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1.
Summary The apparent Michaelis constant (K m) of NADH for muscle-type (M4 isozyme) lactate dehydrogenases (LDHs) is highest, at any given temperature of measurement, for LDHs of cold-adapted vertebrates (Table 1). However, these interspecific differences in theK m of NADH are not due to variations in LDH-NADH binding affinity. Rather, theK m differences result entirely from interspecific variation in the substrate turnover constant (k cat) (Fig. 1; Table 2). This follows from the fact that theK m of NADH is equal tok cat divided by the on constant for NADH binding to LDH,k 1, so that interspecific differences ink cat, combined with identical values fork 1 among different LDH reactions, make the magnitude of theK m of NADH a function of substrate turnover number. The temperature dependence of theK m of NADH for a single LDH homologue is the net result of temperature dependence of bothk cat andk 1 (Figs. 3 and 4). Temperature independentK m values can result from simultaneous, and algebraically offsetting, increases ink cat andk 1 with rising temperature. Salt-induced changes in theK m of NADH also may be due to simultaneous perturbation of bothk cat andk 1 (Table 3). These findings are discussed from the standpoint of the evolution of LDH kinetic properties, particularly the interspecific conservation of catalytic and regulatory functions, in differently-adapted species.  相似文献   

2.
Summary The previous paper (Borgmann, A., and Moon, 1976) suggested that temperature differentially affected the binding of the substrates phosphoenol pyruvate (PEP) and ADP to bat tissue pyruvate kinases (PK) under hibernating and normothermic conditions. Since the regulatory properties of most mammalian type-L PKs are temperature dependent, a study of these properties for the bat enzymes was initiated.M. pectoralis PK ofM. lucifugus is modulator insensitive, although ATP does increase theK m(PEP) slightly (Table 1). This effect is most pronounced at low temperatures for HM-PK (Fig. 1) and may be of some regulatory significance.Modulators affect bat liver PK in a manner analogous to other mammalian type-L enzymes, and marked quantitative differences exist between the NL-and HL-enzymes. TheK m(PEP) is increased only slightly by alanine and ATP (Table 3), althoughV max decreases markedly for NL-PK; fructose-1,6-diphosphate (FDP) decreases theK m(PEP) and overrides the inhibitory action of ATP and alanine (Table 3). As temperature decreases, the proportional change inK m(PEP) with or without effectors is unchanged (Table 3).HL-PK is markedly affected by these modulators. The extent of this interaction, as indicated byn H-values (Table 3) andK i orK a values (Table 2; Figs. 1, 2, 3), between the effectors and the binding of PEP to HL-PK is relatively greater than for the NL-enzyme. However, the temperature sensitivity of these interactions is reduced (Table 3).Therefore, the strategies associated with enzymes of hibernating and normothermic bats are similar to those previously reported by Hochachka and Somero (1973) for certain enzymes of poikilotherms, and argues for the existence of distinct enzyme forms in the two physiological states.Abbreviations ala alanine - FDP fructose-1,6-diphosphate,HL, HM, hibernator liver, and muscle, respectively - LDH lactate dehydrogenase - NL, NM normothermic liver, and muscle, respectively - PEP phosphoenol pyruvate - PK pyruvate kinase  相似文献   

3.
TheV max of the uptake of choline was increased in nerve cell cultures by lowering (from 7.4 to 6.5) or increasing (from 7.4 to 8.1) the pH. In neurons no effect was observed on the value of theK m's of the uptake of either the apparent high or low affinity components. In glial cells only a low affinity component was measured at pH 6.5 and diffusion was observed at pH 8.1. An excess of K+ ions in the incubation medium reproduced the increase inV max observed with changes in pH suggesting a possible dependence of the uptake of choline upon the H+ and OH gradients. Taking into account the characteristics already known of the transport of choline into nerve cells, such a dependence adds new insight in the mechanisms underlying the transport and indicates another possible regulation of choline entry, eventually directed towards the synthesis of acetylcholine.  相似文献   

