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1.
The rate of ethanol elimination in fed and fasted rats can be predicted based on the liver content of alcohol dehydrogenase (EC 1.1.1.1), the steady-state rate equation, and the concentrations of substrates and products in liver during ethanol metabolism. The specific activity, kinetic constants, and multiplicity of enzyme forms are similar in fed and fasted rats, although the liver content of alcohol dehydrogenase falls 40% with fasting. The two major forms of the enzyme were separated and found to have very similar kinetic properties. The rat alcohol dehydrogenase is subject to substrate inhibition by ethanol at concentrations above 10 mM and follows a Theorell-Chance mechanism. The steady-state rate equation for this mechanism predicts that the in vivo activity of the enzyme is limited by NADH product inhibition at low ethanol concentrations and by both NADH inhibition and substrate inhibition at high ethanol concentrations. When the steady-state rate equation and the measured concentrations of substrates and products in freeze-clamped liver of fed and fasted rats metabolizing alcohol are employed to calculate alcohol oxidation rates, the values agree very well with the actual rates of ethanol elimination determined in vivo.  相似文献   

2.
The supply and consumption of metabolites in living cells are catalyzed by enzymes. Here we consider two of the simplest schemes where one substrate is eliminated through Michaelis-Menten kinetics, and where two types of substrates are joined together by an enzyme. It is demonstrated how steady-state substrate concentrations can change ultrasensitively in response to changes in their supply rates and how this is coupled to slow relaxation back to steady state after a perturbation. In the one-substrate system, such near-critical behavior occurs when the supply rate approaches the maximal elimination rate, and in the two-substrate system it occurs when the rates of substrate supply are almost balanced. As systems that operate near criticality tend to display large random fluctuations, we also carried out a stochastic analysis using analytical approximations of master equations and compared the results with molecular-level Monte Carlo simulations. It was found that the significance of random fluctuations was directly coupled to the steady-state sensitivity and that the two substrates can fluctuate greatly because they are anticorrelated in such a way that the product formation rate displays only small variation. Basic relations are highlighted and biological implications are discussed.  相似文献   

3.
Basic amino acid calix[8]arene receptors for tryptase surface recognition have been synthesized. The tetrameric arrangement and the negative charge distribution close to the active sites of the enzyme, have suggested the design of complementary multifunctional receptors that might bind to the active region of the protein blocking the approach of the substrate. Kinetic inhibition analysis on recombinant lung tryptase have showed a time-dependent competitive inhibition with both initial and steady-state rate constants in the nanomolar range.  相似文献   

4.
Rat liver microsomes converted retinol into retinal and retinoic acid. The production of retinal was observed over a range of substrate concentrations (10-100 microM), but retinoic acid was detected only at retinol concentrations of 50 microM or higher. At 50 microM retinol, the rate of microsomal retinal production was 2-fold greater than that of cytosol, but the rate of retinoic acid synthesis was 4-fold less than that of cytosol. Retinal was also converted into retinoic acid by rat liver microsomes, but at a rate 2-5% of that catalyzed by cytosol. Microsomes also interfered with the conversion of retinol and retinal into retinoic acid by rat liver cytosol. A 50% decrease in the cytosolic rates of retinoic acid production from retinol or retinal was caused by microsomal to cytosolic protein ratios of 0.1 and 0.5, respectively. Under the incubation conditions, which included NAD in the medium, addition of microsomes to cytosol did not affect the elimination half-life of retinol or retinoic acid, but did decrease the elimination half-life of retinal by 2-fold. These data show that retinal synthesis from retinol does not necessarily reflect retinoic acid synthesis and suggest that liver microsomes sequester free retinol and convert it into retinal primarily for elimination, rather than to serve as substrate for cytosolic retinoic acid synthesis.  相似文献   

