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PubMed2001

A comparison of the kinetics of low-density lipoprotein oxidation induced by copper or by gamma-rays: influence of radiation dose-rate and copper concentration.

The oxidation of low-density lipoproteins is the first step in the complex process leading to atherosclerosis. The aim of our study was to compare the kinetics of low density lipoprotein oxidation induced by copper ions or by oxygen free radicals generated by 60Co gamma-rays. The effects of copper concentration and irradiation dose-rate on LDL peroxidation kinetics were also studied. The oxidation of LDL was followed by the measurement of conjugated diene, hydroperoxides, and thiobarbituric acid reactive substance formation as well as alpha-tocopherol disappearance. In the case of gamma irradiation, the lag-phase before the onset of lipid peroxidation was inversely correlated to the radiation dose-rate. The radiation chemical rates (nu) increased with increasing dose-rate. Copper-induced LDL peroxidation followed two kinetic patterns: a slow kinetic for copper concentrations between 5-20 microM, and a fast kinetic for a copper concentration of 40 microM. The concentration-dependent oxidation kinetics suggest the existence of a saturable copper binding site on apo-B. When compared with gamma-rays, copper ions act as drastic and powerful oxidants only at higher concentrations (> or = 40 microM).

Adult↗

PubMed1975

Kinetic analysis of phospholipase A2 activity toward mixed micelles and its implications for the study of lipolytic enzymes.

A detailed kinetic scheme is proposed for the action of phospholipase A2 on mixed micelles of phospholipid and surfactant: see article. where E is the enzyme, A is the mixed micelle, and B is the phospholipid substrate in the mixed micelle. This scheme takes into account quantitatively the involvement of the lipid-water interface in the action of this enzyme toward substrate in macromolecular lipid complexes. The kinetic equation for this scheme is derived and four simplifying assumptions which are necessary for its practical application are described. Kinetic data are reported for the action of cobra venom phospholipase A2 (Naja naja naja) on 1,2-dipalmitosyl-sn-glycero-3-phosphorylcholine in mixed micelles with the nonionic surfactant Triton X-100, and these data are analyzed in terms of the kinetic equation presented. At 40 degrees, pH 8.0, and in the presence of 10 mM Ca2+, V was found to be about 4 X 10(3) mumol min(-1) mg of protein(-1). KsA, which is the dissociation constant for the enzyme-mixed micelle complex, is about 5 X 10(-4) M. KmB, the Michaelis constant for the catalytic step, which is (k-2 + k3)/k2, is 1 to 2 X 10(-10) mol cm-2. This kinetic treatment, together with the fact that the mixed micelle system allows the concentration of the substrate in the lipid-water interface to be varied, has made possible the quantitative separation of the association of a lipolytic enzyme with the lipid-water interface (expressed as KsA) and the binding to the substrate in the interface (reflected in the KmB term). The implications of this kinetic scheme for the analysis of phospholipase A2 from other sources acting on other aggregated forms of phospholipid and for the study of other phospholipases and lipases is considered.

Binding Sites↗

PubMed2002

Can electromagnetic radiations induce changes in the kinetics of voltage-dependent ion channels?

Ion channels are protein molecules, which can assume distinct open and closed conformational states, a phenomenon termed ion channel kinetics. The transitions from one state to another depend on the potential energy barrier that separates those two states. Therefore, it is rational to suppose that electromagnetic waves could interact with this barrier and induce changes in the rate transitions of this kinetic process. Our aim is to answer the question: can electromagnetic radiations induce changes in the kinetics of voltage-dependent ion channels? We simulated the effects of the low and high frequency electromagnetic waves on the sodium and potassium channels of the giant axon of Loligo. The key parameter measured was the fractional open time (fv), because it reflects the voltage dependence of the kinetics of channels. The electromagnetic radiations induced the following changes in the kinetics of the potassium and sodium channels: i/ low frequency waves kept the potassium channel 50% of the time open independent on the mean voltage applied through the membrane; ii/ a gradual inhibition of the inactivation on the sodium channel, when the amplitudes of the low frequency waves were increased; iii/ high frequency waves on the potassium channel, decreased both Vo (voltage in which the channel stays 50% open) and the steepness of fv (d fv/dV) as the amplitudes of the waves increased, and iv/ high frequency and low amplitude radiations on the sodium channel decreased the maximum value of fv (in relation to control), while high amplitudes increased this value. In conclusion, high and low frequency electromagnetic radiations were able to change the kinetics of the potassium and sodium channels in a squid giant axon model.

