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1.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,885(1):49-57
High-affinity receptors for α2-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4°C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4·10−4 min−1. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2·10−2 min−1. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca2+ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4°C was rapidly internalized (kint about 3·10−1 min−1) after being warmed to 37°C. Radioactivity released from the cells at 37°C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k−1 about 1·10−1 min−1) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of α2-macroglobulin-proteinase complex into hepatocytes. 相似文献
2.
《Life sciences》1996,58(20):1695-1704
Simultaneous exposure to cocaine and ethanol results in the formation of cocaethylene, an active metabolite of cocaine. The concurrent abuse of both cocaine and ethanol is common during human pregnancy, but the kinetics of elimination and formation of this ethyl ester of cocaine have not been studied during pregnancy in any species. In the late gestation guinea pig (61 to 63 days), cocaethylene, at doses of 2 to 4 mg · kg−1, is rapidly eliminated with a half-life of 29 min and a total body clearance of 77 ml · min−1 · kg−1. It is formed enzymatically by hepatic microsomal preparations from fetal, neonatal and maternal guinea pigs. The maximum rate of cocaethylene production (apparent Vmax) when either ethanol or cocaine are varied while the other substrate is held constant, increases with age, from the late fetal period (65 days gestation, term 70 days) to adulthood. However, the Michaelis-Menten constant (apparent KM) does not change with age. The rapid elimination of cocaethylene, coupled with the slow rate of formation (apparent Vmax of 140 pmol · min−1 · mg microsomal protein−1) and the small amount of plasma analyzed most likely explains the inability to detect cocaethylene in vivo after concomitant cocaine and ethanol administration. 相似文献
3.
Michal Svoboda Monique Lambert Jean Christophe 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,675(1):46-61
(1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p[NH]ppG or GTPγS as nucleotide activator, cholecystokinin as peptide hormone, and GDPβS and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively. The time courses of activation and the degree of activation at steady state (EA/ETOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant k+2, and a deactivation process (rate constant koff) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant k2) and/or the dissociation of the intact nucleotide (rate constant k?1), so that EA/ETOT = k+1/(k+1 + k2 + k?a). (2) The hormone CCK-8 increased the value of k+1 with GTP dose-dependently, from 0.2 to 10.9 min?1. The value of k?1 increased 0.01 to 0.3 min?1 but the value of k2 was unaltered at 7 min?1, so that EA/ETOT increased 15-fold, from 4% to 61%. (3) A cholera toxin pretreatment at 30 μg/ml allowed also a large increase in EA/ETOT with GTP (up to 51%) but the underlying mechanism was different. It consisted of a 14-fold decrease in the koff value of the GTP-activated enzyme (from 7 min? to 0.5 min?1) that corresponded to a reduction in GTPase activity. When testing the system with p[NH]ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of k+1 (from 0.2 to 0.8 min?1) and the occurrence of a significant 0.3 min?1 value for k?1. 相似文献
4.
《Inorganica chimica acta》1987,128(1):87-92
The kinetics of malonate replacement in bis- (malonato)oxovanadate(IV), [VO(mal)2H2O]2−(hereafter water molecule will be omitted), by oxalate has been studied by the stopped-flow method. The reaction was found to consist of two consecutive steps (k1 and k2: first-order rate constants) passing through a mixed ligand complex, [VO(mal)(ox)]2−. The rates for each step depended linearly on the concentrations of free oxalate species, Hox− and ox2−. The second-order rate constants for the replacement by ox2− were much larger in the k1 step than in the k2 step and the activation parameters were determined as follows: ΔH≠= 43.5 ± 5.6 kJ mol−1, ΔS±-53 ± 19 J K−1 mol−1 and ΔH≠= 43.6 ± 0.5 kJ mol−1, δS≠ = -62 ± 2 J K−l mol−1 for the k1 and k2 steps, respectively. The volume of activation was determined to be -0.65 ± 0.75 cm3 mol−1 at 20.2 °C by the high-pressure stopped-flow method for the apparent rate constants. 相似文献
5.
An aldo-keto reductase gene (klakr) from Kluyveromyces lactis XP1461 was cloned and heterologously expressed in Escherichia coli. The aldo-keto reductase KlAKR was purified and found to be NADH-dependent with a molecular weight of approximately 36 kDa. It is active and stable at 30 °C and pH 7.0. The maximal reaction rate (vmax), apparent Michaelis–Menten constant (Km) for NADH and t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a) and catalytic number (kcat) were calculated as 7.63 U mg−1, 0.204 mM, 4.42 mM and 697.4 min−1, respectively. Moreover, the KlAKR has broad substrate specificity to a range of aldehydes, ketones and keto-esters, producing chiral alcohol with e.e. or d.e. >99% for the majority of test substrates. 相似文献
6.
