Kinetic analysis of reversible closed bicyclic enzyme cascades covering the whole course of the reaction

A kinetic analysis of the closed bicyclic enzyme cascades is presented.

• 1.1. It includes the dependence on time from the onset of the reaction, of the concentration of the modified and unmodified enzyme species involved and the time course equations of the modificational fractions of the interconvertible enzymes.
• 2.2. The transient phase equations obtained allow the definition of new regulatory modification properties.
• 3.3. The expressions for concentrations of the unmodified and modified forms of the interconvertible enzymes, as well as those of the fractional modifications in the steady state are derived as particular cases of the general equations.
• 4.4. These steady state expressions coincide with those obtained by other authors.
• 5.5. The analytical results obtained are discussed in relation to the Escherichia coli glutamine syntethase cascade.

     

Purification and some particular kinetic properties of rat spleen cytosolic deoxynucleotidase

• 1.1. Rat spleen cytosolic deoxynucleotidase was purified 40,000-fold to almost homogeneity and had a specific activity of 3000 μmol/min per mg.
• 2.2. Molecular mass of the native enzyme was 45 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the native enzyme comprises two identical 27-kDa subunits.
• 3.3. Specific enzyme activity increases with increasing concentration of enzyme protein and approaches a plateau at high enzyme concentrations.
• 4.4. Enzyme activity increases gradually and nonlinearly with increasing concentration of enzyme in the low concentration range. Above a certain concentration the increase attains a maximal and constant slope.
• 5.5. The kinetic properties can be explained by assuming dissociation of the enzyme into subunits with low or no activity.

     

The characterization of the linoleic acid desaturation and elongation system in ovine placental tissue

• 1.1. The desaturation and elongation of linoleic acid has been studied in homogenates and in subfractions of ovine placental tissue.
• 2.2. The reaction was characterized in terms of pH and temperature optima, time course and protein concentration.
• 3.3. Activity was found to be confined to the 11,000g supernatant fraction of the tissue and the results suggest that the enzymes are membrane bound.
• 4.4. The cytosolic fraction and ATP were required for full activity and the reaction was inhibited by cyanide.
• 5.5. The properties of the reaction are compared with those of other desaturation systems and their implications with regard to possible reaction mechanisms are discussed.

     

Effects of aluminium and pH on calcium fluxes,and effects of cadmium and manganese on calcium and sodium fluxes in brown trout (Salmo trutta L.)

• 1.1. Simultaneous measurement of calcium fluxes in brown trout, at low external [Ca] (20 μ mol 1⁻¹), provided evidence of active uptake of Ca from the medium.
• 2.2. At pH 4.5, calcium influx was inhibited and efflux was stimulated.
• 3.3. Cd and Mn, but not Al, at concentrations within the ranges found in acid waters experiencing fish population decline, inhibited calcium influx. Efflux was unaffected.
• 4.4. Cd and Mn stimulated sodium influx and efflux.

     

Kinetics and stoichiometry of cysteinyldopa formation in the first steps of melanogenesis

• 1.1. Spectra of products obtained during dopa oxidation by mushroom tyrosinase in presence of cysteine or glutathione were recorded for the first minutes of the enzymatic reaction.
• 2.2. Two isosbestic points were defined, indicating the existence of a constant ratio between the disappearance of dopa and the formation of cysteinyl- or glutathione-dopa.
• 3.3. Matrix analysis of these spectra verified that there were two kinetically related absorbing species in solution, these being dopa and either cysteinyldopa or glutathione-dopa.
• 4.4. This stoichiometry (1:1) was confirmed by measuring the lag period in dopachrome accumulation, arising from the presence of thiol.
• 5.5. A kinetic approach has been proposed for the first steps, considered common, in the eumelanin and phaeomelanin biosynthesis pathway, thereby allowing us to establish a quantitative relation between the lag period and thiol concentration.
• 6.6. This relation can be used as a simple kinetic method for thiol evaluation.