Animals↗

PubMedDate not supplied

[Study of kinetic parameters of singlet molecular oxygen in aqueous porphyrin solutions. Effect of detergents and the quencher sodium azide].

The kinetic parameters of porphyrin-photosensitized formation and deactivation of singlet molecular oxygen (1O2) and their dependence on the concentration of the 1O2 quencher sodium azide were investigated in air-saturated water, ethanol, and aqueous micellar solutions of detergents using time-resolved measurements of oxygen phosphorescence under pulsed laser excitation. The lifetimes of 1O2 formation and deactivation and the rate constants of 1O2 quenching by sodium azide were determined. It was shown that, with no azide in the solutions, the rise in phosphorescence intensity after the laser flash corresponded to the kinetics of energy transfer from the porphyrin triplet molecules to oxygen, while the decay kinetics corresponded to the kinetics of 1O2 deactivation. In the presence of detergent, a considerable increase in the 1O2 lifetime was observed, which is likely due to the localization of 1O2 molecules mostly in lipophilic micelles and not in the water phase. If relatively high azide concentrations were used, the lifetime of the porphyrin triplet state did not change but the 1O2 lifetime decreased to values similar to those in living cells. In this case, the inversion of the phosphorescence kinetic phases was observed. The rise corresponded to 1O2 deactivation, and the decay, to the energy transfer from triplet porphyrin to oxygen. The data suggest that, in living cells, 1O2 molecules are also located mainly in lipophilic structures and the 1O2 lifetime determines the kinetics of the phosphorescence rise after the laser pulse.

Detergents↗

PubMed1977

Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.

1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.

Animals↗

PubMed2004

Prognostic aspects of 18F-FDG PET kinetics in patients with metastatic colorectal carcinoma receiving FOLFOX chemotherapy.

UNLABELLED: We evaluated quantitative measurement series (MS) with 18F-FDG and PET and compared different quantification methods for prediction of individual survival in patients with metastatic colorectal cancer receiving chemotherapy with 5-fluorouracil, folinic acid, and oxaliplatin (FOLFOX). METHODS: The study comprised 25 patients. All patients were examined before the onset of FOLFOX therapy and after completion of the first and fourth cycles. SUV, fractal dimension (FD), a 2-compartment model with computation of k1, k2, k3, and k4, and vascular fraction (VB) were used for data evaluation. Survival data served as a reference for the PET data. Discriminant analysis (DA), regression, and best-subset analysis were applied to the data. RESULTS: Twenty of 25 patients died up to 801 d after the first PET study. A cutoff of 1 y (364 d) was used to classify the patients into 2 a priori groups, namely the short- and long-term survival groups. DA was used to predict the 2 categories using SUV and kinetic parameters of 18F-FDG metabolism as predictor variables. SUV provided a correct classification rate (CCR) ranging from 62% to 69%. SUV of the third MS resulted in a CCR of 69% as a single parameter. The best results were yielded by the use of kinetic parameters (k1, k3, VB, and FD) as predictor variables. CCR was 78% using kinetic 18F-FDG parameters of the first and third MS, in comparison with 69% for the corresponding SUVs. A multiple linear regression model was applied to the data to assess the relationship between individual survival and the PET data. The best-subset method revealed a correlation coefficient of 0.850 for the kinetic parameters of the first (k3, k4, VB, and FD) and third (k1, k2, k4, and VB) MS. CONCLUSION: The combination of kinetic parameters of the first and the third MS is acceptable for classification into a short or long survival class. Furthermore, even an individual prognosis of survival can be achieved using kinetic 18F-FDG parameters of the first and third MS.

Algorithms↗

PubMed2005

Effect of soil and a nonionic surfactant on BTE-oX and MTBE biodegradation kinetics.

