共查询到20条相似文献,搜索用时 15 毫秒
1.
J.F. Steffensen K. Johansen C.D. Sindberg J.H. Sørensen J.L. Møller 《Journal of experimental marine biology and ecology》1984,84(2):173-178
Ventilation was measured directly in the hagfish, Myxine glutinosa L., by means of an electro-magnetic blood flowmeter. Ventilatory flow and frequency increased from 0.86 ± 0.27 ml·min?, and 18.2 ± 5.1·min?, respectively, at 7°C to 1.70 ± 0.20 ml·min?, and 70.1 ± 9.5·min? at 15 ·C.Standard oxygen consumption,V?O2, was measured in non-buried hagfish. V?O2 was 0.57 ± 0.17μl O2·g?1·min?1 at 7°C, and 0.85 ± 0.12μl O2·g?1·min?1 at 15°C. 相似文献
2.
The kinetics of hypoxanthine transport were measured in hypoxanthine phosphoribosyltransferase-deficient Novikoff cells by rapid kinetic techniques applying both zero-trans and equilibrium exchange protocols. The data indicate operation of a simple carrier with directional symmetry and equal mobility when substrate loaded and empty. Zero-trans influx and efflux were about equivalent and so were zero-trans influx and equilibrium exchange flux. The apparent Michaelis-Menten constant and maximum velocity were about 500 μM and 100 pmol/s per μl cell H2O, respectively. The time courses of accumulation of radioactively labeled hypoxanthine at a concentration above the Michaelis-Menten constant differed noticeably in zero-trans and equilibrium exchange mode, but computer simulations showed that the difference is predicted by the symmetrical carrier model and does not reflect trans-stimulation. 相似文献
3.
Paul W. Wigler 《Journal of theoretical biology》1981,93(4):997-1008
The efflux of a substrate from preloaded cells may be decelerated by an inhibitor in the external medium or accelerated by a compatible substrate in the external medium. The derivations of rate equations for the initial velocity of the zero-trans reaction, trans efflux inhibition, and accelerated exchange diffusion are described for steady state conditions. The rate constants making up the Michaelis constant for the trans inhibition reaction are the same as the corresponding parameters in the zero-trans reaction. The rate constants making up the Michaelis constant for the accelerated exchange reaction, however, are different from the corresponding parameters in the zero-trans reaction. The rate equation for trans inhibition shows that the velocity constant for recovery of the unloaded carrier may be determined with steady state experimental data. It is suggested that the observed recovery constant is independent of the substrates and trans inhibitors chosen for an assay of a particular carrier system. An experiment is briefly described to show a determination of a tentative value for the recovery constant of the unloaded nucleoside carrier in yeast cells and the apparent inhibition constant for a trans inhibitor. 相似文献
4.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(3):370-376
N-Acetylgalactosamine kinase (GALK2) is a small molecule kinase from the GHMP family which phosphorylates N-acetylgalactosamine at the expense of ATP. Recombinant GALK2 expressed in, and purified from, Escherichia coli was shown to be active with the following kinetic parameters: Michaelis constant for ATP, 14?±?3?μM; Michaelis constant for N-acetylgalactosamine, 40?±?14?μM; and turnover number, 1.0?±?0.1?s?1. The combination of substrate inhibition by N-acetylgalactosamine and α-methylgalactopyranoside acting as an uncompetitive inhibitor with respect to ATP suggested that the enzyme has an ordered ternary complex mechanism in which ATP is the first substrate to bind. The effects of pH on the kinetic parameters provided evidence for ionizable residues playing a role in substrate binding and catalysis. These results are discussed in the context of the mechanisms of the GHMP kinases. 相似文献
5.