Ethanol-Induced Leakage in Saccharomyces cerevisiae: Kinetics and Relationship to Yeast Ethanol Tolerance and Alcohol Fermentation Productivity 总被引:5,自引:5,他引:0
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Ethanol stimulated the leakage of amino acids and 260-nm-light-absorbing compounds from cells of Saccharomyces cerevisiae. The efflux followed first-order kinetics over an initial period. In the presence of lethal concentrations of ethanol, the efflux rates at 30 and 36°C were an exponential function of ethanol concentration: keX = keXmeE (X-Xm), where keX and keXm are the efflux rate constants, respectively, in the presence of a concentration X of ethanol or the minimal concentration of ethanol, Xm, above which the equation was applicable, coincident with the minimal lethal concentration of ethanol. E is the enhancement constant. At 36°C, as compared with the corresponding values at 30°C, the efflux rates were higher and the minimal concentration of ethanol (Xm) was lower. The exponential constants for the enhancement of the rate of leakage (E) had similar values at 30 or 36°C and were of the same order of magnitude as the corresponding exponential constants for ethanol-induced death. Under isothermic conditions (30°C) and up to 22% (vol/vol) ethanol, the resistance to ethanol-induced leakage of 260-nm-light-absorbing compounds was found to be closely related with the ethanol tolerance of three strains of yeasts, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Saccharomyces bayanus. The resistance to ethanol-induced leakage indicates the possible adoption of the present method for the rapid screening of ethanol-tolerant strains. The addition to a fermentation medium of the intracellular material obtained by ethanol permeabilization of yeast cells led to improvements in alcohol fermentation by S. cerevisiae and S. bayanus. The action of the intracellular material, by improving yeast ethanol tolerance, and the advantages of partially recycling the fermented medium after distillation were discussed. 相似文献
7.
《Journal of Fermentation and Bioengineering》1990,69(4):215-219
Factors affecting the rates of plasmid transfer were investigated using Escherichia coli LC102 bearing a conjugative plasmid R100-1 and E. coli DH1. The rate constant of transconjugant increase, kti, was used for presenting the degree of plasmid transmissibility instead of the plasmid transfer efficiency (pte). The rate constant was defined as the specific rate of transconjugant increase (srti, the number of transconjugants per donor per h) divided by the recipient cell concentration. The kti values ranged between 10−10 and 10−15 ml cells−1 h−1, when estimated under various conditions. Moderate liquid agitation had a favorable effect on ktf but agitation rates higher than 33 s−1 (intergrated shear force) greatly decreased the value of kti. The transconjugant-forming activity of the cells growing in continuous culture did not significantly change with the dilution rate, except those growing at dilution rates less than 0.1 h−1. The rate constant kti at temperatures of 10–15°C was as low as the detection limit (10−15 ml cells−1 h−1). 相似文献
8.
《Bioorganic & medicinal chemistry letters》2019,29(17):2452-2454
Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH2 and optimized kinetic parameters (kcat = 0.059 ± 0.008 s−1, Km = 220 ± 50 µM, kcat/Km of 270 M−1s−1). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a kcat value of 0.0038 ± 0.0003 s−1, a Km value of 1100 ± 100 µM and a kcat/Km of 3 M−1s−1. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide. 相似文献
9.
《Environmental Pollution (1970)》1979,18(4):265-274
Lead from automotive sources in roadside soil and vegetation is found to follow a double exponential function of the following form: Pb = A1 e−k1D + A2 e−k2D. The terms A1 and A2 are linear functions of average daily traffic volume. The two exponents are assumed to represent two families of particles of different sizes. The larger particles are deposited within about 5 m of the roadside and are relatively inert in the soil. The smaller particles settle more slowly and are deposited within about 100 m of the roadside. Based on the differences between the relative lead content of the soil and vegetation attributable to the two exponents, the lead contained in the smaller particles is assumed to be more soluble than that of the larger ones. An estimated 72–76% of the historical lead deposited on the soil has been lost from the surface 10 cm of soil. 相似文献
10.