The biodegradation kinetics of BTE-oX and MTBE, mixed all together, in the presence of 905 mg/L VSS of BTEX-acclimated biomass was evaluated. Effects of soil and Tergitol NP-10 in aqueous samples on substrate biodegradation rates were also evaluated. Biodegradation kinetics was evaluated for 36 hours, every 6 hours. MTBE biodegradation followed a first-order one-phase kinetic model in all samples, whereas benzene, toluene and ethylbenzene biodegradation followed a first-order two-phase kinetic model in all samples. O-xylene biodegradation followed a first-order two-phase kinetic model in the presence of biomass only. Interestingly, o-xylene biodegradation was able to switch to a first-order one-phase kinetic model when either soil or soil and Tergitol NP-10 were added. The presence of soil in aqueous samples retarded benzene, toluene and ethylbenzene removal rates. O-xylene and MTBE removal rates were enhanced by soil. The addition of Tergitol NP-10 to aqueous samples containing soil had a positive effect on substrate removal rate in all samples. Substrate percent removals ranged 77-99.8% for benzene, toluene and ethylbenzene. O-xylene and MTBE percent removals ranged 50.1-65.3% and 9.9-43.0%, respectively.

Benzene Derivatives↗

PubMed1991

[Biochemical kinetics of plasmid replication processes].

Two groups of plasmid replication reactions occurring during the growth of cell populations were kinetically characterized: 1) short-term intermediate processes coupled with changes in plasmid concentrations in host cells and occurring in the first few generations; 2) long-term processes resulting in the loss of plasmids and occurring during prolonged cultivation of cells. It was shown that first group processes are determined by the ratios of plasmid replication rates relative to cell growth. Using growing populations of E. coli as examples, it was shown that the shape of kinetic curves reflecting the plasmid copy number depends on the nature of their replicon. In this case plasmids with ColE1 replicon exhibit kinetic behaviour of the first type (temporal loss of plasmid copy number in the cells), whereas plasmids with p15 replicon display the second type behaviour consisting in the decrease of the plasmid copy number followed by its short-term drastic increase. For both types of kinetic behaviour a kinetic description adequately reflecting these processes is given. It is supposed that the loss of plasmids during long-term cultivation of cell populations may be associated with both segregation (lower growth rates of plasmid-containing cells in comparison with cells containing no plasmids) and mutation mechanisms. Kinetic descriptions of both mechanisms are proposed and principles of their discrimination are formulated. Practical recommendations for the prevention of the plasmid loss by the cells that are based on theoretical data are given.

Kinetics↗

PubMed1990

[A kinetic scheme of human neutrophil 5-lipoxygenase activity].

In animal cells arachidonic acid is metabolized via the 5-, 12- and 15-lipoxygenase pathways. The kinetic mechanism of action of plant (soya) and animal (reticulocyte) 15-lipoxygenases is now well established. 5-Lipoxygenase possesses, in all probability, the most complex mechanism of activity regulation. At present several effectors of neutrophil 5-lipoxygenase, both cytosolic and membrane-bound ones, have been identified. The molecular and kinetic mechanisms of action of the enzyme are still open to question. A kinetic scheme of regulation of synthesis of arachidonic acid 5-lipoxygenase metabolites which does not exclude the presence of two binding sites on the enzyme molecule, is proposed. Within the framework of this kinetic scheme the enzyme activator complex may be the active form of the enzyme. There is evidence that the curve for the time dependence of 5-HETE accumulation in neutrophils stimulated by the Ca2+ ionophore A23187 has a maximum, while the corresponding curve for the LTB4 accumulation is a curve with saturation. It was shown that an increase in the concentration of exogenous arachidonate induces the synthesis of 5-HETE, whereas the concentration of LTB4 remains practically unchanged. The results of mathematical analysis of the above kinetic scheme and a comparison of experimental and calculated values suggest that the reaction effector, Ca2+, plays a crucial regulatory role in the observed kinetic dependencies reflecting the formation of two sequential products of 5-lipoxygenase oxidation of arachidonate. In this way Ca2+ strongly influences the first step of the reaction, i.e., 5-HETE formation; its effect on the second reaction step (5-HETE conversion into LTA4) is far less apparent.

