Mechanism of bovine liver S-adenosylhomocysteine hydrolase. Steady-state and pre-steady-state kinetic analysis

The kinetic mechanism of S-adenosylhomocysteine hydrolase was investigated by stopped-flow spectrofluorometry at pH 7.0 and 25 degrees C. Pre-steady-state kinetic steps were identified with chemical steps proposed for the mechanism of this enzyme (Palmer, J.L., and Abeles, R.H. (1979) J. Biol. Chem. 254, 1217-1226). The steady-state kinetic constants for the hydrolysis or synthesis of S-adenosylhomocysteine were in good agreement with those values calculated from the pre-steady-state rate constants. The equilibrium constant for dehydration of 3'-ketoadenosine to 3'-keto-4',5'-dehydroadenosine on the enzyme was 3. The analogous equilibrium constant for addition of L-homocysteine to S-3'-keto-4',5'-dehydroadenosylhomocysteine on the enzyme was 0.3. The elimination of H2O from adenosine in solution had an equilibrium constant of 1.4 (aH2O = 1). Thus, the equilibrium constants for these elimination reactions on the enzyme were probably not perturbed significantly from those in solution. The equilibrium constant for the reduction of enzyme-bound NAD+ by adenosine was 8, and the analogous constant for the reduction of the enzyme by S-adenosylhomocysteine was 4. The equilibrium constant for the reduction of NAD+ by a secondary alcohol in solution was 5 x 10(-5) at pH 7.0. Consequently, the reduction of enzyme-bound NAD+ by adenosine was 10(5)-fold more favorable than the reduction of free NAD+. The magnitude of the first-order rate constants for the interconversion of enzyme-bound intermediates varied over a relatively small range (3-80 s-1). Similarly, the magnitude of the equilibrium constants among enzyme-bound intermediates varied over a narrow range (0.3-10). These results were consistent with the overall reversibility of the reaction.     

Temperature dependence of acetylcholine receptor channels activated by different agonists

The temperature dependence of agonist binding and channel gating were measured for wild-type adult neuromuscular acetylcholine receptors activated by acetylcholine, carbamylcholine, or choline. With acetylcholine, temperature changed the gating rate constants (Q₁₀ ≈ 3.2) but had almost no effect on the equilibrium constant. The enthalpy change associated with gating was agonist-dependent, but for all three ligands it was approximately equal to the corresponding free-energy change. The equilibrium dissociation constant of the resting conformation (K_d), the slope of the rate-equilibrium free-energy relationship (Φ), and the acetylcholine association and dissociation rate constants were approximately temperature-independent. In the mutant αG153S, the choline association and dissociation rate constants were temperature-dependent (Q₁₀ ≈ 7.4) but K_d was not. By combining two independent mutations, we were able to compensate for the catalytic effect of temperature on the decay time constant of a synaptic current. At mouse body temperature, the channel-opening and -closing rate constants are ∼400 and 16 ms⁻¹. We hypothesize that the agonist dependence of the gating enthalpy change is associated with differences in ligand binding, specifically to the open-channel conformation of the protein.     

A bioluminescence method of determining the activity of NAD-dependent hydrogenase]

An analytical multienzyme system composed of NAD-dependent hydrogenase of Alcaligenes eutrophus, and reductase and luciferase from luminous bacteria was studied. The rate of luminescence increase of this system was found to be proportional to hydrogenase activity. The apparent Michaelis constants for NAD and hydrogen were determined (5 and 40 microM, respectively). The pH optimum is 7.5-9.0. Over the NAD concentration range from 20 to 100 microM, the rate of luminescence increase changed by less than 10%. At higher concentrations of NAD a monotonous decreasing of the rate of luminescence increase was observed. The proposed multienzyme system can be used for measuring the hydrogenase activity and hydrogen concentration. The high sensitivity to hydrogen (0.1 nmol in sample) and to hydrogenase (0.5 mU) and specificity of the system enable its application in the development of a biosensor for rapid detection of hydrogen in a medium.     

Role of calcium ions in the regulation of intramitochondrial metabolism. Properties of the Ca2+-sensitive dehydrogenases within intact uncoupled mitochondria from the white and brown adipose tissue of the rat.