5.
D L Anton  R Kutny 《Biochemistry》1987,26(20):6444-6447
S-Adenosylmethionine decarboxylase, a pyruvoyl-containing decarboxylase, is inactivated in a time-dependent process under turnover conditions. The inactivation is dependent on the presence of both substrate and Mg2+, which is also required for enzyme activity. The rate of inactivation is dependent on the concentration of substrate and appears to be saturable. Inactivation by [methionyl-3,4-14C]-adenosylmethionine results in stoichiometric labeling of the protein. In contrast, when either S-[methyl-3H]adenosylmethionine or [8-14C]adenosylmethionine is used, there is virtually no incorporation of radioactivity. Automated Edman degradation of the alpha (pyruvoyl-containing) subunit reveals that substrate inactivation results in the conversion of the pyruvoyl group to an alanyl residue. These data suggest a mechanism of inactivation which involves the transamination of the nascent product to the pyruvoyl group, followed by the elimination of methylthioadenosine and the generation of a 2-propenal equivalent which could undergo a Michael addition to the enzyme. This is the first evidence for a transamination mechanism for substrate inactivation of a pyruvoyl enzyme.  相似文献   

6.
A spectrophotometric assay is proposed to determine the levels of aspartate aminotransferase (AAT) isoenzymes from chicken liver by a steady-state kinetic method which depends on the differential inhibition of these isoenzyme forms by high concentrations of substrate 2-oxoglutarate at pH 6.2. The use of a standard curve permits the determination of the percentage of chicken liver c-AAT and m-AAT isoenzymes. This method yields results in good correlation with those achieved by different extent adipate inhibition and by differential centrifugation.  相似文献   

7.
Apoptosis of infected cells represents a key host defense mechanism against viral infections. The impact of apoptosis on the elimination of hepatitis C virus (HCV)-infected cells is poorly understood. The TRAIL has been implicated in the death of liver cells in hepatitis-infected but not in normal liver cells. To determine the impact of TRAIL on apoptosis of virus-infected host cells, we studied TRAIL-induced apoptosis in a tissue culture model system for HCV infection. We demonstrated that HCV infection sensitizes primary human hepatocytes and Huh7.5 hepatoma cells to TRAIL induced apoptosis in a dose- and time-dependent manner. Mapping studies identified the HCV nonstructural proteins as key mediators of sensitization to TRAIL. Using a panel of inhibitors targeting different apoptosis pathways, we demonstrate that sensitization to TRAIL is caspase-9 dependent and mediated in part via the mitochondrial pathway. Sensitization of hepatocytes to TRAIL-induced apoptosis by HCV infection represents a novel antiviral host defense mechanism that may have important implications for the pathogenesis of HCV infection and may contribute to the elimination of virus-infected hepatocytes.  相似文献   

8.
J P Richard 《Biochemistry》1991,30(18):4581-4585
Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

9.
The first processing event that mouse pre-rRNA undergoes occurs within the external transcribed spacer and is efficiently reproduced in vitro. Analysis with nondenaturing polyacrylamide gels revealed the formation of heparin-resistant complexes of retarded electrophoretic mobility on the substrate rRNA. The specificity of these complexes was demonstrated by their elimination due to competition with processing-competent, but not with processing-incompetent, rRNAs. Furthermore, complex formation, like the processing cleavage, required only 28 nucleotides of rRNA sequence adjacent to the processing site but was stimulated by additional downstream conserved sequences. These processing complexes formed in a time-dependent manner, and once assembled, they were stable to challenge by competitor rRNA and remained on the processed rRNA. Their sedimentation coefficient was approximately 20S. UV cross-linking studies with 4-thiouridine-substituted rRNA have identified six polypeptides, 52 to 250 kilodaltons, that are specifically bound to the rRNA processing substrate.  相似文献   

10.
An equation describing the instantaneous velocity of an ordered bimolecular enzymatic reaction that exhibits inhibition by substrate and product was derived. Using kinetic constant values for horse liver alcohol dehydrogenase, the velocity expression was applied to an open-reaction system. The calculated steady-state surfaces displayed regions of bistability, which further substantiates the link between substrate inhibition and multiple steady states. This general computational approach may be applied to any system that can be described by an instantaneous velocity equation.  相似文献   

