An extended kinetic analysis of valinomycin-induced Rb-transport through monoglyceride membranes

Summary The time course of the current following a voltage jump, which is applied to monoglyceride bilayers in the presence of valinomycin, shows two relaxation times. This is basically in agreement with a simple carrier model which has been described in full detail formerly. Relaxation times and amplitudes allow a calculation of the rate constants of the transport model. The presented data supplement an analysis which was hitherto based only on the slower relaxation process and on information derived from the nonlinearity of currentvoltage characteristics. The additional resolution of the faster relaxation time allowed an approximate determination of the voltage dependence of the translocation rate constant for the carrier-ion-complex and provided evidence for a small voltage dependence of the interfacial reaction. The dependence of the relaxation parameters on the ion concentration in the aqueous phase was interpreted assuming a saturation of the ion concentration at the reaction plane at high bulk concentrations.     

An analysis of extended and exponentially-fed-batch cultures.

Under certain conditions it is shown that an extended culture is equivalent to an exponentially-fed-batch culture, that an exponentially-fed-batch culture (and an extended culture) can be maintained at a steady state and that an exponentially-fed-batch culture may be mimicked by a continuous-flow culture with a constant dilution rate. Operational conditions required to maintain steady states are specified.     

Thermoosmosis through charged membranes. theoretical analysis of concentration dependence

A theoretical equation for thermoosmosis through charged membranes in electrolyte solutions is derived from nonequilibrium thermodynamics. The theory shows that the volume flux through the membrane is proportional to the temperature difference across the membrane. The proportionality constant, i.e., the thermoosmotic coefficient is a function of electrolyte concentration. The electrolyte concentration dependence of the thermoosmotic coefficient calculated is compared with our previous experimental results. Agreement between theory and experiments is satisfactory.     

An elementary kinetic model of energy coupling in biological membranes

The purpose of this work is to contribute to the understanding of the fundamental kinetic properties of the processes of energy coupling in biological membranes. For this, we consider a model of a microorganism that, in its plasma membrane, expresses two electrogenic enzymes (E(1) and E(2)) transporting the same monovalent cation C and electrodiffusive paths for C and for a monovalent anion A. E(1) (E(2)) couples transport C to the reaction S(1)<-->P(1) (S(2)<-->P(2)). We developed a mathematical model that describes the rate of change of the electrical potential difference across the membrane, of the internal concentrations of C and A, and of the concentrations of S(2) and P(2). The enzymes are incorporated via two-state kinetic models; the passive ionic fluxes are represented by classical formulations of electrodiffusion. The microorganism volume is maintained constant by accessory regulatory devices. The model is utilized for stationary and dynamic studies for the case of bacteria employing the electrochemical gradient of Na(+) as energetic intermediate. Among other conclusions, the results show that the membrane potential represents the relevant kinetic intermediate for the overall coupling between the energy donor reaction S(1)<-->P(1) and the synthesis of S(2).     

A kinetic analysis of electron transport across chromaffin vesicle membranes

Some types of secretory vesicles, such as the chromaffin vesicles of the adrenal medulla, have cytochrome b561 which is believed to mediate the transfer of electrons across the vesicle membrane. To characterize the kinetics of this process, we have examined the rate of electron transfer from ascorbate trapped within chromaffin vesicle ghosts to external ferricyanide. The rate of ferricyanide reduction saturates at high ferricyanide concentrations. The reciprocal of the rate is linearly related to the reciprocal of the ferricyanide concentration. The internal ascorbate concentration affects the y intercept of this double-reciprocal plot but not the slope. These observations and theoretical considerations indicate that the slope is associated with a rate constant k1 for the oxidation of cytochrome b561 by ferricyanide. The intercept is associated with a rate constant k0 for the reduction of cytochrome b561 by internal ascorbate. From k0 and standard reduction potentials, the rate constant k-0 for the reduction of internal semidehydroascorbate by cytochrome b561 can be calculated. Under conditions prevailing in vivo, this rate of semidehydroascorbate reduction appears to be much faster than the expected rate of semidehydroascorbate disproportionation. This supports the hypothesis that cytochrome b561 functions in vivo to reduce intravesicular semidehydroascorbate thereby maintaining intravesicular ascorbic acid.     

An instrumented wheel for kinetic analysis of wheelchair propulsion.

An instrumented wheel system for three-dimensional kinetic analysis of upper extremity during wheelchair propulsion has been designed and validated. This system allows the direct measurements of three-dimensional dynamic forces and moments on the handrim during wheelchair propulsion in a laboratory setting as well as in the field. Static loading tests showed a high linearity and little drift (coefficient of determination, r2 > 0.999). Under dynamic loading, the instrumented wheel provided the well-matched measurement forces and moments with the predicted values from the inverse dynamic method using video-based kinematic data (correlation coefficient, p > 0.97). The three-dimensional handrim forces and moments during wheelchair propulsion by a non-disabled subject were demonstrated.     

Translocation of polysialic acid across model membranes: kinetic analysis and dynamic studies.

Transmembrane translocation of polyion homopolymers takes place in the case of polyanionic polysialic acid (polySia), polyanionic polynucleotides and polycationic polypeptides. The purpose of this work was to determine the role of membrane electrical parameters on the kinetics of polyion translocation, the influence of polysialic acid on ion adsorption on positively charged membrane surface and the dynamics of the phospholipid hydrocarbon chains and choline group by using 1H-NMR. The analysis of polyion translocation was performed by using the electrical equivalent circuit of the membrane for the initial membrane potential equal to zero. The changes in polysialic acid flux was up to 75% after 1 ms in comparison with the zero-time flux. Both a decrease of membrane conductance and an increase of polyion chain length resulted in the diminution of this effect. An increase of praseodymium ions adsorption to positively charged liposomes and an increase of the rate of segmental movement of the -CH2 and -CH3 groups, and the choline headgrup of lipid molecules, was observed in the presence of polySia. The results show that the direction of the vectorial polyion translocation depends both on the membrane electrical properties and the degree of polymerization of the polymer, and that polysialic acid can modulate the degree of ion adsorption and the dynamics of membrane lipids.     