1. Increasing concentrations of both Ca2+ and Sr2+ (generated by using EGTA buffers) resulted in 4-fold increases in the initial activity of pyruvate dehydrogenase within intact uncoupled mitochondria from rat epididymal adipose tissue incubated in the presence of the ionophore A23187, ATP, Mg2+ and oligomycin. The k0.5 values (concentrations required for half-maximal effects) for Ca2+ and Sr2+ were 0.54 and 7.1 microM respectively. In extracts of the mitochondria, pyruvate dehydrogenase phosphate phosphatase activity was stimulated about 4-fold by Ca2+ and Sr2+, with k0.5 values of 1.08 and 6.4 microM respectively. 2. NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase appeared to be rate-limiting in the oxidation of threo-Ds-isocitrate and oxoglutarate by uncoupled mitochondria from brown adipose tissue of cold-adapted rats. Ca2+ (and Sr2+) diminished the Km for the oxidation of both threo-Ds-isocitrate and oxoglutarate. The kinetic constants for these oxidations were very similar to those obtained for the activities of NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of the mitochondria. In particular, the k0.5 values for Ca2+ were all in the range 0.2--1.6 microM and Sr2+ was found to mimic Ca2+, but with k0.5 values about 10 times greater. 3. Overall, the results of this study demonstrate that the activities of pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase may all be increased by Ca2+ and Sr2+ within intact mitochondria. In all cases the k0.5 values are close to 1 and 10 microM respectively, as found for the separated enzymes. Experiments on brown-adipose-tissue mitochondria incubated in the presence of albumin suggest that it may be possible to use the sensitivity of the dehydrogenases to Ca2+ as a means of assessing the distribution of Ca2+ across the mitochondrial inner membrane.     

Reduced S-adenosylhomocysteine hydrolase. Kinetics and thermodynamics for binding of 3'-ketoadenosine, adenosine, and adenine.

S-Adenosylhomocysteine hydrolase (SAHase) was resolved into apoenzyme and NAD+ by acidic ammonium sulfate treatment. The apoenzyme was catalytically inactive, but could be reconstituted to active enzyme with NAD+. Reduced SAHase (ENADH) that was prepared by reconstitution of the apoenzyme with NADH was catalytically inactive. ENADH was oxidized by 3'-ketoadenosine to active SAHase. The recovery of activity paralleled the oxidation of enzyme-bound NADH. The association rate constant for ENADH and 3'-ketoadenosine was 6.1 x 10(2) M-1 s-1, and the dissociation rate constant was calculated to be 4 x 10(-7) s-1. This association rate constant was considerably smaller than the association rate constant for adenosine and SAHase (greater than 10(7) M-1 s-1). However, the observed pseudo first-order rate constant for reaction of 3'-ketoadenosine with ENADH (0.6 s-1 with 1 mM 3'-ketoadenosine) approached kcat for the hydrolytic reaction (1.2 s-1). Thus, bound 3'-ketoadenosine probably reacted sufficiently rapidly with ENADH to be considered a kinetically competent intermediate. The dissociation constants of SAHase for adenosine and 4',5'-dehydroadenosine, substrates for the enzyme, were 9 and 14 microM, respectively. In contrast, the dissociation constants of ENADH for 3'-ketoadenosine and 4',5'-dehydro-3'-ketoadenosine, intermediates of the catalytic reaction, were significantly lower with values of 600 and 300 pM, respectively. The equilibrium constant for reduction of enzyme-bound NAD+ in the absence of an adenosine analogue, as estimated from cyanide binding studies, was 10-fold more favorable than that for free NAD+. ENADH was highly fluorescent (emission maximum 428 nm, excitation 340 nm) with a quantum yield that was six times that of free NADH. Since SAHase reduced by adenosine was not highly fluorescent, enzyme-bound intermediates quenched the fluorescence of enzyme-bound NADH. Adenosine and adenine quenched the fluorescence of ENADH. Cyanide formed a complex with SAHase that was analogous to ENADH. Adenine stabilized this complex sufficiently that addition of 65 microM adenine and 25 mM cyanide to SAHase caused total complex formation with loss of over 95% of the catalytic activity.     