11.
N-(1-Methyl)cyclopropylbenzylamine was synthesized and shown to inactivate pig liver mitochondrial monoamine oxidase. Inactivation is time-dependent, concentration dependent, protected by the substrate and product of the enzyme, and is not reversed by exhaustive dialysis. Unlike N-cyclopropylbenzylamine, the adduct formed between N-(1-methyl)cyclopropylbenzylamine and monoamine oxidase is stable to treatment with benzylamine. A mechanism for the inactivation is proposed.  相似文献   

12.
We have examined the steady-state and time-dependent electrical properties of a model membrane system. The model assumes that the directed velocity and energy of ions moving through the membrane are determined by the applied electric field, ionic diffusion forces, and central elastic collisions between ions and membrane molecules. A simple analysis of the steady-state electrical properties of the model yields results identical with ones obtained previously using a more complex analysis procedure. The time-dependent conductance changes of the model in response to a step change in electric field strength when there is solution symmetry display three qualitative patterns dependent on the nature of the ion-membrane molecule interaction. One of the patterns of conductance change is quite similar to that observed in the sodium conductance system of a number of excitable tissues: an initial conductance rise to a maximum (activation) followed by a decay to a final steady-state value (inactivation). However, the correspondence between the time-dependent model behavior and known experimental behavior of excitable systems is only qualitative. We conclude that the classical ion-membrane molecule interactions we consider are not involved in determining time-dependent conductance processes in the excitable systems for which comparison is possible.  相似文献   

13.
A cell-free system consisting of rat liver mitochondria, liver cytosol, lactate, and the substrates intrinsic to the malate-aspartate shuttle was reconstituted for studies of steady-state substrate fluxes and, more specifically, to evaluate further the mechanism of control of the intra- and extramitochondrial steady states of the free NAD+/NADH ratios. Soluble (F1) ATPase or 2,4-dinitrophenol (DNP) were added in varying amounts to alter substrate fluxes and the constant energy state of this 'open' metabolizing system. The steady-state redox segregation (1.36 log NAD+/NADH ratio out vs NAD+/NADH in the mitochondrial matrix) was maximally about 3 kcal, and declined together with the membrane potential (delta psi) and log ATP/ADP, which obtain on imposing an increasing energy load on the system. It is concluded that transmembrane movement of reducing equivalents is coupled to electron transfer through delta psi, mediated by the electrogenic exchange of glutamate and aspartate. When delta psi was high (near State 4), delta G redox was approximately the same as that generated without flux of reducing equivalents [E. J. Davis, J. Bremer, and K. E. Akerman (1980) J. Biol. Chem. 255, 2277-2283], suggesting that delta Gredox is in near thermodynamic equilibrium with delta psi. If the steady-state ATP/ADP ratio was altered with an energy load (F1-ATPase), delta Gredox decreased more steeply than delta psi (tetraphenyl phosphonium-sensitive electrode used to measure delta psi). At comparable ranges of ATP/ADP, both delta Gredox and delta psi decreased more steeply with uncoupler than with an external ADP-regenerating system.  相似文献   

14.
A steady-state biofilm is defined as one that has neither net growth nor decay over time. The model, developed for steady-state-biofilm kinetics with a single substrate, couples the flux of substrate into a biofilm to the mass (or thickness) of biofilm that would exist at steady-state for a given bulk substrate concentration. Based on kinetic and energetic constraints, this model predicts for a single substrate that a steady-state bulk concentration, Smin, exists below which a steady-state biofilm cannot exist. Thus, in the absence of adsorption of bacteria from the bulk water and for substrate concentration below Smin, substrate flux and biofilm thickness are zero. Equations are provided for calculating the steady-state substrate flux and biofilm thickness for S greater than Smin. An example is provided to demonstrate the use of the steadystate model.  相似文献   