Peter G.W. Plagemann Richard Marz Robert M. Wohlhueter Jon C. Graff John M. Zylka 《生物化学与生物物理学报:生物膜》1981,647(1):49-62
6-Mercaptopurine and 6-thioguanine strongly inhibited the zero-trans entry of hypoxanthine into Novikoff rat hepatoma cells which lacked hypoxanthine/guanine phosphoribosyltransferase, whereas 8-azaguanine had no significant effect. 6-Mercaptopurine was transported by the hypoxanthine carrier with about the same efficiency as its natural substrates (Michaelis-Menten constant = 372 ± 23 μM; maximum velocity = 30 ± 0.7 pmol/μl cell H2O per s). 8-Azaguanine entry into the cells, on the other hand, showed no sign of saturability and was not significantly affected by substrates of the hypoxanthine/guanine carrier. The rate of entry of 8-azaguanine at 10–100 μM amounted to only about 5% of that of hypoxanthine transport and was related to its lipid solubility in the same manner as observed for various substances whose permeation through the plasma membrane is believed to be non-mediated. Only the non-ionized form of 8-azaguanine (pKa = 6.6) permeated the cell membrane.Studies with wild type Novikoff cells showed that permeation into the cell was the main rate-determining step in the conversion of extracellular 8-azaguanine to intracellular aza-GTP and its incorporation into nucleic acids. In contrast, 6-mercaptopurine was rapidly transported into cells and phosphoribosylated; the main rate-determining step in its incorporation into nucleic acids was the further conversion of 6-mercaptopurine riboside 5'-monophosphate. 相似文献
6.
Seung-Hye Hong Ho-Phuong-Thuy Ngo Lin-Woo Kang Deok-Kun Oh 《Biotechnology letters》2015,37(4):849-856
A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min?1 mg?1, a K m of 1.8 μM, and a k cat of 1.7 min?1 for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD + as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l?1, and 2,200 mg all-trans-retinal l?1 in the presence of 5 % (v/v) methanol, 1 % (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l?1 after 3 h, with a conversion yield of 27.3 % (w/w) and a productivity of 200 mg l?1 h?1. This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH. 相似文献
7.
Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 μM rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate l-[3H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H+ gradient-stimulated uptake of l-[3H]proline. Uptake was rapid, independent of a Na+ gradient but stimulated by an inwardly directed H+ gradient with maximal uptake occurring at pH 6.0. l-Proline uptake was found to be mediated by a transport system with a Michaelis constant (Kt) of 130 ± 10 μM and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min?1 mg of protein?1. Glycine, l-alanine, and l-tryptophan strongly inhibited l-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H+-amino acid cotransport system PAT2. 相似文献
8.
Uptake of 14C-labelled sucrose and glucose by isolated seed coat halves of pea (Pisum sativum L. cv. Marzia) seeds was measured in the concentration range <0.1 μM to 100 mM. The initial influx of sucrose was strictly proportional to the external concentration, with a coefficient of proportionality (k) of 6.2 μmol·(g FW)?1·min?1·M?1. Sucrose influx was not affected by 10 μM carbonylcyanide m-chlorophenylhydrazone (CCCP), but it was inhibited by 40% in the presence of 2.5 mM p-chloromercuribenzenesulfonic acid (PCMBS). Influx with diffusional kinetics was also observed for glucose (k = 4.8 μmol·(g FW)?1·min ?1·M ?1) and mannitol (k = 5.1 μmol·(g FW)?1·min?1·M?1). For glucose an additional saturable system was found (Km = 0.26 mM, V max = 4.2 nmol·(g FW)?1·min?1), which appeared to be completely inhibited by CCCP and partly by PCMBS. In contrast to the diffusional pathway, uptake by this saturable system was slightly pH-dependent, with an optimum at pH 5.5. The influx of sucrose appears to be by the same pathway as the efflux of endogenous sucrose, which was inhibited by 36% in the presence of 2.5 mM PCMBS (De Jong A, Wolswinkel P, 1995, Physiol Plant 94: 78–86). It is argued that passive transport may be the only mechanism for sucrose transport through the plasma membrane of seed coat parenchyma cells. The estimated permeability coefficient of the plasma membrane for sucrose (P = 3.5·10?7 cm·s?1) is more than 1 × 106-fold higher than that reported for artificial lipid membranes. This relatively high permeability is hypothesized to result from pore-forming proteins that allow the diffusion of sucrose. Furthermore, it is shown that a sucrose gradient across the plasma membrane of the seed coat parenchyma of only 22 mM will suffice to result in the net efflux of sucrose which is required to feed the embryo. 相似文献
9.