Biodegradation Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge

This study focuses on the processes influencing hydrocarbon residue persistence in soil, following land treatment of refinery oily sludge. Treating sludge applied to soil resulted in 70% to 90% degradation of total petroleum hydrocarbon (TPH) during 2 months, regardless of their initial concentrations (9 to 60 g/kg soil). Kinetic analyses performed on TPH degradation, in laboratory and field systems, revealed a degradation pattern characterized by two consecutive first-order kinetics reactions in all experimental settings. The first stage lasted about 3 weeks and was characterized by a temperature dependent rate constant of 0.047 day^-1 at 24°C. That value was comparable to the rate constant obtained when combining the individual rate constants of the saturated, aromatic, asphaltene and polar fractions. The subsequent slower stage rate constant was 0.012 day^-1, insensitive to temperature and to hydrocarbon composition. The transition between the two stages (about 21 days) was independent of the experimental temperature and the biodegradation extent during the first stage. It was concluded that the extent of residual accumulation in the soil was determined by the biodegradation efficiency during the first three weeks of treatment when biological processes dominated. During the following period, abiotic processes leading to reduced bioavailability of the TPH were limiting the degradation rate. Practically, as the first few weeks of treatment determine its efficiency, efforts to enhance the biological activity should be directed to that period.     

The effect of luciferase and NADH:FMN oxidoreductase concentrations on the light kinetics of bacterial bioluminescence

The effects of NADH:FMN oxidoreductase and luciferase concentrations on the light kinetics of the bacterial bioluminescent reaction were investigated. Light emission with low decay rates was obtained by regulating the conversion of NADH to NAD+ by controlling oxidoreductase activity. Constant light emission can be obtained when the oxidoreductase activity is below 2.5 U/1 in the assay system. The luciferase concentration affects the light intensity but it has no effect on the decay rate of light emission. The substrate decanal and the end-products NAD+ and capric acid had no effect on the light kinetics. The Michaelis constants of bacterial luciferase for FMNH2 and decanal were 3 X 10(-6) M and 8 X 10(-7) M, respectively, and those of oxidoreductase for FMN and NADH were 6.1 X 10(-6) M and 1.6 X 10(-5) M, respectively.     

On the origin of the lactate dehydrogenase induced rate effect

To evaluate the ability of lactate dehydrogenase to facilitate the bond making/breaking steps for both the addition of pyruvate enol to NAD (pyruvate adduct reaction) and the normal redox reaction, the ability of the enzyme to facilitate the tautomerization of bound pyruvate is assessed. In addition, the equilibrium constants for the adduct reaction are obtained for both bound and free reactants from the ratio of the rate constants in the forward and reverse reactions (at pH 7). The latter comparison indicates that the enzyme facilitates bond making/breaking in the (forward) pyruvate adduct reaction by a factor of about 10(11) M. Similar comparisons suggest that reactant immobilization accounts for about 1000 M of this 10(11) M rate effect. Since the (pH-independent) rate constant for the ketonization of bound pyruvate enol assisted by the external buffer, imidazolium ion, is 2 X 10(7) M-1 s-1 and the corresponding rate constant for free pyruvate enol, again assisted by imidazolium ion, is 35 M-1 s-1 [Burger, J. W., II, & Ray, W. J., Jr. (1978) Biochemistry 17, 1664], the enzyme facilitates the bond making/breaking steps associated with the conversion of bound HO-C less than to bound O = C less than by a factor of about 10(6)-fold. The product of the above two rate enhancement factors and the rate factor suggested previously for the environmental effect on NAD produced by its binding to lactate dehydrogenase, 100-fold, is 10(11) M, and it accounts for the bond making/breaking effects exerted by the enzyme in the pyruvate adduct reaction. The rate constant for oxidation of ethanol (a model for lactate) by 1-methylnicotinamide (a model for NAD) is about 5 X 10(-12) M-1 s-1 at 25 degrees C in pure ethanol (delta H for this reaction is about 30 kcal/mol). The ratio of the rate constants for E X NAD X Lac----E X NADH X Pyr and the above model reaction is estimated as about 10(14) M in water; i.e., the LDH-induced rate effect is about 10(14) M. The product of the values for the above rate factors for the normal redox reaction is about 10(12) M. Although the value of this product is less certain than that for the adduct reaction, these rate factors do account for much of the LDH-induced rate effect.     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.     