Algorithms↗

PubMed1991

Model development to describe the heterogeneous kinetics of apolipoprotein B and triglyceride in hypertriglyceridemic subjects.

The heterogeneous nature of very low density lipoprotein (VLDL) metabolism in hypertriglyceridemia gives rise to complex kinetics when labeled VLDL are traced. Analysis of such systems benefits from the simultaneous study of several metabolically discrete subfractions which are then integrated. We have studied the kinetics of VLDL and intermediate density lipoprotein (IDL) apoprotein B and triglyceride simultaneously by injecting homologous 125I-labeled VLDL1 and 131I-labeled VLDL2 and [2-3H]glycerol intravenously in three diverse type IV hyperlipoproteinemic subjects. An additional type IV subject received only [2-3H]glycerol. Specific radioactivities were measured in: VLDL1-triglyceride and -apoB, VLDL2-triglyceride and -apoB, and in each corresponding subfraction after further separation into heparin-Sepharose-bound and -unbound fractions. ApoB and triglyceride specific radioactivities were also measured in IDL. Analysis of the kinetics of apoB in the unbound fractions in VLDL1 and VLDL2 showed the presence of two pools of particles, one of which turned over rapidly. The kinetics of apoB in the bound fractions in VLDL1 and VLDL2 were, in contrast, dominated by a large slowly turning over pool of particles that resembled the kinetics of whole VLDL. Evidence of a partial precursor-product relationship between the unbound and bound fractions suggested that the former was richer in nascent-like particles, while the latter contained more remnant particles. However, triglyceride specific radioactivity curves for both unbound and bound fractions showed initial rapid rises and broad peaks, indicating that the bound fraction also contained a substantial proportion of nascent-like particles. Using multicompartmental analysis, a model was constructed to account for the kinetics of both apoB and triglyceride in all fractions of VLDL and in IDL. The model comprises two parallel delipidation pathways that supply a common remnant pool with these features: 1) multiple direct inputs of particles into plasma at VLDL2 and IDL levels; 2) heterogeneous triglyceride precursor pools leading to different rates of labeling of VLDL1 and VLDL2; 3) very substantial delipidation of VLDL2 particles prior to conversion to IDL and; 5) triglyceride production rates somewhat higher than previously reported. The inclusion in the model of the rapidly turning over pool of triglyceride-rich particles, identified in the heparin-unbound fraction, suggests that values for triglyceride production in man have been underestimated.

Aged↗

PubMedDate not supplied

[Kinetics of reactions in polyenzyme systems. III. Electron transport processes].

Kinetic relationships of electron transport processes in biological systems have been analysed. Formal-kinetic description and discrimination of the two main models of electron transport processes in systems of homogeneously distributed carriers and in systems of structurally bound carriers is presented including steady-state and non-steady-state reaction kinetics. Methods of determination of limit-stage rate constant and rate constants for all elementary steps of the reaction are given. Dependencies of the degree of electron carriers reduction on donor and acceptor concentrations and the influence of reduction or oxidation of carriers on the kinetic properties of electron transport chain (electron influence factor) in the system of structurally-bound carriers have been analyzed. On the basis of kinetic models a molecular-kinetic interpretation of activation effect of mitochondrial respiratory chain during the process of electron transport is offered.

Cytochromes↗

PubMed1990

Kinetic measurement of bicarbonate in serum by thiocyanate inhibition of wheat germ phosphoenolpyruvate carboxylase.

We describe a kinetic enzymic method for serum bicarbonate analysis, using wheat germ phosphoenolpyruvate carboxylase (EC 4.1.1.31) coupled through oxaloacetate reduction with NADH in the presence of malate dehydrogenase (EC 1.1.1.37). Inhibition with potassium thiocyanate yielded first-order kinetics with respect to bicarbonate over the concentration range of 0-45 mmol/L. The inhibitor was chosen by evaluating reaction data in the presence of different anions, with use of a monoexponential model. Criteria for first-order kinetics included a constant reaction half-life over the concentration range and SDest for the model comparable with the magnitude of spectrophotometric noise. We compared our kinetic method (y) with an automated ion-selective electrode method (x), obtaining the regression relationship y = 0.97x + 1.2 mmol/L (r = 0.991; n = 77; mean = 25.5 mmol/L; y = 25.3 mmol/L). Within-run precision from duplicates was 3.1% (mean = 25.2 mmol/L; n = 72). Total analytical precision (n = 12) was 9.4% (mean = 15 mmol/L) for the low control and 4.3% (mean = 32 mmol/L) for the high control. We conclude that the kinetic assay allows use of large serum-to-reagent ratios (1:100) and smaller amounts of NADH than an equilibrium assay. The assay is suitable for automated kinetic analysis.