15.
Two forms of glutathione S-aryltransferase were purified from rat liver. The only differences noted between the two forms were in the chromatographic and electrophoretic properties, which permitted the separation of the two species. The molecular weights of the enzyme and its subunits were estimated as about 50000 and 23000 respectively. The steady-state kinetics did no follow Michaelis-Menten kinetics when one substrate concentration was kept constant while the second substrate concentration was varied. Several S-substituted GSH derivatives were tested as inhibitors of the enzymic reaction. The enzyme was inactivated by thiol-group reagents.  相似文献   

16.
1. A method for the isolation of functionally intact mitochondria from lymphocytes is described. It involves digitonin breakage of the plasma membrane, followed by differential centrifugation. The yield was 36 mg of mitochondrial protein/200 g of pig mesenteric lymph node (6 mg of mitochondrial protein/10(9) lymphocytes). The mitochondrial had a respiratory-control ratio of 2--3.5 with succinate as substrate. 2. Ca2+ transport by these mitochondria was investigated. They were able to regulate the extramitochondrial free [Ca2+] very precisely, by buffering any displacements from the steady-state. The exact extramitochondrial free [Ca2+] of this steady-state depended on the conditions of incubation. In a medium designed to resemble the cytoplasmic environment, with added Ca2+, lymphocyte mitochondria maintained a steady-state free [Ca2+] of 0.63 microM (pCa of 6.2). The rates of Ca2+ uptake and efflux under these conditions, with both lymphocyte and liver mitochondria, were very much lower than those in a less complex medium. 3. Lymphocyte mitochondria were shown to possess an Na+-independent Ruthenium Red-insensitive efflux pathway similar to that of liver mitochondria. Ruthenium Red totally inhibited the electrophoretic uniporter. Although Na+ had no effect on the steady-state maintained by lymphocyte mitochondria, they were shown to possess an Na+/H+ antiporter.  相似文献   

17.
E K Fetisova 《Ontogenez》1988,19(5):481-486
Interaction of several lines of epithelial cells was studied in a mixed culture: FBT (bovine fetal trachea), MDSK (dog kidney), IAR-2 (rat liver), MPTR (SV-40 transformed mouse kidney). During mixed cultivation epithelia cells of different types were capable to force out each other from the substrate to full elimination. This capacity correlated with the pattern of cell contacts with the substrate. It is supposed that epithelial cells can form lamellae at the lower surfaces competing for substrate. Those cells which have lamellae with continuous marginal focal contacts and numerous focal contacts eliminate the cells having lamellae with few focal contacts.  相似文献   

18.
The subunit pattern and the steady-state kinetics of cytochrome-c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome-c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus-encoded subunits of the various cytochrome-c oxidase preparations. Tissue homogenates, in which cytochrome-c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome-c oxidase steady-state kinetics. Cytochrome-c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady-state reaction between human ferrocytochrome c and the four human cytochrome-c oxidase preparations revealed large differences for the low-affinity TNmax (maximal turnover number) value, ranging from 77 s-1 for kidney to 273 s-1 for liver cytochrome-c oxidase at pH 7.4, I = 18 mM. It is proposed that the low-affinity kinetic phase reflects an internal electron-transfer step. For the steady-state reaction of human heart cytochrome-c oxidase with human cytochrome c, Km and TNmax values of 9 microM and 114 s-1 were found, respectively, at high ionic strength (I = 200 mM, pH 7.4). Only minor differences were observed in the steady-state activity of the various human cytochrome-c oxidases. The interaction between human cytochrome-c oxidase and human cytochrome-c proved to be highly specific. At high ionic strength, a large decrease in steady-state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady-state TNmax and Km parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome-c oxidase deficiency.  相似文献   

19.
M A Levy  M Brandt  A T Greway 《Biochemistry》1990,29(11):2808-2815
A solubilized preparation of steroid 5 alpha-reductase (EC 1.3.1.30) from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase.  相似文献   

20.
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