Kinetic aspects of regulation of pyruvic decarboxylase

The decarboxylation of pyruvate catalysed by pyruvic decarboxylase (EC 4.1.1.1) from wheat germ is shown to be autocatalytic. Evidence is presented which suggests that the enzyme exists in an active and an inactive form—the latter being converted into the active form in the presence of pyruvate and by low pH. It is suggested that the relatively slow interconversion of the two forms of the enzyme may represent a time buffering system to prevent the decarboxylation of pyruvate in response to transient changes in pH.     

Kinetic aspects of follicle development in the rat

The eighth and ninth generations of follicle growth in rats represent a turning point in development. This stage is characterized by establishing a complete regulatory control based on feedback in follicle development. The feedback between follicles and gonadotropin secretion regulates a number of follicles maturing for ovulation. Gonadotropins seem to play a permissive, and not a directive part in regulation of follicle development in the eighth and ninth generations. By this stage of development, possibilities for theca and granule cells become sharply limited. Whereas expression of many maturation features may be hastened or hindered by changes in hormonal status, the result of development cannot be changed. Although only last generations of theca and granule cells exhibit mature functional features, their precursors seem to become committed to a single direction of development at early stages of follicle development. Neither the stage when precursor cells become irreversibly committed to differentiation into granule or theca cells, neither regulatory factors which determine this process have been identified yet. We suppose that precursor cells become committed to thecal tissue compartment when the follicle is at a primordial stage of development. Precursors of all the follicle components may already be assembled into one unit by the beginning of follicle growth. Accumulation of sufficient number of precursor cells around the primordial follicle may serve as a signal for follicle growth initiation. We think that the understanding of follicle postnatal growth and development should be based on understanding of origins, destiny, and possibilities of cells which form ovary and its compartments. First generations of follicle growth seem to be most promising for future research.     

Kinetic analysis and mechanistic aspects of autoxidation of catechins

A peroxidase-based bioelectrochemical sensor of hydrogen peroxide (H(2)O(2)) and a Clark-type oxygen electrode were applied to continuous monitoring and kinetic analysis of the autoxidation of catechins. Four major catechins in green tea, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, and (-)-epigallocatechin gallate, were used as model compounds. It was found that dioxygen (O(2)) is quantitatively reduced to H(2)O(2). The initial rate of autoxidation is suppressed by superoxide dismutase and H(+), but is independent of buffer capacity. Based on these results, a mechanism of autoxidation is proposed; the initial step is the one-electron oxidation of the B ring of catechins by O(2) to generate a superoxide anion (O(2)(*-)) and a semiquinone radical, as supported in part by electron spin resonance measurements. O(2)(*-) works as a stronger one-electron oxidant than O(2) against catechins and is reduced to H(2)O(2). The semiquinone radical is more susceptible to oxidation with O(2) than fully reduced catechins. The autoxidation rate increases with pH. This behavior can be interpreted in terms of the increase in the stability of O(2)(*-) and the semiquinone radical with increasing pH, rather than the acid dissociation of phenolic groups. Cupric ion enhances autoxidation; most probably it functions as a catalyst of the initial oxidation step of catechins. The product cuprous ion can trigger a Fenton reaction to generate hydroxyl radical. On the other hand, borate ion suppresses autoxidation drastically, due to the strong complex formation with catechins. The biological significance of autoxidation and its effectors are also discussed.     

Kinetic aspects of l-quinuclidinyl benzilate interaction with muscarinic receptor

l-Quinuclidinyl benzilate is undoubtedly the most widely used radioactive reporter ligand for studies on muscarinic receptor. In the present Commentary the kinetic aspects of the interaction of this ligand with muscarinic receptor are summarized. On the basis of these results a kinetic mechanism has been proposed involving consequential isomerization of the receptor-ligand complex and cooperative regulation of this process by the excess of the ligand. In addition, the data give evidence of at least two different types of binding sites on the receptor. Owing to the solubilization of the receptor protein there occur remarkable changes in its kinetic properties. The kinetic analysis points to inadequacy of the simple one-step equilibrium binding scheme for l-quinuclidinyl benzilate interaction with muscarinic receptor, which may explain the apparently contradictory data in the literature, such as the large scattering of the K_d values and the different regularities described in the case of the receptor-ligand complex dissociation reaction. That points to the conclusion that l-quinuclidinyl benzilate is an “inconvenient” ligand for receptor studies, which call for true equilibrium conditions of the system.     