Bicarbonates↗

PubMed1990

Kinetic asymmetry of renal Na+-L-lactate cotransport. Characteristic parameters and evidence for a ping pong mechanism of the trans-stimulating exchange by pyruvate.

Brush border vesicles prepared from horse renal cortex were used to study the kinetic properties of the Na+-L-lactate carrier on the outer and inner faces of the membrane. Two methods were applied for these measurements (in the absence of an electrical gradient): a direct method using influx and efflux kinetics, and an indirect method applied to trans-stimulated influx kinetics using membrane vesicles preloaded with various pyruvate concentrations (the latter enabled us to observe simultaneously the inner and outer carrier properties). Kinetic parameters obtained by the first method have shown that under sodium lactate chemical gradient, the carrier efficiency (estimated by the ratio of k = Vm/Km) is higher for the influx than efflux, a mechanism indicating a kinetic asymmetry of the transport. This difference remains at chemical equilibrium of solute concentration. The similarity of outer and inner affinity of sodium permits one to conclude that the kinetic asymmetry of the sodium lactate transport is related to the lactate-carrier interaction and not to that of the sodium-carrier. The second method using the pyruvate trans-activation effect (under sodium chemical equilibrium) has shown an affinity of lactate (Kt(out) = 1.1 mM), about 15 times higher for the carrier in the extracellular orientation than that of pyruvate for the carrier in the intracellular orientation (Kt(pyr) = 36 mM). This method has demonstrated a ping pong mechanism for the trans-activation exchange which accounts for a selective pore carrier model like a gated channel. These asymmetric properties are related to the AS glide sequential model (A and S being Na+ and lactate, respectively) proposed previously for the Na-L-lactate cotransport and to a different accessibility of the organic solute but not of the sodium on the two membrane faces.

Animals↗

PubMedDate not supplied

[Purification, molecular multiplicity and kinetic properties of "biosynthetic" L-threonine dehydratase from E. coli K-12].

"Biosynthetic" L-threonine dehydratase was purified to homogeneous state with yield 29% of total activity from E. coli K-12. The cells were disrupted by means of ultra sound. Nucleic acids and nucleoproteins were precipitated with protamine sulphate, the proteins were fractioned with (NH4)2SO4, by gel filtration through Sephadex G-25 followed by chromatography on DEAE-cellulose using stepways elution by changing the pH-values. The homogenity of the enzyme was shown by polyacrylamide gel disc electrophoresis in the presence of dodecylsulphate. The enzyme consists of equal subunits having a molecular weight about 57000. The polyacrylamide gel disc electrophoresis had shown that the native enzyme consists of a set of oligomeric forms. The multiplisity of molecular organization of the enzyme was relfected in complicated kinetic behavior: at pH greater than 9 on the plots of initial reaction rate (upsilon) versus initial substrate concentration ([S]0) there were four inflexion points (two intermediate plateaux) the position and deepness of which depended on enzyme concentration. Kinetic properties of the highly purified enzyme and the enzyme in crude cell extracts at pH 9.3 and 7.4 were identical. At pH 8,3 on the upsilon versus [S]0 plots appeared two inflexion points (one intermediate plateau), the position of which practically did not depend on enzyme concentration in the reaction mixture but strongly depended on the enzyme concentration in the stock solution. Repeated polyacrylamide gel disc electrophoresis of several oligomeric forms isolated by the first electrophoresis had shown that oligomeric forms underwent a slow polymerization. It is suggested that "biosynthetic" L-threonine dehydratase from E. coli K-12 is a set of multiple oligomeric forms having different kinetic parameters. Probably, each form of the enzyme has a "simple" kinetics characterized by hyperbolic or sigmoidal shape of upsilon versus [S]0 plots. The rate of equilibrium between the oligomeric forms is small in comparison with the enzyme reaction velosity, that lead to the complex kinetic curves appearing as a result of summing up the kinetics inherent to the individual forms.

