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1.
S. Brennan J. Holder J. Baldwin 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,110(4):689-695
Lactate dehydrogenase (LDH) was purified from the siphon muscle of the intertidal ascidian Pyura stolonifera. The enzyme is unique among chordate LDHs but resembles some bacterial and platyhelminth LDHs in being activated by fructose 1,6-bisphosphate (FBP). Concentrations of FBP in the range 5μM to 0.5 mM increase Vmax of the pyruvate reductase reaction by 130% to 210%, and decrease Km pyruvate 5 to 11 fold and Km NADH 2.5 to 5 fold. The enzyme is also activated by inorganic phosphate, but requires a 50 fold higher concentration to attain the maximum activation achieved by 0.5 mM FBP. Of a range of metabolites tested, including other glycolytic sugar phosphates, only FBP and inorganic phosphate activated the enzyme. FBP activation was not observed with 16 representative vertebrate LDH homotetramers, but did occur to a limited extent with LDH from an echinoderm. LDH was the only pyruvate reductase enzyme detected in P. stolonifera siphon muscle, and its activity was much greater than that of phosphorylase or phosphofructokinase. The LDH reaction is utilized by P. Stolonifera during prolonged siphon closure on exposure to air when lactate, but not succinate, accumulates in the siphon muscle. While the ascidian enzyme provides the first example of a FBP activated LDH from a chordate, it remains to be determined if this unusual property has any role in metabolic regulation. 相似文献
2.
A number of kinetic parameters of the lactate dehydrogenases of three rumen bacterial species (Peptostreptococcus productus, Propionibacterium acnes and Actinomyces viscosus), the rumen ciliate Isotricha prostoma and mixed rumen microorganisms (MRM) with respect to NADH, pyruvate, fructose-1,6-diphosphate (FDP) as well as the effects of several nucleotide phosphates were studied.Partially purified LDH of Peptostr. productus had the same kinetic parameters as in crude cell free extracts. Values for Km, determined by Michaelis-Menten kinetics with pyruvate as the substrate, were in the same range for all lactate dehydrogenases. After feeding a cow, changes in the apparent Km and Vmax values for NADH of the total LDH activity in MRM were followed.It is suggested that of the factors studied the ratio NADH/NAD(H) and ATP are the most important regulatory factors for the lactate dehydrogenases of mixed rumen microorganisms.The investigation were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO) 相似文献
3.
Kenneth B. Storey 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1985,155(3):339-345
Summary Pyruvate kinases from flight muscle and fat body of the cockroach,Periplaneta americana, were purified to homogeneity. The two tissues contained different forms of the enzyme which were separable by starch gel electrophoresis and isoelectric focusing (pI=5.75 for flight muscle and 6.15 for fat body). Both enzymes had molecular weights of 235,000±20,000.Flight muscle pyruvate kinase displayed Michaelis-Menten kinetics with respect to both ADP and P-enolpyruvate withK
m values of 0.27 and 0.04 mM, respectively.K
m for Mg2+ was 0.60 mM andK
a for K+ was 15 mM. The enzyme was weakly inhibitied by four compounds, ATP, arginine-P,l-alanine and citrate with apparentK
i values of 3.5, 15, 20 and 24 mM, respectively. Competitive inhibition by 3 mM ATP or 10 mM arginine-P raised theK
m for P-enolpyruvate to 0.067 or 0.057 mM. Fructose-1,6-P2 did not activate the enzyme but reversed inhibitions by ATP and arginine-P.Fat body pyruvate kinase showed sigmoidal kinetics with respect to P-enolpyruvate with S0.5=0.32 mM andn
H=1.43.K
m values for ADP and Mg2+ were 0.30 and 0.80 mM, respectively with aK
a for K+ of 10 mM. ATP andl-alanine were inhibitors of the enzyme; 2 mM ATP raised S0.5 for P-enolpyruvate to 0.48 mM while 3 mMl-alanine increased S0.5 to 0.84 mM. Neither citrate nor arginine-P inhibited the enzyme but citrate affected the enzyme by reversingl-alanine inhibition. Fat body pyruvate kinase was strongly activated by fructose-1,6-P2 with an apparentK
a of 1.5 M. Fructose-1,6-P2 at 0.1 mM reduced S0.5 for P-enolpyruvate to 0.05 mM andn
H to 1.0.Flight muscle and fat body pyruvate kinases from the cockroach show properties analogous to those of the muscle and liver forms of mammalian pyruvate kinase. Fat body pyruvate kinase is suited for on-off function in a tissue with a gluconeogenic capacity. Strong allosteric control with a feed-forward activation by fructose-1,6-P2 is key to coordinating enzyme function with glycolytic rate. The function of flight muscle pyruvate kinase in energy production during flight is aided by a lowK
m for P-enolpyruvate, weak inhibitor effects by high energy phosphates and deinhibition of these effects by fructose-1,6-P2. 相似文献
4.