4.
Summary The effect of lowering intracellular pH on the membrane potential (E m ) of rat thymic lymphocytes was studied using the potential-sensitive dyebis-oxonol. Cells were acid loaded by addition of the electroneutral K+/H+ exchanging ionophore nigericin. Acidification to pH 6.3 in Na+-free solution resulted in a biphasic change inE m : an early transient hyperpolarization followed by a sustained depolarization. These changes were associated with a rise in cytosolic free Ca2+ ([Ca2+] i ). The hyperpolarization was eliminated when the change in [Ca2+] i was prevented using BAPTA, an intracellular Ca2+ chelator. Moreover, a similar hyperpolarization was elicited by elevation of [Ca2+] i at physiological pH i using ionomycin, suggesting involvement of Ca2+-activated K+ channels. In contrast, the depolarization phase could not be mimicked by raising [Ca2+] i with ionomycin. However, intracellular BAPTA effectively inhibited the acidificationinduced depolarization. Inhibition was also obtained by extracellular addition of EGTA or dithiothreitol, even when the external free Ca2+ concentration remained unaltered. These observations suggested a possible role of contaminating trace metals. Cytosolic acidification is envisaged to induce intracellular accumulation of one or more trace metals, which induces the observed changes inE m . Accordingly, similar changes inE m can be induced without acidification by the addition of small amounts of Cu2+ to the medium. The ionic basis of theE m changes induced by acidification and the significance of these observations are discussed.  相似文献   

5.
To explain the six-banded pattern obtained upon electrophoresis of the soluble form of malate dehydrogenase (sMDH, EC 1.1.1.37) from the characiform Hoplias malabaricus, a recent locus duplication of its A isoform (sMDH-A*), in addition to its sMDH-B* isoform, was proposed. Klebe’s serial dilutions carried out using skeletal muscle, heart and liver extracts showed that the A1 and A2 subunits have the same visual end-points, indicating that these A-duplicated genes have a nondivergent pattern. Since there is no evidence of polyploidy in the Erythrinidae family, the MDH-A* loci have probably evolved from regional gene duplication. While these sMDH-A* loci encode nondivergent thermostable isoforms, the sMDH-B* encodes a thermolabile one. Thermostable sMDHs differ from the thermolabile sMDHs in that they have a higher Km of oxaloacetate. Liver, muscle and heart unfractionated sMDH levels at three different temperature and two pH regimens were analysed and the results showed that, in the adaptative temperature range of Hoplias, the variation in Km under conditions of constant pH (imidazole buffer) was less (approximately threefold) than that measured in the presence of temperature-dependent pH imidazole buffer (sevenfold). Estimation of the ratio of both isoforms in these tissues by Klebe’s method showed that, in unfractionated liver – where Km values were the highest and the minimum Km was obtained at 30^C (both for temperature-dependent pH and constant-pH imidazole buffer) – the duplicate A (thermostable, A1 and A2) and B (thermolabile) subunits were detected in a ratio of 2:1. On the other hand, in muscle extracts – in which the lowest Km values were measured, with the minimum Km at 10–20^C (temperature-dependent pH and constant-pH imidazole buffer, respectively) – a ratio of two thermolabile to one thermostable subunits was observed.  相似文献   

6.
An NAD-dependent D-2-hydroxyacid dehydrogenase (EC 1.1.1.) was isolated and characterized from the halophilic Archaeon Haloferax mediterranei. The enzyme is a dimer with a molecular mass of 101.4 ± 3.3 kDa. It is strictly NAD-dependent and exhibits its highest activity in 4 M NaCl. The enzyme is characterized by a broad substrate specificity 2-ketoisocaproate and 2-ketobutyrate being the substrates with the higher Vmax/Km. When pyruvate and 2-ketobutyrate were the substrates the optimal pH was acidic (pH 5) meanwhile for 2-ketoisocaproate maximum activity was achieved at basic pH between 7.5 and 8.5. The optimum temperature was 52 ºC and at 65 ºC there was a pronounced activity decrease. This new enzyme can be used for the production of D-2-hydroxycarboxylic acid.  相似文献   

