cases. The extrapolated partition ratios were 4 and 9 without and with 10 mM glutathione, respectively. Consistent with mechanism-based inactivation, the loss of 7-EFC O-deethylase activity was irreversible, was not due to product inhibition, was saturable, and could be slowed by including increasing concentrations of competing substrate. However, the inactivated P450 2B1 was still able to metabolize substrate if iodosobenzene was used as an alternate oxidant. Inactivation of 2B1 with either N-[14C]-7-benzyl-1-aminobenzotriazole (BBT) or N-benzyl-1-amino-[14C]-2,3-benzotriazole resulted in the incorporation of covalent radiolabel into the apoprotein. The stoichiometry of labeled metabolite adduct to protein was approximately 0.4:1 in both cases. Identification of metabolites revealed the formation of 1-aminobenzotriazole, benzotriazole, benzaldehyde, and a new metabolite (27) during catalysis of BBT by P450 2B1. Together, these data suggest that P450 2B1 could be inactivated and labeled by more than one metabolite. 相似文献
13.
The presumptive first step in the Rad51-promoted formation of joint molecules is binding of the protein to ssDNA in the presence of ATP and Mg2+. In this paper, we report that Rad51's ability to bind DNA is rapidly inactivated when incubated at 30-37 degrees C but is stabilized by the presence of ATP and Mg2+. Although unable to promote binding to DNA, ATP-gamma-S also prevents inactivation of Rad51 at 37 degrees C. AMP-P-N-P lacks this property, while ADP protects partially but only at 5-10 times higher concentrations than ATP. These observations correlate with the dissociation constant of those nucleotides for Rad51 determined by equilibrium dialysis. Rad51 binds ATP and ATP-gamma-S with a 1:1 stoichiometry and Kds of 21 and 19 microM, respectively. The presence of DNA significantly increases the affinity of Rad51 for ATP, while DNA has a smaller effect on the affinity of ATP-gamma-S. Competition binding studies show that ADP and AMP-P-N-P bind with a 5- and 55-fold lower affinity, respectively, than ATP. The CD spectrum of Rad51 with negative double minima at around 210 and 222 nm is characteristic of an alpha-helical protein. Upon binding ATP and Mg2+, the CD spectrum is altered in the regions 194-208 and 208-235 nm, changes that are indicative of a more structured state; this change does not occur with Rad51 that has been inactivated at 37 degrees C. We surmise that the active conformation is more resistant to inactivation at elevated temperature. Our data suggest that one of the roles of ATP and Mg2+ in Rad51-mediated strand exchange is to induce the proper protein structure for binding the two DNA substrates. 相似文献
14.
Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by beta-adrenergic signals, leading to a rise in intracellular 3',5'-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 +/- 0.08. A change in assay temperature from a euthermic value (37 degrees C) to one typical of hibernating body temperature (5 degrees C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 degrees C to 8.7 at 5 degrees C, (b) K(m) values at 37 degrees C for Mg.ATP (49.2 +/- 3.4 microM) and for two phosphate acceptors, Kemptide (50.0 +/- 5.5 microM) and Histone IIA (0.41 +/- 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 degrees C, and (c) inhibition by KCl, NaCl and NH4Cl was reduced. However, temperature change had little or no effect on K(m) values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 degrees C and activation energies that were 29.1 +/- 1.0 kJ/mol for temperatures > 9 degrees C and 2.3-fold higher at 68.1 +/- 2.1 kJ/mol for temperatures < 9 degrees C, whereas the rabbit enzyme showed a breakpoint at 17 degrees C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 +/- 0.18 at 37 degrees C to 2.19 +/- 0.07 at 5 degrees C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with 32P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 degrees C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor. 相似文献
15.
A strain of Aspergillus flavus isolated from an agricultural soil in Egypt produced a gluycoamylase which when purified had a molecular weight of 51,300 +/- 800 Daltons. The optimum pH for activity was 4 and the optimum temperature was 60 degrees C. The enzyme was stable at 70 degrees C for 15 min but denatured at 90 degrees C over 30 min. The Km value with soluble starch was 2.85 mg ml-1, and 10 mM HgCl2 inhibited the enzyme. It was possible to store the enzyme for at least 1 year at -20 degrees C without significant loss in activity. 相似文献
16.
The stability of ferulic acid esterase III (FAE-III) from Aspergillus niger was examined using chemical and thermal denaturation. Thermal denaturation was irreversible and the loss of activity was dependent on pH. At 60 degrees C and pH 6.0, the rate constant of unfolding was 0.76 10(-3)/s, and the change in free energy of irreversible inactivation, deltaG*, was 101.9 kJ/mol. Sinapic acid, a product of the reaction of methyl sinapate with FAE-III, reduced the rate of unfolding (0.66 10(-3)/s at 0.1 mM sinapic acid). Chemical denaturation was performed using guanidine hydrochloride. FAE-III was very sensitive to this denaturant, and the midpoint of unfolding was 1.38 M guanidine hydrochloride at 30 degrees C, pH 6.0. The stability of FAE-III is compared to other enzymes. 相似文献
17.