Mario Klimacek Regina Kratzer Margarete Szekely 《Biocatalysis and Biotransformation》2013,31(2-4):194-201
The 2.2 Å X-ray crystal structure of Candida tenuis xylose reductase (AKR2B5) bound with NADP+ reveals that Phe-114 contributes to the substrate binding pocket of the enzyme. In the related human aldose reductase (AKR1B1), this phenylalanine is replaced by a tryptophan. The side chain of Trp was previously implicated in forming a hydrogen bond with bound substrate or inhibitor. The apparent Michaelis constant of AKR2B5 for xylose (Km≈90 mM) is 60 times that of AKR1B1, perhaps because critical enzyme–substrate interactions of Trp are not available to Phe-114. We, therefore, prepared a Phe-114→Trp mutant (F114W) of AKR2B5, to mimic the aldose reductase relationship in xylose reductase. Detailed analysis of the kinetic consequences in purified F114W revealed that the Km values for xylose and xylitol at pH 7.0 and 25°C were increased 5.1- and 4.4-fold, respectively, in the mutant compared with the wild-type. Turnover numbers (kcat) of F114W for xylose reduction and xylitol oxidation were half those of the wild-type. Apparent dissociation constants of NADH (KiNADH=44 µM) and NAD+ (KiNAD+=177 µM) were increased 1.6- and 1.4-fold in comparison with values of KiNADH and KiNAD+ for the wild-type, respectively. Catalytic efficiencies (kcat/Km) for NADH-dependent reduction of different aldehydes were between 3.1- and 31.5-fold lower than the corresponding kcat/Km values of the wild-type. Therefore, replacement of Phe-114 with Trp weakens rather than strengthens apparent substrate binding by AKR2B5, suggesting that xylose reductase exploits residue 114 in a different manner from aldose reductase. 相似文献
5.
Michael Guppy P. W. Hochachka 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1979,129(2):185-191
Summary The skipjack tuna maintains its red skeletal musculature well above ambient temperatures while the temperature of the heart is within 1°C of that of the water. These two tissues exhibit tissue specific forms of pyruvate kinase. The red muscle has one form while the heart has two.TheK
m(PEP) of the red muscle enzymes rises with temperature, within the normal temperature range of the tissue. The affinity of the major form of the heart enzyme for phosphoenolpyruvate is relatively independent of temperature over the physiological temperature range.K
m(ADP) values are temperature independent for both enzymes.Inhibition by alanine of both enzymes is temperature dependent and rises with temperature. The same is true of ATP inhibition of the heart enzyme, but ATP inhibition of the red muscle enzyme is temperature independent. Fructose diphosphate reverses alanine and ATP inhibition at all temperatures.With both enzymes, temperature affects substrate affinities and the sensitivity of the enzyme to metabolite effectors. These effects can be rationalized in terms of physiological significance only in the case of the red muscle enzyme.List of abbreviations ADP
adenosine diphosphate
- ATP
adenosine triphosphate
- EDTA
ethylene diamine tetra acetic acid
- FDP
fructose 1,6 diphosphate
- LDH
lactate dehydrogenase
- NADH
nicotinamide adenine dinucleotide (reduced)
- NAD
nicotinamide adenine dinucleotide (oxidized)
- PEP
phosphoenol pyruvate 相似文献
6.