Kinetic aspects of virus targeting by nanoparticles in vivo

One of the suggested ways of the use of nanoparticles in virology implies their association with and subsequent deactivation of virions. The conditions determining the efficiency of this approach in vivo are now not clear. Herein, I propose the first kinetic model describing the corresponding processes and clarifying these conditions. My analysis indicates that nanoparticles can decrease concentration of infected cells by a factor of one order of magnitude, but this decrease itself (without feedback of the immune system) is insufficient for full eradication of infection. It can, however, induce delay in the progress of infection, and this delay can help to form sufficient feedback of the immune system.

     

Chlorophyllase in citrus leaves. Kinetic aspects of the reaction

The reaction: Chlorophyll + Enzyme → Chlorophyllide + Phytol follows a first order kinetics with regard to the quantity of enzyme, when it is saturated by substrate. K_m and V_m were determined from the average reaction rates for the substrates: Chlorophylla andb, pheophytina andb, methylchlorophyllidea and methylpheophorbide a: The lowest K_m corresponded to chlorophylla and the highest V_m to methylpheophorbidea. For a substrate of chlorophyll (a + b), K_m and V_m were determined also using the initial reaction rates. “Enzyme efficiency” was calculated using both methods of determination. The reaction products partially inhibit the hydrolytic process.     

Kinetic and thermodynamic aspects of lipid translocation in biological membranes

A theoretical analysis of the lipid translocation in cellular bilayer membranes is presented. We focus on an integrative model of active and passive transport processes determining the asymmetrical distribution of the major lipid components between the monolayers. The active translocation of the aminophospholipids phosphatidylserine and phosphatidylethanolamine is mathematically described by kinetic equations resulting from a realistic ATP-dependent transport mechanism. Concerning the passive transport of the aminophospholipids as well as of phosphatidylcholine, sphingomyelin, and cholesterol, two different approaches are used. The first treatment makes use of thermodynamic flux-force relationships. Relevant forces are transversal concentration differences of the lipids as well as differences in the mechanical states of the monolayers due to lateral compressions. Both forces, originating primarily from the operation of an aminophospholipid translocase, are expressed as functions of the lipid compositions of the two monolayers. In the case of mechanical forces, lipid-specific parameters such as different molecular surface areas and compression force constants are taken into account. Using invariance principles, it is shown how the phenomenological coefficients depend on the total lipid amounts. In a second approach, passive transport is analyzed in terms of kinetic mechanisms of carrier-mediated translocation, where mechanical effects are incorporated into the translocation rate constants. The thermodynamic as well as the kinetic approach are applied to simulate the time-dependent redistribution of the lipid components in human red blood cells. In the thermodynamic model the steady-state asymmetrical lipid distribution of erythrocyte membranes is simulated well under certain parameter restrictions: 1) the time scales of uncoupled passive transbilayer movement must be different among the lipid species; 2) positive cross-couplings of the passive lipid fluxes are needed, which, however, may be chosen lipid-unspecifically. A comparison of the thermodynamic and the kinetic approaches reveals that antiport mechanisms for passive lipid movements may be excluded. Simulations with kinetic symport mechanisms are in qualitative agreement with experimental data but show discrepancies in the asymmetrical distribution for sphingomyelin.     

Kinetic aspects of the role of phospholipids in D-beta-hydroxybutyrate dehydrogenase activity

Interactions of phospholipids with D-beta-hydroxybutyrate dehydrogenase (BDH), a lecithin-requiring enzyme, have been studied by a kinetic approach. The process of reactivation of BDH by phospholipids, which follows a second-order mechanism, reveals that (1) at least 2 mol of lecithins is essential for the reactivation of the enzyme, and (2) the enzyme contains two dependent binding sites for lecithins. The graphic representation of the time course of reactivation shows a latent phase which decreases when there is an increase in the amount of phospholipids. A Scatchard plot treatment of the reactivation kinetic data reveals the presence of two classes of phospholipid binding sites, which exhibit high and low affinities related to the binding of four and two lecithin molecules, respectively. The effect of temperature on BDH activity and on the inactivation of the apoenzyme with N,N'-dicyclohexylcarbodiimide (a specific carboxyl reagent) or with phenylglyoxal (a specific arginine reagent) shows a break at 22-24 degrees C, indicating a slight structural change in the enzyme-active site around this temperature. In addition, the variations in enzyme kinetic parameters, according to the nature of phospholipids, are in agreement with conformational changes related to the nature and to the fluidity state of phospholipids. However, the apparent NAD+ binding constant does not depend on the phospholipid's fluidity.     