7.
For the purpose of producing pyruvate from -lactate by enzymatic methods, four microorganism strains that produce lactate oxidase (LOD) were screened and isolated from many soil samples. Among them, strain SM-6, which showed high potential for pyruvate production, was chosen for further research. Physiological studies and 16S rDNA relationship reveal that SM-6 belongs to Pseudomonas putida. The optimized pH and temperature of the enzyme-catalyzed reaction were pH 7.2, and 39 °C, respectively. Low-concentration EDTA (1 mM) could improve the stability of pyruvate and conversion ratio of lactate oxidase. Vmax and Km value for -lactate were 2.46 μmol/(min mg) protein and 9.53 mM, respectively. On preparation scale, cell-free extract from SM-6, containing 300 mg/l of crude enzyme (4037 U/ml lactate oxidase), could convert 66% of 116 mM of -lactate into 76.6 mM pyruvate in 18 h, and 82% of substrate was transformed after 48 h, giving 95.0 mM (10.5 mg/ml) of pyruvate. The ratio of product to biocatalyst was 34.8:1 (g/g).  相似文献   

8.
The H+-ATPase activities of root and leaf plasma membranes from tobacco (Nicotiana tabacum) have been characterized with respect to Vmax, Km for ATP, pH dependence and activation involving the C-terminal autoinhibitory domain. With root plasma membranes, addition of lysophosphatidylcholine (lyso-PC) resulted in the expected increase in Vmax, a decrease in Km(ATP), and a shift in pH optimum to a more alkaline pH, typical for activation via the C-terminal inhibitory domain. With leaf plasma membranes, however, Km(ATP) was relatively low and the pH optimum was around pH 7.0 before the addition of lyso-PC and did not change upon addition of the activator, although Vmax increased twofold. Similar results were obtained with the in vivo activator fusicoccin. The results obtained with the leaf plasma membranes show that Vmax may be regulated independently of Km(ATP) and pH optimum, and suggest the presence of at least two regulatory sites within the C-terminal autoinhibitory domain of the H+-ATPase.  相似文献   

9.
M T Record 《Biopolymers》1967,5(10):993-1008
The theory developed in the previous paper to discuss changes in electrostatic free energies in polynucleotide order–disorder transitions is extended to cases where one or more of the participating species is titrated to some degree α. It is shown that, for any class of transition, the melting temperature Tm at constant pH is a linear function of the logarithm of the monovalent counterion concentration M, that at high salt the logarithm of the depression of the melting temperature by pH titration is proportional to the pH change, and that the stability of the ordered form as measured by its melting temperature at neutral pH, is a monotonic function of the quantity pHm – pK, where pHm and pK are the pH of melting and the monomer base pK, both measured under similar conditions of temperature and ionic strength. For the transition from double helix to coil, the dependences of Tm and dTm/d log M on pH are determined experimentally and compared with the qualitative predictions of the theory. It is found that dTm/dlog M, a measure of – ΔF?el (the negative of the electrostatic free energy change in the transition), decreases with increasing pH. In acid solution, where the coil is more extensively prolonated than the helix, the change in electrostatic free energy in the transition is larger than at neutral pH. Conversely, in alkali the electrostatic five energy change is smaller than at neutral pH. Hence (dTm/d log M)acid > (dTm/d log M neutral) > (dTm/d log M)alkali. At Suffeciently high pH, dTm/d log M is observed to become negative, indicating that the electrostatic free energy change is positive in the transition of this region. Date from the literature on the ionic strength dependence of the melting temperature for the acid helices of poly rA, poly rC, and poly dC are also considered from the standpoint of the theory.  相似文献   