Joseph F. Siebenaller George N. Somero 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1979,129(4):295-300
Summary The pressure sensitivities of substrate (pyruvate) and cofactor (NADH) binding and catalytic rate of purified muscle-type (M4) lactate dehydrogenases (LDH, EC 1.1.1.27; NAD+: lactate oxidoreductase) from shallow- and deep-living teleost fishes were compared. The LDH's of the shallow species are significantly more pressure-sensitive than the LDH's of the deep-living fishes. The apparent Michaelis constant (K
m)1 of pyruvate of the deep-living species' LDH's is pressure-insensitive over the entire pressure range used in these studies, 1 to 476 atmospheres (Fig. 1). For the LDH's of the shallow species, theK
m of pyruvate increases significantly between 1 and 68 atmospheres, and then remains stable up to 476 atmospheres. TheK
m of NADH displays a much higher pressure sensitivity. For the LDH's of the deep species, theK
m of NADH increases slightly (approximately 32%) between 1 and 68 atmospheres, and then remains stable up to 476 atmospheres (Fig. 1). TheK
m of the shallow species' LDH's rises sharply (approximately 113%) between 1 and 68 atmospheres, and then continues to increase at a slower rate up to 476 atmospheres. This marked inhibition of cofactor binding by pressure for the shallow species' LDH's may be of sufficient magnitude to seriously impair the function of these LDH's at pressures typical of those encountered by the deeper-living species.Pressure effects on optimal velocity, measured under high (optimal) concentrations of pyruvate and NADH, were generally lower for the LDH's of the deep species (Table 1).These results indicate that M4-LDH's of shallow water fishes are not pre-adapted for function at deepsea pressures, and that the reduction of pressure sensitivities ofK
m's and catalysis may be a ubiquitous feature of adaptation to life at depth. The virtually identical pressure responses of M4-LDH's from deepliving teleosts belonging to four different families represents a striking example of convergent evolution at the molecular level. 相似文献
7.
M. Boll 《Archives of microbiology》1969,66(4):374-388
Summary Glutathione reductase (NADPH1: glutathione oxidoreductase (EC 1.6.4.2) was purified 70 fold from Rhodospirillum rubrum by ammonium sulfate fractionation, gelfiltration with Sephadex and chromatography on DEAE-cellulose. The optimum pH of the reaction is 7.5–8.2 K
m
values of 8.4×10–6 M for NADPH and 5.8×10–5 M for GSSG were determined. The kinetic data indicate a bisubstrate reaction mechanism. The prosthetic group is FAD (K
m
1.1×10–6M). The flavin can be completely dissociated from the enzyme, and 70% of the original activity can subsequently be restored by FAD. The molecular weight was determined with a calibrated column Sephadex G-200 and found to be approximately 63,000. The enzyme is inhibited reversibly by several anions. With iodide the inhibition is competitive with respect to GSSG. Sulfhydryl reagents (N-ethylmaleinimide, p-chlormercuribenzoate) strongly inhibit the enzyme when it is present in the reduced state. The enzyme is reduced by low concentrations of NADPH and by higher concentrations of NADH. GSSG protects the enzyme against this inhibition. The enzyme is reversibly inhibited by incubation with NADPH or NADH.