Kinetic aspects of macroscopic fluctuations in solutions of liver alcohol dehydrogenase

Kinetic analysis of macroscopic fluctuations in alcohol dehydrogenase reaction was carried out. The integral form of Michaelis-Menten equation was used. It is shown that reaction rate fluctuations to be observed in experiment are connected with kinetic constants of relative affinity of ADG to NAD and NAD X H.     

Kinetic aspects of alkaline phosphatase refolding in the presence of alpha-cyclodextrin

To get a better understanding of the molecular aspects of protein folding, the refolding kinetic behavior of guanidine hydrochloride-denatured alkaline phosphatase (ALP) was studied in the presence of alpha-cyclodextrin (alpha-CD) through two different approaches: the dilution additive and the artificial chaperone-assisted methods. It was found that alpha-CD enhanced the recovered activity more than 50% via both approaches while decreased the refolding rate, perhaps due to engaging the hydrophobic patches of the protein in a rigid conformation. In contrast, detergents used in the artificial chaperone method increased the refolding rate significantly. A comparison of the rate constants for the refolding and the activity recovery of denatured ALP in the presence of various concentrations of CD and different kinds of detergents showed that they do not progress in a synchronized pattern. This may be attributed to continuous structural rearrangements in the protein long after the return of enzyme activity. These observations are discussed in terms of kinetic and structural aspects of the refolding pathway.     

Enzyme peptide synthesis and semisynthesis: Kinetic and thermodynamic aspects

The hydrolysis/synthesis equilibrium of the peptide bond is governed by the relative magnitudes of the corresponding Gibbs' energies of hydrolysis to non-ionized products and of their ionization. The positive energy change in peptide hydrolysis to non-ionized products is the thermodynamic basis for the acyl and leaving group specificity of proteinases. With a proteinase of suitable specificity, some peptide bonds can be synthesized by a thermodynamically controlled enzyme aminolysis of specific acylamino or peptide acids; any peptide bond can be formed by a kinetically controlled enzyme aminolysis of the corresponding acylamino or peptide esters.     

Mass cultivation of Medicago sativa growing on lactose: Kinetic aspects

Summary Batch cultures of Medicago sativa cells have been carried out in the dark under aerobic conditions using lactose as the sole carbon source. The stoichiometric analysis has been correlated with both the oxygen demand and the cell productivity in an oxygen-limited cultivation. The minimum oxygen transfer has been estimated to be 12.5 h^–1, i.e., 0.3 v.v.m; this initial aeration rate led to cell necrosis. Starting with a low oxygen transfer coefficient k_L·a and increasing the air flow rate during the course of fermentation gave an exponential growth phase. The maximum specific growth rate was 0.012 h^–1 and the growth yield was 0.43 g.d.w./g. of lactose. On the basis of the mass-balance relation the maintenance coefficient and the maximum growth yield have been calculated.     

Kinetic aspects of inhibition of the phytopathogenic fungi growth by rhizosphere bacteria

Kinetics of growth inhibition of fungi Fusarium and Bipolaris caused by bacteria Pseudomonas sp. V-6798 and Azotobacter chroococum V-2272 D on dense nutrient media, both in single-crop system and by coinoculation, was demonstrated. The speed of fungal colonies growth as a function of bacteria concentration in inoculate was shown to be in accordance with the Ierysalimskii modified equation. The degree of antagonistic activity was suggested to be assessed by the constant of inhibition (K _i ) and residual rate of fungi growth. Constant of inhibition of fungal growth by bacteria varied within 10–100 cells/ml for observed species. More effective fungistatic influence of bacterial strains in combined culture was observed. Parameters reported in the present study allow comparing the degree of bacteria antifungal activity in vitro. Suggested screening method could be used for selection of bacteria as activity biofungicide and while selecting biomedication for defined plant pathogen disruption.