10.
Summary The rate of transport of amine ions intoChara australis internodes is studied by measuring changes in membrane current when amine solutions are presented to voltage-clamped cells. The dependence of this rate on ion concentration is investigated for a series of alkyl-amine ions: methyl-, ethyl-, isopropyl-, dimethyl-, trimethyl- and tetramethylammonium. A Michaelis-Menten relationship is displayed by all except tri- and tetramethylammonium, where currents are irregular and difficult to reproduce. Evidence suggests that the different ions cross the plasmalemma via a common uniport.K M values for this porter increase as the amine ion becomes more highly substituted. TheV m values are similar for all amines and lie within the range 10 to 100 mA m–2 (for cell potential at –200 mV). The changes inK M indicate that hydrogen bonding may be involved in the binding interaction.V m varies with external pH in a way which suggests that an ionizable group on the transport protein with pKa5.8 directly affects the transport rate.K M is independent of external pH over the range 4.5 to 10.5  相似文献   

11.
Jesús A. Marcos, Dolores de Arriaga, Félix Busto, and Joaquín Soler11997. Functional Characteristics of Pyruvate Transport inPhycomyces blakesleeanus. Fungal Genetics Biology25, 204-215. A saturable and accumulative transport system for pyruvate has been detected inPhycomyces blakesleeanusNRRL 1555(−) mycelium. It was strongly inhibited by α-cyano-4-hydroxycinnamate. -Lactate and acetate were competitive inhibitors of pyruvate transport. The initial pyruvate uptake velocity and accumulation ratio was dependent on the external pH. TheVmaxof transport greatly decreased with increasing pH, whereas the affinity of the carrier for pyruvate was not affected. The pyruvate transport system mediated its homologous exchange, which was essentially pH independent, and efflux, which increased with increasing external pH. The uptake of pyruvate was energy dependent and was strongly inhibited by inhibitors of oxidative phosphorylation and of the formation of proton gradients. Glucose counteracted the inhibitory effect of the pyruvate transport produced by inhibitors of mitochondrial ATP synthesis. Our results are consistent with a pyruvate/proton cotransport inP. blakesleeanusprobably driven by an electrochemical gradient of H+generated by a plasma membrane H+-ATPase.  相似文献   

12.
Considerable evidence indicates that acetaldehyde is released from the leaves of a variety of plants. The conventional explanation for this is that ethanol formed in the roots is transported to the leaves where it is converted to acetaldehyde by the alcohol dehydrogenase (ADH) found in the leaves. It is possible that acetaldehyde could also be formed in leaves by action of pyruvate decarboxylase (PDC), an enzyme with an uncertain metabolic role, which has been detected, but not characterized, in cottonwood leaves. We have found that leaf PDC is present in leaf veins and petioles, as well as in non-vein tissues. Veins and petioles contained measurable pyruvate concentrations in the range of 2 mM. The leaf vein form of the enzyme was purified approximately 143-fold, and, at the optimum pH of 5.6, the Km value for pyruvate was 42 μM. This Km is lower than the typical millimolar range seen for PDCs from other sources. The purified leaf PDC also decarboxylates 2-ketobutyric acid (Km = 2.2 mM). We conclude that there are several possible sources of acetaldehyde production in cottonwood leaves: the well-characterized root-derived ethanol oxidation by ADH in leaves, and the decarboxylation of pyruvate by PDC in leaf veins, petioles, and other leaf tissues. Significantly, the leaf vein form of PDC with its high affinity for pyruvate, could function to shunt pyruvate carbon to the pyruvate dehydrogenase by-pass and thus protect the metabolically active vascular bundle cells from the effects of oxygen deprivation.  相似文献   

13.
W/O微乳液中糖化酶的研究   总被引:2,自引:0,他引:2  
在十六烷基三甲基溴化铵/正戊醇/异辛烷/水体系中,选取W/O型微乳液作为糖化酶的微环境,研究了其催化活性.并与其在水溶液中进行了比较.结果表明,在微乳液中,糖化酶催化淀粉水解反应的最佳反应温度和pH比在水溶液中低,反应的最大速度Vm和米氏常数Km比其在水溶液中分别提高了近6倍和3倍.  相似文献   