Zusammenfassung Glutathionreduktase wurde aus Rhodospirillum rubrum mit Ammoniumsulfatfraktionierung, Gelfiltration mit Sephadex und Chromatographie an DEAE-Cellulose 70 fach angereichert. Das pH Optimum der Reaktion liegt bei 7,5–8,2. K m -Werte: 8,4·10–6 M für NADPH und 5,8·10–5 M für GSSG. Aus den kinetischen Daten ergibt sich für das Enzym ein Bisubstratreaktionsmechanismus. Die prosthetische Gruppe ist FAD (K m 1,1·10–6 M). Das Flavin kann vollständig vom Enzymprotein abdissoziiert werden, durch erneute Zugabe von FAD können etwa 70% der ursprünglichen Aktivität zurückerhalten werden. Das Molekulargewicht, bestimmt durch Gelfiltration mit einer kalibrierten Säule Sephadex G-200, ist ca. 63000. Das Enzym wird durch verschiedene Anionen reversibel gehemmt. Bei J– ist die Hemmung kompetitiv mit GSSG. Sulfhydrylreagentien (N-Äthylmaleinimid und p-Chlomercuribenzoat) sind potente Inhibitoren, wenn das Enzym im reduzierten Zustand vorliegt. Das Enzym kann bereits durch niedrige Konzentrationen an NADPH sowie durch höhere Konzentrationen an NADH reduziert werden. GSSG schützt das Enzymprotein gegen die Hemmung durch Sulfhydryl-reagentien. Das Enzym wird durch Inkubation mit NADPH und NADH reversibel gehemmt.相似文献
8.
9.
The influence of Ca homoionic clay minerals (montmorillonite, illite, and kaolinite) on the activity,K
m
, andV
m
values of acid phosphatase was examined in model experiments. At each substrate (p-nitrophenyl phosphate) level tested, the addition of increasing amounts of clays (50, 100, and 150 mg, respectively) decreased the activity and increased theK
m
value from 1.43×10–3
m PNP (in the soluble state) to 82.3×10–3M (montmorillonite), 8.02×10–3
m (kaolinite), and 7.65×10–3
m (illite) at the 150 mg level. The maximum enzyme reaction velocity (V
m
) remained nearly constant at different amounts of kaolinite and illite, but increased remarkably with rising quantities of montmorillonite. Apparently, the substrate affinity of sorbed acid phosphatase is significantly lower with montmorillonite than with kaolinite or illite. This may be ascribed to an intensive sorption of both substrate and enzyme to the surface as well as to interlattice sites of montmorillonite. 相似文献
10.
Reiner Gromes Harry Schwartz Martin Heinrich Walther Johannssen 《Applied microbiology and biotechnology》1991,35(4):491-495
Summary Several yeast strains were assayed for occurence of nitrate reductase after growth in a defined medium with nitrate as the sole nitrogen source, Candida boidinii DSM 70026, showing the highest specific activity, was further investigated. The procedures for yeast fermentation and nitrate reductase purfication are described in detail. Nitrate reductase from this yeast was characterized as NAD(P)H: nitrate oxidoreductase (E.C.1.6.6.2). The enzyme activity with NADH (NADPH) was highest at pH 7.0 (7.1) and 30° C (25° C). The values of K
m determinations with NADH/NADPH were both 4 × 10–4 mol/l; values for the substrate inhibition constant (K
i) were 6 × 10–4 mol/l. The molecular mass of the native enzyme was estimated by gel permeation chromatography to be approximately 350 kDa.
Offprint requests to: R. Gromes 相似文献
11.
The lactic dehydrogenase (l-lactate: NAD oxidoreductase, EC 1.1.1.27, LDH)of Trichomonas gallinae was characterized and some of its regulatory properties studied. Electrophoretic analysis, with specific enzymatic staining of crude and dialyzed cell-free extracts and dialyzed ammonium sulfate fractions, all revealed a single band of enzymatic activity suggesting only one molecular form of the enzyme. The pH optima were found to be the following: 7.0 in the pyruvate to lactate direction and 9.0 in the reverse direction. Thermal inactivation studies showed a narrow temperature optimum peaking at 35 C. The Km values for all four reaction components were determined and found to be: NADH, 70 μm; pyruvate, 88 μm; NAD, 65 μm; and l-lactate, 4.6 mM. T. gallinae LDH was absolutely specific for NAD, NADH, l-lactate, and pyruvate. The enzyme exhibited negative cooperativity, with both NADH and l-lactate, as evidenced by curvilinear Lineweaver-Burk kinetics and Hill coefficients of less than one. Several glycolytic intermediates lowered the Km of NADH with variable effects on the Km of pyruvate. The regulation of LDH by glycolytic intermediates is discussed. 相似文献
12.