     

Mitochondria from the ventricle of the marine clam,Mercenaria mercenaria: Substrate preferences and effects of pH and salt concentration on proline oxidation

• 1.1. Mitochondria with high respiratory control ratios (RCR) have been isolated from the ventricle of the marine clam Mercenaria mercenaria.
• 2.2. Proline is the preferred substrate of the mitochondria of the ventricle based on state 3 rates.
• 3.3. Pyruvate, ornithine and succinate are oxidized at rates 3/4 that of proline.
• 4.4. α-Glycerophosphate was oxidized at rates 1/2 that of proline.
• 5.5. The pH optimum for proline oxidation lies between 6.5 and 7.5 based on RCR and ADP/O and between 7.0 and 7.4 based on state 3 rates.
• 6.6. KCl concentrations between 250 and 450 mM gave optimal values for the oxidation of proline based on RCR and state 3 rates.
• 7.7. KCl concentration had little effect on ADP/O between 100 and 850 mM.

     

Phosphatidylethanolamine: Ceramide-ethanolaminephosphotransferase activity in synaptic plasma membrane vesicles. Influence of some cations and phospholipid environment on transferase activity. Further proof of its location

• 1.1. Synaptic plasma membrane vesicles (SPMV) from rat brain synthesized ceramide-phosphoethanolamine (SpE), an analogue of sphingomyelin (SpC) from phosphatidylethanolamine (PE) and ceramide.
• 2.2. This reaction was catalyzed by PE: ceramide-phosphotransferase.
• 3.3. The presence of PC did not modify the SpE synthesis and PI and PS at twice PE concentration seemed to be activators; only PG was an inhibitor at all concentrations.
• 4.4. Some cations (Mg²⁺, Mn²⁺) were without effect, while Ca²⁺ increased transferase activity, so was interesting to study.
• 5.5. Transferase was compared with sialidase (external enzyme).
• 6.6. Kinetics other than those already performed by us were undertaken in order to confirm its location.

     

Chemical intermediates in α-methylnoradrenaline oxidation by tyrosinase—II. Kinetic study of process

• 1.1. The pathway for α-methylnoradrenaline oxidation to α-methylnoradrenochrome by tyrosinase. has been studied as a system of various chemical reactions coupled to an enzymatic reaction.
• 2.2. A theoretical and experimental kinetic approach was proposed for such a system, we named this type of mechanism as a mechanism enzymatic-chemical-chemical (E₂CC).
• 3.3. Rate constants for the implied chemical steps at different temperature and pH values, were evaluated from measurement of the lag period, arising from the accumulation of aminochroine, that took place when α-methylnoradrenaline was oxidized at acid pH.
• 4.4. The thermodynamic activation parameters of the chemical steps, the deprotonation and the internal cyclization of o-quinone into leukoaminochrome, were also calculated.

     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

Effect of temperature on the kinetics of the yeast AMP deaminase

• 1.1. The temperature dependence of the kinetics of the yeast AM P deaminase was examined using the purified enzyme and the permeabilized yeast cells.
• 2.2. The increase in the enzyme affinity for the substrate AMP was accompanied by the decrease in the maximal velocity with the decreasing temperature in the absence and presence of ATP.
• 3.3. The apparent Km for AMP was lowest at 15–20°C, and the affinity was decreased below and above this temperature.
• 4.4. The rate of the AMP deaminase reaction remained constant over a wide range of temperature in the presence of physiological AMP concentrations.
• 5.5. The temperature dependent change in kinetic properties of AMP deaminase may contribute to the control of the yeast glycolytic flux under the condition of lower temperature environments.

     

The effect of d2o on glycolysis by rat hepatocytes

• 1.1. The effect of incorporating D₂O into the incubation medium on glycolysis and gluconeogenesis by hepatocytes from fasted rats was examined.
• 2.2. The substitution by heavy water, D₂O, at concentrations from 10 to 40%, stimulated glucose uptake, lactate production and CO₂ yields from glucose. At 10 mM glucose, 40% D₂O doubled glucose uptake, increased CO₂ production by 40%, and increased lactate production by 350%.
• 3.3. The stimulation of lactate production decreased at higher glucose concentrations, but was still substantial even at 80 mM glucose.
• 4.4. There was no effect on CO₂ production above glucose concentrations of 30 mM.
• 5.5. Ten percent D₂O showed little inhibition of lactate uptake, its oxidation and gluconeogenesis. At 40% D₂O the inhibition ranged from 10 to 20%.
• 6.6. No effect of D₂O on the rate of glucokinase or glucose-6-phosphatase was observed.
• 7.7. The concentration of fructose, 2,6-P was not affected by D₂O