Substrate specificity of the ultraviolet-endonuclease from Micrococcus luteus. Endonucleolytic cleavage of depurinated DNA 总被引:5,自引:0,他引:5
The ultraviolet-endonuclease isolated from Micrococcul luteus, specific for pyrimidine dimers, is able to attack not only ultraviolet-irradiated DNA (leading to 3'OH-5'PO4 single-strand breaks) but also superhelical covalently-closed circular DNA of phage lambda damaged by heating at 70 degrees C, pH 5.93. The number of endonuclease-sensitive defects in the DNA corresponds to the number of alkalilabile bonds (apurinic sites) induced by heating. Competition between ultraviolet-induced lesions and apurinic sites for ultraviolet-endonuclease is demonstrated; the affinity of the enzyme for pyrimidine dimers is about three times that for apurinic sites. Both activities of the ultraviolet-endonuclease are inactivated at 50 degrees C at the same rate. The ultraviolet-endonuclease is able to reduce the infectious activity of depurinated lambda DNA towards Ca2+-treated uvr+ and uvr A Escherichia coli cells. It is concluded that both pyrimidine dimers and apurinic sites can be recognized by one and the same enzyme (the ultraviolet-endonuclease). 相似文献
18.
Inorganic pyrophosphatase (EC 3.6.1.1.) has been isolated from the archaebacterium Methanobacterium thermoautotrophicum (strain delta H). The enzyme was purified 850-fold in three steps to electrophoretic homogeneity. The soluble pyrophosphatase consists of four identical subunits: the molecular mass of the native enzyme estimated by gel filtration was approx. 100 kDa and denaturing polyacrylamide gel electrophoresis gave a single band of 25 kDa. The enzyme also may occur as an active dimer formed by dissociation of the tetramer. The pyrophosphate showed an optimal activity at 70 degrees C and a pH of 7.7 (at 60 degrees C) and was not influenced by dithiothreitol, sodium dithionite or potassium chloride. The enzyme was very specific for pyrophosphate (PPi) and Mg2+. Magnesium could be partially replaced by Co2+ (15%). The reaction was inhibited for 60% by 1 mM Mn2+ in the presence of 24 mM Mg2+. In addition, the enzyme was inhibited by potassium fluoride (50% at 0.9 mM). Kinetic analysis revealed positive co-operativity for both Mg2+ and PPi with Hill coefficients of 3.3 and 2.0, respectively. Under the experimental conditions at which the enzyme was present as its dimer, the apparent Km of PPi and magnesium were determined and were approx. 0.16 mM and 4.9 mM, respectively; Vmax was estimated at about 570 U/mg. 相似文献
19.
Enzymatically isolated myocytes from ferret right ventricles (12-16 wk, male) were studied using the whole cell patch clamp technique. The macroscopic properties of a transient outward K+ current I(to) were quantified. I(to) is selective for K+, with a PNa/PK of 0.082. Activation of I(to) is a voltage-dependent process, with both activation and inactivation being independent of Na+ or Ca2+ influx. Steady-state inactivation is well described by a single Boltzmann relationship (V1/2 = -13.5 mV; k = 5.6 mV). Substantial inactivation can occur during a subthreshold depolarization without any measurable macroscopic current. Both development of and recovery from inactivation are well described by single exponential processes. Ensemble averages of single I(to) channel currents recorded in cell-attached patches reproduce macroscopic I(to) and indicate that inactivation is complete at depolarized potentials. The overall inactivation/recovery time constant curve has a bell-shaped potential dependence that peaks between -10 and -20 mV, with time constants (22 degrees C) ranging from 23 ms (-90 mV) to 304 ms (-10 mV). Steady-state activation displays a sigmoidal dependence on membrane potential, with a net aggregate half-activation potential of +22.5 mV. Activation kinetics (0 to +70 mV, 22 degrees C) are rapid, with I(to) peaking in approximately 5-15 ms at +50 mV. Experiments conducted at reduced temperatures (12 degrees C) demonstrate that activation occurs with a time delay. A nonlinear least-squares analysis indicates that three closed kinetic states are necessary and sufficient to model activation. Derived time constants of activation (22 degrees C) ranged from 10 ms (+10 mV) to 2 ms (+70 mV). Within the framework of Hodgkin-Huxley formalism, Ito gating can be described using an a3i formulation. 相似文献
20.
Cholesterol domains and transport have been well-studied in non-neuronal membranes in contrast to neuronal membranes. The purpose of the experiments reported in this paper was to determine: (1) exchangeable and non-exchangeable cholesterol domains or pools were present in brain synaptosomal membranes; (2) effects of hydrolysis of sphingomyelin on cholesterol pools, that has previously been shown to alter membrane cholesterol in non-neuronal membranes and; (3) sphingomyelin hydrolysis and enzyme activity. Cholesterol pools were determined using cholesterol exchange between radiolabeled small unilamellar vesicles and mouse synaptosomes. Activity of Ca(2+)+Mg(2+)-ATPase and Na(+)+K(+)-ATPase were measured in synaptosomal membranes following treatment with sphingomyelinase. The size of the exchangeable pool of synaptosomal membrane cholesterol was approx 50% of total membrane cholesterol when measured at 37 degrees C. The t1/2 of cholesterol exchange at 37 degrees C in synaptosomes was approx 10 h. Lowering the incubation temperature to 25 degrees C, significantly reduced the size of the exchangeable pool and significantly increased the t1/2 of cholesterol exchange. Sphingomyelinase treatment of synaptosomes significantly slowed cholesterol exchange but did not modify the size of the exchangeable pool of cholesterol. Ca(2+)+Mg(2+)-ATPase activity was significantly inhibited by sphingomyelinase treatment as compared to Na(+)+K(+)-ATPase activity. Cholesterol domains were described in neuronal tissue and the size and kinetics of those pools were altered by temperature-induced changes in fluidity and hydrolysis of sphingomyelin. Sphingomyelinase-induced changes in Ca(2+)+Mg(2+)-ATPase activity were not affected by hydrolysis of sphingomyelin but appeared to be associated with a reduction in cytofacial phosphatidylinositol. 相似文献