Pyruvate kinase variants of the Alaskan king-crab. Evidence for a temperature-dependent interconversion between two forms having distinct and adaptive kinetic properties 总被引:1,自引:1,他引:0
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George N. Somero 《The Biochemical journal》1969,114(2):237-241
1. Pyruvate kinase of Alaskan king-crab leg muscle exists in two kinetically distinct forms, each of which displays a different temperature-dependence in the Km for phosphoenolpyruvate. 2. A `cold' variant of the enzyme has hyperbolic kinetics and exhibits a minimal Km for substrate at 5°. At physiological concentrations of phosphoenolpyruvate the `cold' enzyme is active only below 10°. A `warm' pyruvate kinase has a minimal Km for substrate at about 12°. This enzyme displays sigmoidal kinetics and is likely to be inactive, at physiological substrate concentrations, at temperatures below 9°. 3. The combined activities of these two pyruvate kinases yield highly temperature-independent rates of catalysis, at physiological substrate concentrations, over the range of habitat temperatures encountered by the organism, namely 4–12°. 4. The two variants of pyruvate kinase do not appear to be isoenzymes in the conventional sense. Electrophoretic and electrofocus analyses revealed only single peaks of activity. 5. The results suggest that the `warm' pyruvate kinase and the `cold' pyruvate kinase are formed by a temperature-dependent interconversion of one protein species. This interconversion has major adaptive significance: as the temperature is lowered the `warm' enzyme is converted into the `cold' enzyme; the opposite situation obtains when the temperature is raised. Temperature changes thus mimic the effects noted for fructose 1,6-diphosphate on certain mammalian pyruvate kinases. 相似文献
13.
Saburova E. A. Avseenko N. V. Simonova N. B. Elfimova L. I. Pronina N. A. Semenenko V. E. 《Russian Journal of Plant Physiology》2000,47(6):761-771
The dependence of the catalytic properties of lactate dehydrogenase (LDH, EC 1.1.1.27) from a halophilic alga Dunaliella salina, a glycophilic alga Chlamydomonas reinhardtii, and from porcine muscle on glycerol concentration, medium pH, and temperature was investigated. Several chemical properties of the enzyme from D. salina differentiated it from the LDH preparation obtained from C. reinhardtii and any homologous enzymes of plant, animal, and bacterial origin. (1) V
max of pyruvate reduction manifested low sensitivity to the major intracellular osmolyte, glycerol. (2) The affinity of LDH for its coenzyme NADH dropped in the physiological pH region of 6–8. Above pH 8, NADH virtually did not bind to LDH, while the enzyme affinity for pyruvate did not change considerably. (3) The enzyme thermostability was extremely low: LDH was completely inactivated at room temperature within 30 min. The optimum temperature for pyruvate reduction (32°C) was considerably lower than with the enzyme preparations from C. reinhardtii (52°C) and porcine muscle (61°C). (4) NADH greatly stabilized LDH: the ratio of LDH inactivation constants in the absence of the coenzyme and after NADH addition at the optimum temperature in the preparation from D. salina exceeded the corresponding indices of LDH preparations from C. reinhardtii twelve times and from porcine muscle eight times. The authors believe that these LDH properties match the specific metabolism of D. salina which is set at rapid glycerol synthesis under hyperosmotic stress conditions. The increase of cytoplasmic pH value produced in D. salina by the hyperosmotic shock can switch off the terminal reaction of the glycolytic pathway and thus provide for the most efficient utilization of NADH in the cycle of glycerol synthesis. As LDH is destabilized in the absence of NADH, this reaction is also switched off. In the course of alga adaptation to the hyperosmotic shock, glycerol accumulation and the neutralization of intracellular pH stabilize LDH, thus creating the conditions for restoring the complete glycolytic cycle. 相似文献
14.