Pre-steady-state kinetic studies on cytoplasmic sheep liver aldehyde dehydrogenase

Stopped-flow experiments in which sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was rapidly mixed with NAD(+) and aldehyde showed a burst of NADH formation, followed by a slower steady-state turnover. The kinetic data obtained when the relative concentrations and orders of mixing of NAD(+) and propionaldehyde with the enzyme were varied were fitted to the following mechanism: [Formula: see text] where the release of NADH is slow. By monitoring the quenching of protein fluorescence on the binding of NAD(+), estimates of 2x10(5) litre.mol(-1).s(-1) and 2s(-1) were obtained for k(+1) and k(-1) respectively. Although k(+3) could be determined from the dependence of the burst rate constant on the concentration of propionaldehyde to be 11s(-1), k(+2) and k(-2) could not be determined uniquely, but could be related by the equation: (k(-2)+k(+3))/k(+2) =50x10(-6)mol.litre(-1). No significant isotope effect was observed when [1-(2)H]propionaldehyde was used as substrate. The burst rate constant was pH-dependent, with the greatest rate constants occurring at high pH. Similar data were obtained by using acetaldehyde, where for this substrate (k(-2)+k(+3))/k(+2)=2.3x10 (-3)mol.litre(-1) and k(+3) is 23s(-1). When [1,2,2,2-(2)H]acetaldehyde was used, no isotope effect was observed on k(+3), but there was a significant effect on k(+2) and k(-2). A burst of NADH production has also been observed with furfuraldehyde, trans-4-(NN-dimethylamino)cinnamaldehyde, formaldehyde, benzaldehyde, 4-(imidazol-2-ylazo)benzaldehyde, p-methoxybenzaldehyde and p-methylbenzaldehyde as substrates, but not with p-nitrobenzaldehyde.     

Role of phosphorus in activated sludge

Monod's kinetic model was used to correlate the specific growth rate of mixed activated sludge with the limiting substrate of phosphorus for both batch and continuous-flow culture systems. In the batch reactor system, the specific growth rate varied from 0.092 to 0.617 h(-1) and the saturation constant changed from 25.5 to 117.5 when the COD: P ratio was controlled within the range of 10 to 788 and at the temperature 25+/- 0.5 degrees C. An inverse relationship between specific growth rate and cell yield was found. the maximum specific growth rate and the saturation constant obtained from this study were equal to 0.64 h(-1) and 0.378mg/L, respectively. In the completely mixed continuous-flow culture system, it was found that the substrate utilization, biological solids production, and sludge composition were markedly affected by the source of phosphorus available in the wastewater. The phosphorus-limited activated sludge is normally high in carbohydrate content and low in protein content. Also, sludge organisms growth under the severely restricted phosphorus condition usually possess a large capsule. These capsulated carbohydrate-like substances can be converted to cellular protein if the source of phosphorus is added. The values of cell yield in the continuous-flow activated sludge system are predictable by the use of kinetic constants that are generated from batch culture studies.     

Properties of NAD binding by synaptic membranes of rat brain

The binding of [14C]NAD to rat brain synaptic membranes is reversible and depends on incubation time, temperature and protein concentration in the reaction mixture. The value of the rate constant for [14C]NAD binding to the synaptic membranes at 24 degrees C (kl) is 1.1 X 10(-6) M-1 S-1, the rate constant for dissociation of the [14C]NAD-receptor complex (k-1) is 3.3 X 10(-3) S-1. The value of the constant for the ligand dissociation from this complex (Kd) is 3.0 nmole. Treatment of the experimental results in the Scatchard plots for the equilibrium binding of [14C]NAD to the synaptic membranes demonstrated that the receptor sites with high and low affinities for the ligand (Kd1 = 3.3 nmol, Kd2 = 14.4 nmole) and with binding capacities of 44 and 77 pmole of [14C]NAD, respectively. It was found that the synaptosomal membrane components which bind the labelled NAD have a protein nature. Data from [14C]NAD and [nicotinamide-3H]NAD binding suggest that brain synaptic membranes bind NAD at the nicotinamide and adenylic moieties.     