Karsten Junker 《生物化学与生物物理学报:生物膜》1980,597(2):399-410
Reduced glucose transport across the plasma membrane and reduced phosphorylation may both be responsible for the early inhibitory effect of physiological concentrations of glucocorticoids on glucose uptake by rat thymocytes.The early inhibitory effects of glucocorticoids (5 · 10?7 M dexamethasone) on glucose consumption and 14CO2 formation from d-[U-14C]glucose were reproduced.The total uptake curve of 4.8 μM was biexponential with of 1.1 min and 36 min, respectively, the rapid part comprising about 50% of the equilibrated intracellular water space. The latency of the effect of 5 · 10?7 M dexamethasone on uptake ranged from 15 to 100 min and the inhibition varied from 15 to 55% independently of the lag period. The effect of 3-O-methylglucose concentration on the initial uptake by steroid-responsive cell preparations was tested after 45 min of preincubation with or without 5 · 10?7 M dexamethasone. In 12 experiments dexamethasone reduced V from 1.36 ± 0.16 mmol · min?1 · l?1 cell water to 0.81 ± 0.10 mmol · min?1 · l?1 cell water with insignificant change of Km (6.0 mM versus 5.9 mM). Dexamethasone had similar effect after 90 or 120 min.The variabilities of control cell transport capacity, the lag period and the magnitude of the dexamethasone effect could not be accounted for by changes in pH, effects of cell density, concentrations of albumin, ethanol, nucleosides, pyruvate or correlated to age and sex of the rats. In conclusion the inhibition of glucocorticoids on glucose consumption by thymocytes appears to be an inhibited plasma membrane transport capacity. 相似文献
10.
11.
《生物化学与生物物理学报:生物膜》1986,862(2):387-398
The kinetic features of glucose transport in human erythrocytes have been the subject of many studies, but no model is consistent with both the kinetic observations and the characteristics of the purified transporter. In order to reevaluate some of the kinetic features, initial rate measurements were performed at 0°C. The following kinetic parameters were obtained for fresh blood: zero-trans efflux Km = 3.4 mM, Vmax = 5.5 mM/min; infinite-trans efflux Km = 8.7 mM, Vmax = 28 mM/min. For outdated blood, somewhat different parameters were obtained: zero-trans efflux Km = 2.7 mM, Vmax = 2.4 mM/min; infinite-trans efflux Km = 19 mM, Vmax = 23 mM/min. The Km values for fresh blood differ from the previously reported values of 16 mM and 3.4 mM for zero-trans and infinite-trans efflux, respectively (Baker, G.F. and Naftalin, R.J. (1979) Biochim. Biophys. Acta 550, 474–484). The use of 50 mM galactose rather than 100 mM glucose as the infinite-trans sugar produced no change in the infinite-trans efflux Km values but somewhat lower Vmax values. Simulations indicate that initial rates were closely approximated by the experimental conditions. The observed time courses of efflux are inconsistent with a model involving rate-limiting dissociation of glucose from hemoglobin (Naftalin, R.J., Smith, P.M. and Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235–249). The results presented here support the adequacy of the carrier model to account for the kinetics. 相似文献
12.
Guangfei Liu Jiti Zhou Congcong Chen Jing Wang Ruofei Jin Hong Lv 《Applied microbiology and biotechnology》2013,97(17):7935-7942
Geobacter metallireducens was found to be capable of decolorizing several azo dyes with different structures to various extents. Pyruvate, ethanol, acetate, propionate, and benzoate could support 66.3?±?2.6?93.7?±?2.1 % decolorization of 0.1 mM acid red 27 (AR27) in 40 h. The dependence of the specific decolorization rate on AR27 concentration (25 to 800 μM) followed Michaelis–Menten kinetics (K m?=?186.9?±?1.4 μΜ, V max?=?0.65?±?0.02 μmol?mg protein?1 h?1). Enhanced AR27 decolorization was observed with the increase of cell concentrations ranging from 7.5 to 45 mgL?1. AR27 decolorization by G. metallireducens was retarded by the presence of goethite, which competed electrons with AR27 and was reduced to Fe(II). The addition of low concentrations of humic acid (1?100 mgL?1) or 2-hydroxy–1,4-naphthoquinone (0.5?50 μM) could improve the decolorization performance of G. metallireducens. High-performance liquid chromatography analysis suggested reductive pathway to be responsible for decolorization. This was the first study on azo dye decolorization by Geobacter strain and might improve our understanding of natural attenuation and bioremediation of environments polluted by azo dyes. 相似文献
13.