14.
Acclimation of loach to relatively low and high temperatures gives rise to changes in the properties of LDH from oocytes. The minimum of apparent Km values for pyruvate of LDH from immature oocytes of fish acclimated to low temperature was registered at low assay temperatures, whereas adaptation to high temperatures leads to the enzyme showing a minimum at high temperatures. In thermal acclimation of fish the Km minimum value drifts during 15 days. During oocyte maturation, the Km minimum drifts during 40 hr. The differences in the kinetic features of LDH from oocytes both at thermal acclimation and during oocyte maturation are not associated with the appearance of new isozymes.  相似文献   

15.
A pyruvate, orthophosphate dikinase (EC 2.7.9.1) has been isolated from Acetobacter aceti grown on pyruvate as the only source of carbon and energy. The enzyme was purified 65-fold, and its molecular weight was determined to be about 330,000 by gel filtration.The optimum pH was 8.0 in the forward direction [phosphoenolpyruvate (PEP) formation] and 7.1 for the backward reaction (pyruvate production). In both directions Mg2+ was required (forward K m 1.70 mM; reverse K m 0.87 mM) and no other divalent cation was able to replace it. The K m values for pyruvate, ATP, and Pi were 27 M, 0.20 mM, and 0.83 mM, respectively, in the forward direction. The K m values for PEP, AMP, and PPi were 0.13 mM, 6 M, and 62 M, respectively, for the reverse reaction. The substrate-product pairs pyruvate-PEP, ATP-AMP, Pi-PPi were competitive inhibitors to each other in both directions. These product inhibition studies suggest for the enzyme from A. aceti nonclassical three-site Tri (Uni Uni) Ping-Pong kinetics.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - SDS sodium dodecyl sulphate - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl2, 1 mM glutathione  相似文献   

16.
(1) Pyruvate kinase type M2 from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of Mr = 224 000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M2, excepts that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K0.5 value for phosphoenolpyruvate is 0.26 mM (nH = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM (nH = 1.06). 1 μM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phosphoenolpyruvate half-maximally. The Km value for ADP at 1 mM phosphoenolpyruvate is 0.4 mM. The Km value for other nucleoside diphosphates increases in the order ADP<GDP<IDP<UDP. (3) No evidence for an interconversion of pyruvate kinase type M2 from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M2 from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047–1055) we suggest that the interconvertible form of pyruvate kinase type M2 may represent a separate form of the pyruvate kinase type M2 family.  相似文献   