Free energy of hydrolysis of tyrosyl adenylate and its binding to wild-type and engineered mutant tyrosyl-tRNA synthetases

The equilibrium constant for the formation of tyrosyl adenylate and pyrophosphate from ATP and tyrosine in solution has been measured by applying the Haldane relationship to wild-type and three mutant tyrosyl-tRNA synthetases from Bacillus stearothermophilus. The formation constant (=[Tyr-AMP] [PPi]/[ATP] [Tyr]) at pH 7.78, 25 degrees C, and 10 mM MgCl2 is (3.5 +/- 0.5) X 10(-7). This corresponds to a free energy of hydrolysis of tyrosyl adenylate at pH 7.0 and 25 degrees C of -16.7 kcal mol-1. All necessary rate constants had been determined previously for the calculations apart from the dissociation constant of tyrosyl adenylate from its enzyme-bound complex. This was measured by taking advantage of the 100-fold difference in hydrolysis rates of the tyrosyl adenylate when sequestered by the enzyme and when free in solution. These are technically difficult measurements because the dissociation constants are so low and the complexes unstable. The task was simplified by using mutants prepared by site-directed mutagenesis. These were designed to have different rate and equilibrium constants for dissociation of tyrosyl adenylate from the enzyme-bound complexes. The dissociation constants were in the range (3.5-38) X 10(-12) M, with that for wild type at 13 X 10(-12) M. The four enzymes all gave consistent data for the formation constant of tyrosyl adenylate in solution. This not only improves the reliability of the measurement but also provides confirmation of the reliability of the measured kinetic constants for the series of enzymes.     

怀槐细胞悬浮培养的结构化动力学模型

为探明怀槐细胞生长、异黄酮染料木素合成与底物消耗间的关系,建立了怀槐细胞悬浮培养的结构化动力学模型。模型预测分析了胞内外的蔗糖代谢、胞内结构组分变化、胞内中间组分的变化、细胞呼吸损失以及胞内外异黄酮染料木素的合成情况。模型各参数灵敏度的分析表明kb1、kb2和kp是最为灵敏的参数,其调节10%时,目标函数变化的最大比例分别达12.8%、4.61%和2.54%,其它参数对目标函数变化的影响均小于0.5%。该模型预测值与实验值具有较好的吻合性。     

Transformation using (x + 0.5) to stabilize the variance of populations

Transformation is required to achieve homo-scedasticity when we perform ANOVA to test the effect of factors on population abundance. The effectiveness of transformations decreases when the data contain zeros. Especially, the logarithmic transformation or the Box–Cox transformation is not applicable in such a case. For the logarithmic transformation, 1 is traditionally added to avoid such problems. However, there is no concrete foundation as to why 1 is added rather than other constants, such as 0.5 or 2, although the result of ANOVA is much influenced by the added constant. In this paper, I suggest that 0.5 is preferable to 1 as an added constant, because a discrete distribution defined in {0, 1, 2, . . .} is approximately described by a corresponding continuous distribution defined in (0, ≧) if we add 0.5. Numerical investigation confirms this prediction. Received: October 16, 1998 / Accepted: June 10, 1999     

Kinetic and structural characteristics of the inhibition of enoyl (acyl carrier protein) reductase by triclosan.

Triclosan is used widely as an antibacterial agent in dermatological products, mouthwashes, and toothpastes. Recent studies imply that antibacterial activity results from binding to enoyl (acyl carrier protein) reductase (EACPR, EC 1.3.1.9). We first recognized the ability of triclosan to inhibit EACPR from Escherichia coli in a high throughput screen where the enzyme and test compound were preincubated with NAD(+), which is a product of the reaction. The concentration of triclosan required for 50% inhibition approximates to 50% of the enzyme concentration, indicating that the free compound is depleted by binding to EACPR. With no preincubation or added NAD(+), the degree of inhibition by 150 nM triclosan increases gradually over several minutes. The onset of inhibition is more rapid when NAD(+) is added. Gel filtration and mass spectrometry show that inhibition by triclosan is reversible. Steady-state assays were designed to avoid depletion of free inhibitor and changes in the degree of inhibition. The results suggest that triclosan binds to E-NAD(+) complex, with a dissociation constant around 20-40 pM. Triclosan follows competitive kinetics with respect to NADH, giving an inhibition constant of 38 pM at zero NADH and saturating NAD(+). Uncompetitive kinetics are observed when NAD(+) is varied, giving an inhibition constant of 22 pM at saturating NAD(+). By following regain of catalytic activity after dilution of EACPR that had been preincubated with triclosan and NAD(+), the rate constant for dissociation of the inhibitor (k(off)) is measured as 1.9 x 10(-4) s(-1). The association rate constant (k(on)) is estimated as 2.6 x 10(7) s(-1) M(-1) by monitoring the onset of inhibition during assays started by addition of EACPR. As expected, the ratio k(off)/k(on) = 7.1 pM is similar to the inhibition constants from the steady-state studies. The crystal structure of E. coli EACPR in a complex with coenzyme and triclosan has been determined at 1.9 A resolution, showing that this compound binds in a similar site to the diazaborine inhibitors. The high affinity of triclosan appears to be due to structural similarity to a tightly bound intermediate in catalysis.     