14.
Δ53β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ53β hydroxy steroids into the active Δ43-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for Δ53β HSD we have analysed the initial velocity rates (Vo) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ53β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/Vo versus 1/S plot, the x intercept being positive. Using a 1/Vo versus 1/S2 plot the Vmax was determined to be 1·0 ± 0·08 μmol min?1 mg?1 CL (n = 6). The Hill constant was 2·7 ± 0·02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 ± 1 μmoles (n = 6). In the corpora lutea cells of the proestrous ovary, the Vmax for DHEA transformation was unchanged (0·95 ± 0·04 μmol min?1 mg?1, n = 3) whilst the S0·5 was significantly increased to 27 ± 0·1 (p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 (n = 3). NAD+ binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis-Menten model.1 Extending the analysis of NAD+ binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent Vmax and Km being 0·84 ± 0·04 μmol min?1 mg?1 CL (n = 3) and 27 ± 7 μmol 1?1 (n = 3) respectively. The Hill constant was 1·1 ± 0·03 (n = 3), indicating no co-operativity of co-factor binding. 相似文献
15.
1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O2 evolution by envelope-free chloroplasts and oxaloacetate-dependent O2 evolution by intact chloroplasts. The half maximal concentration () for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml?1.2. Ionophore A23187 also induces inhibition of CO2 and 3-phosphoglycerate-dependent O2 evolution by intact chloroplasts in the presence of 3 mM MgCl2. The half maximal concentrations () for inhibition of O2 evolution are 3 μM and 5 μM respectively · 100 μg?1 chlorophyll · ml?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg?1 chlorophyll · ml?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts. 相似文献
16.
《Neurochemistry international》1987,11(1):89-93
The uptake and accumulation of N-methyl-4-phenylpyridinium ion (MPP+), a neurotoxin produced by oxidation of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), into PC12h pheochromocytoma cells were examined. Concentration gradients of MPP+ were established at its low concentrations of 10 to 100 nM. Uptake of MPP+ into PC12h cells was mediated by saturable, carrier mediated transport systems with two different kinetic properties; a high-affinity and low-capacity system and a low-affinity and high-capacity system. The apparent Km values of these two systems were obtained to be 254.4 ± 96.5 nM and 23.1 ± 6.9 μM, respectively, and the maximal uptake velocity was obtained to be 8.47 ± 1.72 and 28.6 ± 5.2 pmol/min/mg protein, respectively. The uptake by a high-affinity system was mediated by a carrier system common to dopamine and noradrenalin and MPTP itself proved to be taken up by this system, which was further confirmed by the inhibition of the MPP+ uptake by nomifensine and mazindol. The uptake was inhibited by metabolic inhibitors, such as carbonyl cyanide m-chlorophenyl hydrazone, sodium cyanide and 2,4-dinitrophenol, and the uptake was inhibited by ouabain and nigercin. By subcellular fractionation, MPP+ taken up was found to be localized mainly in cytosol fraction, but a definite amount of MPP+ was found also in mitochondrial fraction. 相似文献
17.