17.
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   

18.
Kinetic properties of homogeneous preparations of pig kidney and pig muscle pyruvate kinases (EC 2.7.1.40) were studied. Both isozymes showed a hyperbolic relationship to ADP with an apparent Km of 0.3 mm. K+ and Mg2+ were necessary for the activity of both isozymes, and their dependences on these cations were similar. The muscle isozyme expressed Michaelis-Menten type of kinetics with respect to phosphoenolpyruvate, and the apparent Km was the same (0.03 mm) from pH 5.5 to pH 8.0. In contrast, the dependence on phosphoenolpyruvate changed with pH for the kidney isozyme. It showed similar properties to the muscle isozyme at pH 5.5–7.0 (apparent Km of 0.08 mm), while two apparent Km values for this substrate were present at pH 7.5–8.0, one low (0.1 mm) and one high (0.3–0.6 mm). At pH 7.5, fructose 1,6-bisphosphate converted the kidney isozyme to a kinetical form where only the lower apparent Km for phosphoenolpyruvate was detected. On the other hand, in the presence of alanine or phenylalanine the kidney pyruvate kinase showed only the higher Km for this substrate. At low phosphoenolpyruvate levels both isozymes were inhibited by phenylalanine, and half-maximal inhibition was found at 0.3 and 2.2 mm for the kidney and muscle isozymes, respectively. At a 5 mm concentration of the substrate only the kidney isozyme was inhibited, the apparent Ki being the same. Alanine inhibited the kidney isozyme (apparent Ki at 0.3 mm, irrespective of substrate concentration). No effect was seen on the muscle isozyme. Fructose 1,6-bisphosphate was an activator of the kidney isozyme at phosphoenolpyruvate concentrations below 1.0 mm It also counteracted the inhibition by alanine or phenylalanine of this isozyme. ATP inhibited both isozymes, and this inhibition was not counteracted by fructose 1,6-bisphosphate. The kidney isozyme showed both a high and a low apparent Km for phosphoenolpyruvate in the presence of ATP. The influence of the effectors on the activity of both isozymes varied markedly with pH, except for the action of ATP. At low substrate concentrations, however, the inhibitor action of ATP on the muscle enzyme was diminished around pH 7.5, in contrast to higher or lower pH values. Alanine or phenylalanine were more effective as inhibitors at higher pH values, and fructose 1,6-bisphosphate stimulated the kidney isozyme only at pH levels above pH 6.5. The influence of activators and inhibitors on the regulation of the kidney and muscle pyruvate kinases is discussed.  相似文献   

19.
The efficacy and mechanism of -dendrotoxin (DTX) block of K+ channel currents in Vicia stomatal guard cells was examined. Currents carried by inward- and outward-rectifying K+ channels were determined under voltage clamp in intact guard cells, and block was characterized as a function of DTX and external K+ (K+) concentrations. Added to the bath, 0.1-30 nM DTX blocked the inward-rectifying K+ current (IK,in), but was ineffective in blocking current through the outward-rectifying K+ channels (IK,out) even at concentrations of 30 nM. DTX block was independent of clamp voltage and had no significant effect on the voltage-dependent kinetics for IK,in, neither altering its activation at voltages negative of –120 mV nor its deactivation at more positive voltages. No evidence was found for a use dependence to DTX action. Block of IK,in followed a simple titration function with an apparent K1/2 for block of 2.2 nM in 3 mm K o + . However, DTX block was dependent on the external K+ concentration. Raising K+ from 3 to 30 mm slowed block and resulted in a 60–70% reduction in its efficacy (apparent K i = 10 mm in 10 nm DTX). The effect of K+ in protecting I K,in was competitive with DTX and specific for permeant cations. A joint analysis of IK,in block with DTX and K+ concentration was consistent with a single class of binding sites with a K d for DTX of 240 pm. A K d of 410 m for extracellular K+ was also indicated. These results complement previous studies implicating a binding site requiring extracellular K+ (K1/2 1 mm) for IK,in activation; they parallel features of K+ channel block by DTX and related peptide toxins in many animal cells, demonstrating the sensitivity of plant plasma membrane K+ channels to nanomolar toxin concentrations under physiological conditions; the data also highlight one main difference: in the guard cells, DTX action appears specific to the K+ inward rectifier.We thank J.O. Dolly (Imperial, London) and S.M. Jarvis (University of Kent, Canterbury) for several helpful discussions. This work was supported by SERC grant GR/H07696 and was aided by equipment grants from the Gatsby Foundation, the Royal Society and the University of London Central Research Fund. G.O. was supported by an Ausbildungsstipendium (OB 85/1-1) from the Deutsche Forschungsgemeinschaft. F.A. holds a Sainsbury Studentship.  相似文献   

20.
Enzymes catalyzing the synthesis and subsequent transformation of α-acetolactate (AcL)—acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)—were isolated and partially purified from the cells of lactic acid bacteriaLactococcus lactis ssp.lactis biovar.diacetylactis, strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358 300 U/mg protein (9% yield). The preparation of AcLDC., purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparentK m, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic and the affinity of the enzyme for its substrate was high (K m = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.  相似文献   

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