Escherichia coli↗

PubMed1978

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Kinetic characterization of wild type and feedback-resistant forms of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous wild type and feedback-resistant forms of the biosynthetic sn-glycerol 3-phosphate (glycerol-P) dehydrogenase of Escherichia coli (EC1.1.1.8) were subjected to two-substrate kinetic analysis. The kinetics of the NADPH-dependent reduction of dihydroxyacetone phosphate (dihydroxyacetone-P) and of the NADP-dependent oxidation of glycerol-P indicate that these reactions proceed by a sequential mechanism. Glycerol-P was a competitive inhibitor with respect to dihydroxyacetone-P for both enzymes. The wild type and feedback-resistant glycerol-P dehydrogenases had Ki values for glycerol-P of 4.4 micrometer and 43 micrometer, respectively. Therefore, the sensitivity of the wild type activity and reduced sensitivity of the feedback-resistant activity, both noted previously in crude extracts, were inherent properties of the enzymes. The patterns of product inhibition for both enzymes were identical, and the difference in the inhibition constants for glycerol-P occurred without significant alteration of any other kinetic constant determined. Kinetic mechanisms consistent with the patterns of product inhibition violated Haldane relationships and other kinetic relationships. These discrepancies suggest that glycerol-P inhibition occurs at a site distinct from the active site. The pH dependencies of the Km for dihydroxyacetone-P and the Ki for glycerol-P were markedly different suggesting the existence of an allosteric site. The addition of glycerol-P in the presence of NADPH stabilized both enzymes against thermal inactivation. Half-maximal stabilization was provided by 5 micrometer and 50 micrometer glycerol-P for the wild type and feedback-resistant enzymes, respectively. These kinetic data, considered in conjunction with previous physiologic and genetic data, indicate that the synthesis of glycerol-P is regulated in vivo by glycerol-P inhibition of the glycerol-P dehydrogenase. The data suggest that glycerol-P inhibition occurs at an allosteric, regulatory site.

Dihydroxyacetone Phosphate↗

PubMed1988

Kinetic analysis of the Ca2+-dependent, membrane-bound, macrophage phospholipase A2 and the effects of arachidonic acid.

The kinetics of the Ca2+-dependent, alkaline pH optimum, membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line were studied using various phosphatidylcholine (PC) and phosphatidylethanolamine (PE) substrates. This enzyme exhibits "surface dilution kinetics" toward PC in Triton X-100 mixed micelles, and the "dual phospholipid model" was found to adequately describe its kinetic behavior. With substrate in the form of sonicated vesicles, the dual phospholipid model should give rise to Michaelis-Menten type kinetics. However, the hydrolysis of dipalmitoyl-PC, 1-palmitoyl-2-oleoyl-PC, and 1-stearoyl-2-arachidonoyl-PC vesicles exhibited two distinct activities. Below 10 microM, the data appeared to follow Michaelis-Menten behavior, while at higher concentrations, the data could best be fit to a Hill equation with a Hill coefficient of 2. These PCs had Vmax values for the low substrate concentration range of 0.2-0.6 nmol min-1 mg-1 and Km values of 1-2 microM. At the high substrate concentration range, the Vmax values were between 5 and 7 nmol min-1 mg-1. PC containing unsaturated fatty acids had an apparent Km, determined from the Hill equation, of about 15 microM, while the apparent Km of dipalmitoyl-PC was 0.6 microM. When 70% glycerol was included in the assays, a single Michaelis-Menten curve was obtained for both dipalmitoyl-PC and 1-stearoyl,2-arachidonoyl-PC. Possible explanations for these kinetic results include reconstitution of the membrane-bound phospholipase A2 in the phospholipid vesicle or the enzyme has tow distinct phospholipid binding function. The kinetics for both dipalmitoyl-PC and dipalmitoyl-PE hydrolysis in vesicles was very similar, indicating that the enzyme does not greatly prefer one of these head groups over the other. The enzyme also showed no preference for arachidonoyl containing phospholipid. Enzymatic activity toward PC containing saturated fatty acids was linear to about 15% hydrolysis while the hydrolysis of PC containing unsaturated fatty acids was linear to only about 5%. This loss of linearity was due to inhibition by released unsaturated fatty acids. Arachidonic acid was found to be a competitive inhibitor of dipalmitoyl PC hydrolysis with a K1 of 5 microM. This tight binding suggests a possible in vivo regulatory role for arachidonic acid. Three compounds of the arachidonic acid cascade, prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2, showed no inhibition of enzymatic activity.