Abstract: A previous study of the metabolism of 6-[18F]-fluoro-l -3,4-dihydroxyphenylalanine (FDOPA) in rats pretreated with carbidopa contained information amenable to kinetic analysis. Using these data, tracer transfer coefficients and metabolic rate constants were estimated. After intravenous injection, FDOPA in circulation was O-methylated (kD0 = 0.055 min?1), and the metabolite (O-methyl-FDOPA) escaped from plasma with a rate constant (kM?1) of 0.01 min?1. The initial clearance of FDOPA to striatum (KD1) was 0.07 ml g?1 min?1, and the equilibrium distribution volume (VDe) was 0.67 ml g?1. The initial clearance of O-methyl-FDOPA to striatum (KM1) was 0.08 ml g?1 min?1, and the equilibrium distribution volume (VMe) was 0.75 ml g?1. The rate constant of FDOPA decarboxylation (kD3) was 0.17 min?1 in striatum. The elimination of 6-[18F]fluorodopamine (FDA) from striatum suggested an apparent rate constant for monoamine oxidase activity (k′7) of 0.055 min?1. 6-[18F]Fluorohomovanillic acid (FHVA) was formed from 6-[18F]fluoro-l -3,4-dihydroxyphenylacetic acid with a rate constant (k11) of 0.083 min?1, and FHVA was eliminated from striatum (k9) with a rate constant of 0.12 min?1. The steady-state concentration ratios of FDA and its metabolites were shown to be functions of these rate constants. 相似文献
11.
《Bioorganic & medicinal chemistry letters》2014,24(1):173-176
l-Arabinitol 4-dehydrogenase (LAD) from Hypocrea jecorina (HjLAD) was cloned and overexpressed in Escherichia coli BL21 (DE3). The kinetics of l-arabinitol oxidation by NAD+, catalyzed by HjLAD, was studied within the pH range of 7.0–9.5 at 25 °C. The turnover number (kcat) and the catalytic efficiency (kcat/Km) were 4200 min−1 and 290 mM−1 min−1, respectively. HjLAD showed the highest turnover number and catalytic efficiency among all previously characterized LADs. In further application of HjLAD, rare l-sugar l-xylulose was produced by the enzymatic oxidation of arabinitol to give a yield of approximately 86%. 相似文献
12.
d-Allose was considered as a kind of rare sugars with testified potential medicinal and agricultural benefits. l-Rhamnose isomerase (L-RI, EC 5.3.1.14), an aldose-ketose isomerase, played a significant part in producing rare sugar. In this article, a thermostable d-allose-producing L-RI was characterized from a thermotolerant bacterium, Thermobacillus composti KWC4. The recombinant L-RI was activated obviously in the presence of Mn2+ with an optimal pH 7.5 and temperature 65 °C. The Michaelis-Menten constant (Km), turnover number (kcat) and catalytic efficiency (kcat/Km) for l-rhamnose were 33.8 mM, 1189.8 min−1 and 35.2 min−1 mM−1, respectively. At a higher temperature, Mn2+ played a pivotal role in strengthening the thermostability of T. composti L-RI. The differential scanning calorimetry (DSC) results showed the denaturing temperature (Tm) of T. composti L-RI was increased by 3 °C in presence of Mn2+. Although the T. composti L-RI displayed the optimum substrate as l-rhamnose, it could also effectively catalyze the isomerization between d-allulose and d-allose. When the reaction reached equilibrium, the sole product d-allose was produced from D-alluose by T. composti L-RI. 相似文献
13.
《Inorganica chimica acta》1986,123(3):175-179
The palladium(II)-promoted hydrolysis of the methyl esters of glycyl-L-leucine, glycyl-L-alanine and L-alanylglycine have been studied at 25 °C and I=0.1 M in the pH range 4–5. At a 1:1 metal to ligand ratio the peptide esters act as tridentate ligands, donation occurring via the terminal amino group, the deprotonated amide nitrogen, and the carbonyl group of the ester. Due to the high Lewis acidity of Pd(II) rapid hydrolysis of the ester function by water and hydroxide ion occurs. Rate constants kOH and kH2O have been obtained for base hydrolysis and water hydrolysis of the coordinated peptide esters at 25 °C. The rate constants for base hydrolysis are 3.4 X 106 M−1 s−1 (L-alaglyOMe), 6.4 X 106 M−1 s−1 (gly-L-alaOMe) and 2.3 X 107 M−1 s−1 (gly-L-leuOMe). Base hydrolysis of the coordinated peptide esters is at least 106 times that of the free unprotonated ligand. Activation parameters have been obtained for both water and base hydrolysis of the Pd(II) complex of methyl L-alanylglycinate and possible mechanisms for the hydrolyses are considered. 相似文献
14.