The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD+ specific and oxaloacetate can partially substitute for pyruvate. The K
m
app
for NAD+ is 14 M and 60 M at low and high NAD+ concentrations, respectively. The K
m
app
for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K
m
app
for NH
4
+
varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N2-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N2-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH
alanine dehydrogenase
- DEAE
diethylamino ethyl cellulose
- EDTA
ethylenediamine tetraacetic acid
- GDH
glutamic dehydrogenase
- GS
glutamine synthetase
- GOT
aspartate-glutamate aminotransferase
- NAD+
nicotinamide adenine dinucleotide
- NADH
reduced nicotinamide adenine dinucleotide
- NADP+
nicotinamide adenine dinucleotide phosphate
- NADPH
reduced nicotinamide adenine dinucleotide phosphate
- SDS
sodium dodecyl sulphate
- Tris
tris(hydroxymethyl) aminomethane 相似文献
15.
The initial rates of ATP synthesis catalyzed by tightly coupled Paracoccus denitrificans plasma membrane were measured. The reaction rate was hyperbolically dependent on the substrates, ADP and inorganic phosphate
(Pi). Apparent K
m values for ADP and Pi were 7–11 and 60–120 μM, respectively, at saturating concentration of the second substrate (pH 8.0, saturating Mg2+). These values were dependent on coupling efficiency. The substrate binding in the ATP synthesis reaction proceeds randomly:
K
m value for a given substrate was independent of the concentration of the other one. A decrease of electrochemical proton gradient
by the addition of malonate (when succinate served as the respiratory substrate) or by a decrease of steady-state level of
NADH (when NADH served as the respiratory substrate) resulted in a proportional decrease of the maximal rates and apparent
K
m values for ADP and Pi (double substitution, ping-pong mechanism). The kinetic scheme for ATP synthesis was compared with that described previously
for the proton-translocating ATP hydrolysis catalyzed by the same enzyme preparation (T. V. Zharova and A. D. Vinogradov (2006)
Biochemistry, 45, 14552–14558). 相似文献
16.
Shinji Nagata Yuki Kobayashi Sanae Shinkawa Reina Katoh Toshihisa Ohshima Haruo Misono 《Journal of Molecular Catalysis .B, Enzymatic》2003,23(2-6):223
A halophilic NAD+-dependent 2-aminobutyrate dehydrogenase (EC1.4.1.1) was purified to homogeneity from a crude extract of an extreme halophile, Halobacterium saccharovorum DSM 1137, with a 30% yield. The enzyme had a molecular mass of about 160 kDa and consisted of four identical subunits. It retained more than 70% of the activity after heating at 60 °C for 1 h and kept it at 30 °C for 8 months in the presence of 2 M NaCl. The enzyme showed maximum activity in the presence of 2 M RbCl or KCl. The enzyme required NAD+ as a coenzyme and used
-2-aminobutyrate,
-alanine, and
-norvaline as substrates. The best substrate was
-2-aminobutyrate. The optimum pH was 9.3 for the oxidative deamination of
-2-aminobutyrate and 8.6 for the reductive amination of 2-ketobutyrate. The Michaelis constants were 1.2 mM for
-2-aminobutyrate, 0.16 mM for NAD+, 0.012 mM for NADH, 0.78 mM for 2-ketobutyrate, and 500 mM for ammonia in the presence of 2 M KCl. The Km values for the substrates depended on the concentration of KCl, and the Km values decreased under high salt conditions. 相似文献
17.
《Journal of Fermentation Technology》1988,66(6):615-623
A yeast growing at 48°C was isolated from soil and the strain was identified as Cryptococcus lactativorus. The aldose reductase which the strain produced was purified 114-fold with an overall recovery of 36%. The stability of the enzyme was higher than that of other aldose reductases. The half life of the enzyme was 800 h and 14 h at 30°C and 50°C, respectively. The enzyme showed the best activity with d-xylose. l-Sorbose and d-fructose were also reduced by the enzyme. The enzyme was active with both NADPH and NADH as a conenzyme, and the activity with NADH was 1.25 times higher than that with NADPH. The Kmapp value for d-xylose was 8.6 mM and the Vmaxapp was 20.8 units/mg NADH was used as a coenzyme. The Kmapp values for NADPH and NADH were 6μM and 170 μM, respectively, when d-glucose was used as a substrate. 相似文献
18.