     

Characteristics of tubulin aggregation by tubulin-binding proteins from brain and by synthetic polycations

• 1.1. A basic fraction from brain cytosol was found to contain two or more tubulin-binding proteins, able to induce aggregation of tubulin accompanied by hydrolysis of GTP.
• 2.2. In some respects tubulin aggregation by the brain proteins was similar to its aggregation by polylysine.
• 3.3. The burst of GTP hydrolysis accompanying tubulin aggregation by polylysine had the following characteristics: enhanced by salt and abolished by low temperature; not stoichiometric with the amount of tubulin precipitated and actually maximal at relatively low polylysine concentration; uncoupled temporally from aggregation, which occurred over a much shorter interval.

     

Relations between fatty acid synthesis,pyruvate concentration and cell concentration of suspensions of isolated rat hepatocytes

• 1.1. The cell concentration of suspensions of isolated rat hepatocytes affects both the rate of pyruvate accumulation in the incubation medium and the rate of fatty acid synthesis.
• 2.2. At low cell concentrations pyruvate accumulation is directly related to the cell concentration but levels off at higher concentrations even when maximum pyruvate concentrations in the medium are not yet reached.
• 3.3. The rate of fatty acid synthesis in the 30–60-min incubation interval is proportional to the cell concentration. In contrast, the rate of fatty acid synthesis during the 0–30-min incubation period decreases with increasing cell concentrations and subsequently becomes independent of the cell concentration.

     

Kinetic mechanism of the molecular forms of chicken liver mitochondrial malate dehydrogenase

• 1.1. The reaction kinetic mechanism (pH 7.4) of the molecular forms of chicken liver m-MDH is of the ordered bi-bi ternary complex type with the existence of the E-oxaloacetate, E-L-malate, E-NAD⁺ oxaloacetate, E-NADH-l-malate, E-NAD⁺-NADH, E-NAD⁺-NAD⁺, E-NADH-NAD⁺ and E-NADH-NADH abortive complexes.
• 2.2. The saturating concentration values of the substrates are notably modified, in certain cases, in the presence of the reaction products.

     

Kinetic analysis of the michaelis-menten mechanism in which the substrate and the product are unstable

• 1.1. A kinetic analysis of the Michaelis-Menten mechanism for the case in which both the substrate and the product are unstable, either spontaneously or as the result of the addition of a reagent, has been made.
• 2.2. The explicit time course equations of the immediate product and the species into which it subsequently is transformed have been derived under the conditions of rapid equilibrium and limiting substrate concentration.
• 3.3. The validity of these equations has been checked using numerical simulations.
• 4.4. The kinetic data analysis which we suggest is based on the time progress curves of the product or, in the case in which the product accumulation cannot be monitored experimentally, on the time progress curve of the species into which the immediate product is transformed.
• 5.5. This analysis allows the determination of the rate and the equilibrium constants if adequate experimental results are available.
• 6.6. We have chosen a numerical example, with which we illustrate the procedure of the kinetic data analysis by simulating some curves with assumed experimental errors.

     

Soluble and immobilized RP. palustris rhodanese—II. Some particular kinetic behaviour

• 1.1. Kinetic studies were carried out on the soluble and immobilized Rhodanese.
• 2.2. The soluble enzyme showed a typical Michaelis-Menten behaviour, an inhibitory effect was observed at high thiosulphate and cyanide concentrations.
• 3.3. The product sulphite was also an inhibitor, instead thiocyanate increased the enzyme velocity when it was added to the incubation mixture.
• 4.4. A ping-pong mechanism was proposed for Rp. palustris Rhodanese with a stable (free enzyme: E) and an unstable (sulfur substituted enzyme: ES) kinetic enzyme form.
• 5.5. The insolubilized Rhodanese presented an unusual kinetic behaviour, with sigmoid shape substrate profiles and non-linear double reciprocal plots.
• 6.6. From the empirical Hill equation, positive cooperativity (n>1) was found for both substrates.