The Effect of Acetylcholine Release on Choline Fluxes in Isolated Synaptic Terminals 总被引:11,自引:9,他引:2
Abstract: As in intact tissues, choline influx into synaptosomes is enhanced after a period of depolarization induced release of acetylcholine. The activation of uptake is dependent on the presence of Ca2+ and inhibited by high Mg2+ concentrations in the medium during depolarization. Choline transport in erythrocytes was not activated by prior treatment with potassium. The permeability constant of the synaptosome membrane to choline was found to be 2.7 × 10?8 cm·s?1 and to acetylcholine 1.8 ′ 10?8 cm·s?1. Choline influx has been studied after pre-loading synaptosomes with choline. Different radiolabels were used to measure efflux of preloaded choline and influx simultaneously. Isotopic dilution in flux studies was estimated and corrected for. Influx was stimulated by high internal concentrations of choline, and efflux similarly stimulated by high outside concentrations of choline. The maximal influx and efflux at saturating opposite concentrations of choline were equal with a value of about 500 pmol·min?1 per mg synaptosomal protein. A reciprocating carrier would explain the equality of the maximal influx and efflux. Acetylcholine competes with choline for binding to the carrier but is itself hardly transported. Increased acetylcholine concentrations were shown to inhibit both choline influx and efflux from the trans position. Raising intrasynaptosomal acetylcholine concentrations by pre-loading abolished the stimulation of influx by prior depolarization. It is proposed that high concentrations of acetylcholine immobilize the carrier on the inside of the synaptic membrane. The stimulation of choline influx consequent upon depolarization is caused by release of ACh which results in relief of this immobilisation. The enhanced supply of choline achieved by this mechanism is likely to be important in maintaining stores of the acetylcholine in vivo. 相似文献
18.
Hubert Felle 《生物化学与生物物理学报:生物膜》1980,602(1):181-195
Green thallus cells of the aquatic liverwort, Riccia fluitans, are rapidly depolarized in the presence of 1–20 μM NH4Cl and 5–100 μM CH3NH3Cl, respectively. Simultaneously, the membrane conductance is increased from 0.41 to 1.2 S · m?2. Uptake of [14C]methylamine is stimulated by increasing [K+]o and inhibited by increasing [Na+]o or [H+]o, is highly voltage sensitive, and saturates at low amine concentrations.Double-reciprocal plots of (a) maximal membrane depolarization and (b) methylamine uptake vs. external amine concentration give apparent Km values of 2 ± 1 μM ammonia and 25–50 μM methylamine; Km values for changes in conductance and membrane current are greater and voltage dependent. Whereas the amine transport into the cell is strongly inhibited by CN?, the amine efflux is stimulated.The current-voltage characteristics of the ammonia transport are represented by a sigmoid curve with an equilibrium potential of ?60 mV, and this is understood as a typical carrier curve with a saturation current of about 70 mA · m?2. It is further concluded that the evidently carrier-mediated transport is competitive for the two amines tested, and that ammonia and methylamine are transported in the protonated form as NH4+ and CH3NH3+ into the cytoplasm. 相似文献
19.
Methylamine uptake in nitrogen-starved Chlorella pyrenoidosa Beij. follows Michaelis-Menten kinetics: maximum uptake is about 1.6 nmol μl?1· cells · min?1, half-saturation occurs at 4 μM methylamine, and the slope in the range where uptake is proportional to concentration is 0.4 nmol μl?1· min?1·μM?1. In cells grown in the presence of a non-limiting nitrogen concentration, methylamine uptake is directly proportional to concentration up to at least 0.5 mM, and the slope is 1/500 that for starved cells. Similar uptake kinetics have been reported for Penicillium chrysogenum and attributed to an inducible “ammonium permease.” Apparently, a similar permease occurs in algae. 相似文献
20.
RicharD. T. Mayer A. C. Chen J. R. Deloach 《Archives of insect biochemistry and physiology》1983,1(1):1-15
Particulate fractions (10,000g) from pupae of Stomoxys calcitrans transfer [14C]-mannose from GDP-[14C]-mannose to dolichol monophosphate and proteins. Production of the mannosyl lipid was inhibited by Mn2+, UDP, GMP, GDP, and EDTA. The insect growth regulator diflubenzuron had no effect on mannosyl transferase activity. Dolichol monophosphate and Mg2+ stimulated mannosyl transferase activity. The mannosyl lipid product was identified as mannosyl-phosphoryl-dolichol (Man-P-Dol). The apparent Km and Vmax values for the formation of Man-P-Dol using GDP-[14C]-Man while holding dolichol phosphate constant were 2.4 ± 0.9 μM and 9.4 ± 2.3 pmol Man-P-Dol·min?1·mg?1 protein, respectively. The apparent Km and Vmax values using dólichol phosphate while holding GDP-Man constant were 2.2 ± 1.2 μM and 18.5 ± 1.7 pmol Man-P-Dol·min?1·mg?1 protein. 相似文献