Procedures are described for isolating highly purified porcine liver pyruvate and α-ketoglutarate dehydrogenase complexes. Rabbit serum stabilized these enzyme complexes in mitochondrial extracts, apparently by inhibiting lysosomal proteases. The complexes were purified by a three-step procedure involving fractionation with polyethylene glycol, pelleting through 12.5% sucrose, and a second fractionation under altered conditions with polyethylene glycol. Sedimentation equilibrium studies gave a molecular weight of 7.2 × 106 for the liver pyruvate dehydrogenase complex. Kinetic parameters are presented for the reaction catalyzed by the pyruvate dehydrogenase complex and for the regulatory reactions catalyzed by the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. For the overall catalytic reaction, the competitive Ki to Km ratio for NADH versus NAD+ and acetyl CoA versus CoA were 4.7 and 5.2, respectively. Near maximal stimulations of pyruvate dehydrogenase kinase by NADH and acetyl CoA were observed at NADH:NAD+ and acetyl CoA:CoA ratios of 0.15 and 0.5, respectively. The much lower ratios required for enhanced inactivation of the complex by pyruvate dehydrogenase kinase than for product inhibition indicate that the level of activity of the regulatory enzyme is not directly determined by the relative affinity of substrates and products of catalytic sites in the pyruvate dehydrogenase complex. In the pyruvate dehydrogenase kinase reaction, K+ and NH+4 decreased the Km for ATP and the competitive inhibition constants for ADP and (β,γ-methylene)adenosine triphosphate. Thiamine pyrophosphate strongly inhibited kinase activity. A high concentration of ADP did not alter the degree of inhibition by thiamine pyrophosphate nor did it increase the concentration of thiamine pyrophosphate required for half-maximal inhibition. 相似文献
19.
G B Van den Berg H Vaandrager-Verduin T J Van Berkel J F Koster 《Archives of biochemistry and biophysics》1982,219(2):277-285
The measurement of the time dependency of the activity of rat liver fructose 1,6-bisphosphatase shows that the enzyme under certain conditions exhibits kinetic hysteretics. After addition of the substrate, the enzyme is initially in a state characterized by a “high” Km of about 2 μm. During the reaction the enzyme is converted in a slow process to a low Km form (Km is about 0.5 μm). The transition is accompanied by a decrease in V. It is concluded that the hysteretic behavior is caused by binding of the Zn2+ substrate complex to the enzyme. The earlier reported effect of glucagon treatment on the activity of fructose 1,6-bisphosphate (O. D. Taunton, F. B. Stifel, H. L. Greene, and R. H. Herman (1974) J. Biol. Chem.249, 7228–7239) was reinvestigated, taking into account the hysteretic behavior. Under conditions where the pyruvate kinase activity is decreased by glucagon injection, no activity change of fructose 1,6-bisphosphatase is observed. It can be suggested that for studies concerning the effects of incubation or hormone treatment on fructose 1,6-bisphosphatase, the complex kinetics of the rat liver enzyme has to be taken into account. 相似文献
20.
Giovanni Testore Sebastiano Colombatto Francesca Silvagno Stefano Bedino 《The international journal of biochemistry & cell biology》1995,27(11):1201-1210
Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD+-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. Km for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD+ is affected by the aldehyde present at the active site: Km for NAD+ is 70 μM with ABAL, 200 μM with isobutyraldehyde and capronaldehyde, and>800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD+ activates the enzyme (Kact 500 μM) while NADH competes for the regulatory site (Kin 70 μM). In the presence of high NAD+ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (Kact 10 mM). The data show that the enzyme is probably an E3 aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines. 相似文献