I.Kofi Adzamli Henry Ong Wah Kim A.Geoffrey Sykes George V. Buxton 《Journal of inorganic biochemistry》1982,16(4):311-317
The reaction of parsley 2Fe-2S ferredoxin in the normal oxidized state with eaq? generated by pulse radiolysis techniques has been studied at ~25°C, pH 7–8, I = 0.10 M (NaClO4). Rate constants ke (eaq? decay) and kp (protein absorbance change) are the same, second-order rate constant 9.7 × 109 M?1 sec?1. The reaction exhibits close to 100% efficiency. With 8Fe-8S ferredoxin from Clostridium pasteurianum under identical conditions it now appears that kp (although sometimes significantly smaller) is equal to ke. Varying efficiencies are also observed with this protein depending on the batch used. The reasons for such variable behavior are not fully understood. With oxidized and reduced forms of Chromatium v. high-potential iron-sulfur protein (HIPIP), ke and kp are essentially the same, but the highest efficiency observed is only ~50%. The prevailing pattern is therefore that rate constants ke and kp are generally in step for proteins having a single (or identical) active site(s). When the active site is buried as with HIPIP the efficiency of the reaction appears to decrease. 相似文献
15.
Yuliana Raynova Lyuba Doumanova Krassimira Nikolova Idakieva 《The protein journal》2013,32(8):609-618
The oxygen-transporting protein, hemocyanin (Hc), of the garden snail Helix aspersa maxima (HaH) was isolated and kinetically characterized. Kinetic parameters of the reaction of catalytic oxidation of catechol to quinone, catalyzed by native HaH were determined: the V max value amounted to 22 nmol min?1 mg?1, k cat to 1.1 min?1. Data were compared to those reported for other molluscan Hcs and phenoloxidases (POs). The o-diphenoloxidase activity of the native HaH is about five times higher than the activity determined for the Hcs of the terrestrial snail Helix pomatia and of the marine snail Rapana thomasiana (k cat values of 0.22 and 0.25 min?1, respectively). The K m values obtained for molluscan Hcs from different species are comparable to those for true POs, but the low catalytic efficiency of Hcs is probably related to inaccessibility of the active sites to potential substrates. Upon treatment of HaH with subtilisin DY, the enzyme activity against substrate catechol was considerably increased. The relatively high proteolytically induced o-diPO activity of HaH allowed using it for preparation of a biosensor for detection of catechol. 相似文献
16.
《Bioorganic & medicinal chemistry》2016,24(21):5592-5601
An amino acid based and bidentate Schiff base, (E)-methyl 2-((2-oxonaphthalen-1(2H)-ylidene)methylamino)acetate (ligand), was synthesized from the reaction of glycine-methyl ester hydrochloride with 2-hydroxy-1-naphthaldehyde. Characterization of the ligand was carried out using theoretical quantum–mechanical calculations and experimental spectroscopic methods. The molecular structure of the compound was confirmed using X-ray single-crystal data, NMR, FTIR and UV–Visible spectroscopy, which were in good agreement with the structure predicted by the theoretical calculations using density functional theory (DFT). Antimicrobial activity of the ligand was investigated for its minimum inhibitory concentration (MIC) to several bacteria and yeast cultures. UV–Visible spectroscopy studies also shown that the ligand can bind calf thymus DNA (CT-DNA) electrostatic binding. In addition, DNA cleavage study showed that the ligand cleaved DNA without the need for external agents. Energetically most favorable docked structures were obtained from the rigid molecular docking of the compound with DNA. The compound binds at the active site of the DNA proteins by weak non-covalent interactions. The colorimetric response of the ligand in DMSO to the addition of equivalent amount of anions (F−, Br−, I−, CN−, SCN−, ClO4−, HSO4−, AcO−, H2PO4−, N3− and OH−) was investigated and the ligand was shown to be sensitive to CN− anion. 相似文献
17.