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Characterization of NaK ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (de Man)

• 1.1. The specific activity of Na-K ATPase was determined from the microsomal preparation of gills dissected from adult Macrobrachium rosenbergii.
• 2.2. Maximal ATPase activity was achieved at a substrate concentration of 0.5 mM ATP.
• 3.3. Optimal enzyme activity was obtained at pH of 7.5.
• 4.4. The Arrhenius plot of Na-K ATPase activity revealed a marked discontinuity at 30°C. “Mg” ATPase activity did not exhibit a marked discontinuity.
• 5.5. The E_a for Na-K ATPase and “Mg” ATPase was 14.6 kCal/mole and 9.31 kCal/mole respectively. Q₁₀ values for Na-K ATPase was 2.34 and for “Mg” ATPase 1.65.
• 6.6. ATPase activity and gill homogenate protein concentration exhibited a linear relationship up to 130 μg protein/ml.
• 7.7. Na-K ATPase activity was inhibited by 10⁻³ M ouabain. It was equally inhibited by the removal of K⁺ from the reaction medium.

     

Regulation by progesterone and pregnenolone of dimeric aldehyde dehydrogenase from rat testis cytoplasm

• 1.1. Aldehyde dehydrogenase from rat testis cytosol has been purified to electrophoretic homogeneity. With an isoelectric point of 9.5, the enzyme appears a dimer with a subunit molecular weight of 52,500.
• 2.2. The influence of pregnenolone and progesterone on the kinetic behaviour has been investigated using valeraldehyde as substrate.
• 3.3. The kinetic data were fitted to a modified version of the Monod-Wyman-Changeux model and the fitting procedure resulted in a good correspondence between theoretical and experimental reaction rates over a wide range of valeraldehyde concentrations.
• 4.4. According to the model, the dimeric enzyme is in equilibrium between two confonnational states R and T. The R state displays higher affinity for valeraldehyde, but lower catalytic power. In the absence of substrates and effectors the [T]/[R] ratio is near to 1.
• 5.5. Pregnenolone and progesterone activate the enzyme by stabilizing the more active state T and by increasing the catalytic power of the R state. The increase of activity is counteracted by the inhibition exerted by both steroids on the T state.

     

Effects of low environmental pH and temperature on hatching and metabolic rates in embryos of Anax junius drury (Odonata: Aeshnidae) and the role of hypoxia in the hatching process

• 1.1. Studies were conducted in order to determine the combined effects of low environmental pH and temperature on embryonic survival capacity and metabolic rates in the dragonfly, Anax junius Drury. Studies were also conducted to assess the effects of hypoxia on hatching success as well as to investigate the role of hypoxia as a possible physiological triggering mechanism for hatching.
• 2.2. At water temperatures of 10–30°C, an environmental pH value of 3.0 was extremely limiting and significantly reduced hatching success.
• 3.3. Over a pH range of 3.0–5.0, a water temperature of 30°C was found to be severely limiting. Over a pH range of 6.0–7.0, hatching success was greater than 80% at test temperatures ranging from 10 to 25°C.
• 4.4. Embryos of A. junius exhibited a greater tolerance to markedly low environmental pH (3.0) than that previously reported for fish and amphibians, although survival capacity was less than 10%.
• 5.5. An environmental pH value of 3.0 has a significant detrimental effect on embryonic development. Survivorship and developmental rate increase significantly over a pH range of 4.0–5.0.
• 6.6. Oxygen consumption rates were lowest for fertilized eggs exposed to a pH of 3.0 at all test temperatures (10–30°C). Metabolic rates increased significantly at pH 4.O.
• 7.7. Embryos hatch successfully under hypoxic conditions in both aqueous and nonaqueous media. Results suggest that hypoxia acts as a triggering mechanism for hatching in this aquatic insect.