采后预冷处理和贮藏温度对台湾绿竹笋鲜笋老化的影响

研究采后预冷处理和贮藏温度对台湾绿竹笋鲜笋老化的影响。结果表明,采后预冷处理和贮藏温度对台湾绿竹笋的老化速度、保鲜期和失重率有显著影响。采后预冷处理抑制鲜笋的老化,降低其老化率,延长保鲜期。(5±0.5) ℃恒温箱贮藏下,鲜笋的老化率、失重率低于(30±0.5) ℃恒温箱贮藏,贮藏期则相反。贮藏3、6、9、12 d,(5±0.5) ℃恒温贮藏下的鲜笋失重率均低于1%;(30±0.5) ℃恒温贮藏下,鲜笋失重率分别为26.32%、38.10%、48.04%、55.23%。     

Properties of passive binding of calcium to endoplasmic reticulum from adipocytes.

Calcium binding to isolated adipocyte microsomes enriched in endoplasmic reticulum has been characterized. Binding was concentration-dependent, saturable, and totally dissociable. Steady state was reached within 20 min at all calcium concentrations tested. Three apparent classes of binding sites were identified in kinetic and steady state studies using calcium concentrations from 1 muM to 10 mM. The affinity constants (and maximum binding capacities) as determined by computer analysis for the three classes were 2.1 X 10(5) M-1 (0.28 nmol of calcium/mg of protein), 1.3 X 10(4) M-1 (1.1 nmol/mg), and 1.3 X 10(2) M-1 (35 nmol/mg). The dissociation rate constants for the high and intermediate affinity classes of sites were 1.6 X 10(-3) S-1, respectively, and the association rate constant for the high affinity sites was 8 X 10(2) M-1 S-1. The affinity constant calculated from the rate constants was 5.0 X 10(5) M-1 for the high affinity sites in agreement with the value obtained in studies at steady state. The three classes of binding sites were specific for calcium. Magnesium was a noncompetitive inhibitor of calcium binding to all three classes of sites with a Ki of 9 to 12 mM. Calcium binding at 1 muM calcium was 50% inhibited by 18 muM La3+, 600 muM Sr2+, or 2.7 mM Ba2+. These data represent the first analysis of passive calcium binding to endoplasmic reticulum from nonmuscular cells and the first report of corresponding rate constants for either endoplasmic or sarcoplasmic reticulum. The characteristics of the binding are consistent with the properties of calcium transport by endoplasmic reticulum of adipocytes. The characteristics and specificity of the calcium binding constitute further evidence that endoplasmic reticulum plays an important role in cellular calcium homeostasis.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

A simple steady state kinetic model for alcohol dehydrogenase. Use of reaction progress curves for parameters estimation at pH 7.4

A simple rate equation for alcohol dehydrogenase was obtained by assuming independent binding sites for ethanol and NAD+ and fully competitive inhibition by the products of the reaction, acetaldehyde and NADH. A random binding order was also assumed. The rate equation is described by six parameters: four association constants (two for the substrates and two for the products of the reaction), Vf for the forward direction, and the equilibrium constant of the reaction. The six parameters were determined at pH 7.4 by numerical analysis of progress curves of reactions started with different concentrations of ethanol and NAD+. The parameters for alcohol dehydrogenase partially purified from rat liver were: Km for ethanol = 0.746 mM, Km for NAD+ = 0.0563 mM, Km for acetaldehyde = 7.07 microM, Km for NADH = 4.77 microM and Keq = 2.36 X 10(-4). The computed values allowed a very good simulation of the experimental progress curves and little variation was observed in the kinetic parameters when the reactions were started in the presence of either NADH or acetaldehyde.