Algorithms↗

PubMed1987

The kinetics of assembly of normal and variant human oxyhemoglobins.

The kinetics of assembly have been monitored spectrophotometrically for normal and variant human oxyhemoglobins in 0.1 M Tris, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4, at 21.5 degrees C. Oxyhemoglobin versus oxy chain static difference spectra were performed and revealed subtle but significant absorption changes in both the visible and Soret regions. Kinetic experiments were performed by rapidly mixing equivalent (in heme) concentrations of alpha and beta A chains and following the change in absorbance at 583 nm with time. Over a protein concentration range of 10-100 microM in heme prior to mixing, these time courses were homogeneous and followed first-order kinetics, yielding a value of 0.069 s-1 for the apparent rate constant of dissociation of oxygenated beta A chain tetramers. Under these conditions, the overall assembly of oxyhemoglobins S (beta 6Glu----Val) and N-Baltimore (beta 95Lys----Glu) were also governed by the rates of dissociation of their respective oxygenated beta S and beta N-Baltimore chain tetramers with the apparent first-order rate constants of 0.044 and 0.15 s-1, respectively. In the Soret region, the alpha, beta monomer combination reaction could be observed if the protein concentration (heme basis) was lowered and if protein nonequivalency (beta chain exceeded alpha chain concentration) mixing experiments were performed. A kinetic oxyhemoglobin A, oxy-alpha, oxy-beta A monomer difference spectrum could be generated, and simple second-order kinetics were observed (415 nm) yielding rate constants of 2.3, 3.3, and 4.8 X 10(5) M-1 s-1 for the assembly of oxyhemoglobins S, A, and N-Baltimore, respectively. To our knowledge, this is the first kinetic study to reveal significant differences between the rate of association of alpha and beta monomers of hemoglobin A and those of two distinctly charged hemoglobin variants.

Humans↗

PubMed1987

Quantitation of endotoxin in products using the LAL kinetic turbidimetric assay.

The data presented here show the kinetic turbidimetric LAL assay to be a highly quantitative and effective method for determining endotoxin concentrations in products. The assay allows for the accurate assessment of inhibiting or enhancing effects in products when related to a LRW standard curve. However, designating some products as inhibitors or enhancers can be both misleading and erroneous unless qualified as to the dilution and/or endotoxin concentration. Our results demonstrate that some products can yield both inhibiting and enhancing results when related to water. Due to the enhanced resolution of the kinetic turbidimetric assay, these complicating inhibition/enhancing effects can usually be avoided by diluting to the WED. Alternatively, products could be related to a PSC in which the endotoxin response is defined and quantified within the product itself. The practicality of a PSC, however, depends upon the pass/fail limit established, the "cleanliness" of the product used as a standard and the degree of product "lot to lot" variability. Current FDA Guidelines consider a PSC valid providing the value of the "unspiked" product extrapolated from the regression line of the PSC is less than 10% of lambda, the lowest endotoxin concentration used to construct the standard. All products in which LAL kinetics have not been previously analyzed will require a characterization similar to that used with the four products described. From these data, the optimal methodology for kinetically testing the product (dilution to a WED or generation of a PSC) can be determined. Although analysis of endotoxin in a product will always require a characterization of the kinetics of the LAL-endotoxin-product reaction, subsequent testing should be rapid and straightforward. More importantly, the kinetic turbidimetric assay allows the user to quantitatively assess product endotoxin levels with a degree of precision greater than that of any other methodology currently in use.

Endotoxins↗