《Inorganica chimica acta》1988,152(2):91-99
The reactions of PtCl2en or cis-Pt(NH3)2Cl2 and their aqua species with adenine and adenosine were studied by means of ion-pair HPLC. From the chromatograms, it was found that the first binding site of Pt(II) was the N(7) site of adenine under both acidic and neutral conditions. The rates of Pt(II) binding at the (N7) site of adenosine and deoxyadenosine were measured. The rate constants, k1, were obtained for the reactions of PtCl2en or cis-Pt(NH3)2Cl2 with adenosine and deoxyadenosine at pH 3 and 7 over the temperature range 9–25 °C. The k1 values were 6.8–7.7 × 10−4 dm3 mol−1 s−1 at 25 °C. For the aqua species, the rate of [cis-Pt(NH3)2ClH2O]+ with adenosine N(7) was measured. The rate constants, k2 which were found to be smaller than those of hydrolysis, kh, were calculated at pH 3 over the temperature range 25–40 °C. The k2 value obtained at 25 °C was 1.1 × 10−2 dm3 mol−1 s−1, 15 time larger than k1. The activation parameters were also calculated. 相似文献
18.
Numerous physiological aldehydes besides glucose are substrates of aldose reductase, the first enzyme of the polyol pathway which has been implicated in the etiology of diabetic complications. The 2-oxoaldehyde methylglyoxal is a preferred substrate of aldose reductase but is also the main physiological substrate of the glutathione-dependent glyoxalase system. Aldose reductase catalyzes the reduction of methylglyoxal efficiently (kcat=142 min−1 and kcat/Km=1.8×107 M−1 min−1). In the presence of physiological concentrations of glutathione, methylglyoxal is significantly converted into the hemithioacetal, which is the actual substrate of glyoxalase-I. However, in the presence of glutathione, the efficiency of reduction of methylglyoxal, catalyzed by aldose reductase, also increases. In addition, the site of reduction switches from the aldehyde to the ketone carbonyl. Thus, glutathione converts aldose reductase from an aldehyde reductase to a ketone reductase with methylglyoxal as substrate. The relative importance of aldose reductase and glyoxalase-I in the metabolic disposal of methylglyoxal is highly dependent upon the concentration of glutathione, owing to the non-catalytic pre-enzymatic reaction between methylglyoxal and glutathione. 相似文献
19.
《Inorganica chimica acta》1987,138(1):61-65
The kinetics and mechanism of a linear trihydroxamic acid siderophore (deferriferrioxamine B, H4DFB+) ligand exchange with Al(H2O)63+ to form mono(deferriferrioxamine B)aluminum(III) (Al(H2O)4H3DFB)3+ have been investigated at 25 °C over the [H+] range 0.001−1.0 M and I = 2.0 M (HClO4/NaClO4) by 27Al NMR. Kinetic results are consistent with Al(H2O)4(H3DFB)3+ formation and dissociation proceeding through a parallel path mechanistic scheme involving Al(H2O)63+(k2/k−1) and Al(H2O)5(OH)2+(k2/k−2) where k1 = 0.13 M−1 s−1, k−1 = 8.7 × 10−3 M−1 s−1, k2 = 2.7 × 103 M−1 s−1, and k−2 = 9.6 × 10−4 s−1. Relative complex formation rates at Al(H2O)63+ and Al(H2O)5OH2+, and comparison with kinetic data for a series of synthetic hydroxamic acids, suggest that an interchange mechanism is operative. These results are also discussed in relation to kinetic data for the corresponding iron(III)-deferriferrioxamine B system. 相似文献
20.
《Bioorganic & medicinal chemistry》2014,22(20):5569-5577
AurF catalyzes the N-oxidation of p-aminobenzoic acid to p-nitrobenzoic acid in the biosynthesis of the antibiotic aureothin. Here we report the characterization of AurF under optimized conditions to explore its potential use in biocatalysis. The pH optimum of the enzyme was established to be 5.5 using phenazine methosulfate (PMS)/NADH as the enzyme mediator system, showing ∼10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a Km of 14.7 ± 1.1 μM, a kcat of 47.5 ± 5.4 min−1 and a kcat/Km of 3.2 ± 0.4 μM−1 min−1. PMS/NADH and the native electron transfer proteins showed significant formation of the p-hydroxylaminobenzoic acid intermediate, however H2O2 produced mostly p-nitrobenzoic acid. Alanine scanning identified the role of important active site residues. The substrate specificity of AurF was examined and rationalized based on the protein crystal structure. Kinetic studies indicate that the Km is the main determinant of AurF activity toward alternative substrates. 相似文献