Natural feruloyl monoglyceride macrocycles as protecting factors against free-radical damage of lipidic membranes

Nine novel natural feruloyl monoglyceride (MGs) macrocycles have been isolated from the leaves of Carex distachya, an herbaceous plant growing in the Mediterranean maquis. All the structures have been elucidated on the basis of their spectroscopic features, especially two-dimensional NMR (DQ-COSY, TOCSY, NOESY, ROESY, HSQC, HMBC, HSQC-TOCSY) and ESI-MS. All the compounds have been assayed as protecting factors against the radical damage of the lipids by using different antioxidant tests.     

An analysis of the autophosphorylation of rabbit and human erythrocyte membranes.

The autophosphorylation of rabbit and human erythrocyte membranes has been studied under various experimental conditions. The phosphopeptides of the erythocyte membranes were identified using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by ratioautography. The pattern of phosphorylatiion of membrane components differs with respect to the phosphoryl donor used (ATP or GTP) and to the pH at which the reaction is carried out. Both species appear to contain at least two distinct membrane-bound protein kinases. The human erythrocyte membrane contains a cyclic adenosine 3'5'-monophosphate (cyclic AMP)-dependent protein kinase and several substrates for this kinase. Only ATP can be used as a phosphoryl donor for this kinase. In contrast, the rabbit erythrocyte membrane does not contain a cyclic AMP dependent protein kinase but does contain a kinase which utilizes only ATP as the phosphoryl donor and is specific for certain endogenous substrates at low pH. Both the human and rabbit erythrocyte membranes contain a kinase which utilizes GTP, perhaps also ATP, as the phosphoryl donor. The substrates of these kinases are similar in both species.     

A kinetic analysis of protein transport through the anthrax toxin channel

Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF(N), the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF(N) molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF(N)s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF(N) bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF(N) bound. We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.     

Thickness dependence of monoglyceride bilayer membrane conductance.

We have studied the conductance properties of unmodified monoglyceride membranes as a function of monoglyceride chain length. As membrane thickness decreases from 31 to 20 nm, the steepness of the current-voltage (I-V) curve increases from 80 mV per e-fold current increase to 52 mV per e-fold current increase. The zero-voltage conductance increases more than 1,000-fold and the apparent activation energy of conductance decreases from 18.4 to 14.2 kcal/mol. We have analyzed our results using both the Nernst-Planck equation and absolute rate theory. Both approaches are consistent with our results and give consistent values for the parameters describing the I-V curves. We conclude that both the surface ion concentration and the distance from the surface of the membrane at which the energy of an ion rises appreciably above its value in solution (position of the barrier) are invariant with thickness.     

Dimeric-like kinetic cooperativity of the bacteriorhodopsin molecules in purple membranes.

The kinetics of the absorption changes accompanying the photocycle of bacteriorhodopsin (BR) strongly depend on the intensity of the exciting short laser pulse. The decrease in the flash intensity dependence of the M kinetics after different extents of bleaching of the purple membranes by hydroxylamine proves the existence of a cooperative interaction between the photocycling BR molecules. The yield of the slow component of the M decay (M(s)) is a quadratic function of the extent of the fraction cycling. The slope of the relative weight of M(s) versus the fraction cycling is 0.5. This slope indicates a dimeric-like cooperative interaction, although the structural units of the purple membranes are the trimers of the BR molecules. For the most probable cooperative mechanism an asymmetric trimeric interaction is suggested, which accounts for the apparently dimeric features. A photocycling molecule may influence only one of its two neighbors in the trimer. From this asymmetric feature a deformative interaction is expected to be the cooperative mechanism, which would be an allosteric regulating mechanism in the purple membrane.     

Movement of monoglyceride derived from hydrolysis of fluorescence-labeled lyso platelet-activating factor by lysophospholipase C through plasma membranes of porcine kidney epithelial cell line LLC-PK1

To investigate the mechanisms of the release of lyso platelet-activating factor (PAF), an alkyl ether-linked lysophosphatidylcholine, from the kidney epithelial cell line LLC-PK1, the cell monolayer was incubated with a fluorescence-labeled lysoPAF analog, Bodipy-lysoPAF, on either the basolateral or apical side. The fluorescent lipids in the culture media mixed with or without bovine serum albumin at a final concentration of 2% were analyzed by thin layer chromatography. In both cases, two major bands, assignable to Bodipy-lysoPAF and Bodipy-monoglyceride (MG), were detected in the culture medium to which Bodipy-lysoPAF had been added, whereas the culture medium at the opposite side exhibited only the major band of Bodipy-MG. Our results suggest that lysoPAF was degraded by high ecto-lysophospholipase C activity. The possible physiological significance of this metabolic pathway is discussed.     

An extended dynamic model of oxidative phosphorylation.

The presented model based on an earlier one (Korzeniewski, B. and Froncisz, W. (1989) Studia Biophys. 132, 173-187) simulates concentration changes in time of chemical compounds and thermodynamic forces during respiration of cell suspension in a closed chamber. A set of differential equations solved numerically describes the utilization of oxygen up to anaerobiosis and the behaviour of the system after a sudden pulse of oxygen. Flux control coefficients for most important reactions (enzymes) of oxidative phosphorylation were calculated. A good qualitative and (when a direct comparison is possible) quantitative agreement with experimental results can be observed. The following conclusions can be drawn from the simulation: (1) Wilson's steady state model is not in contradiction with sharing of the control over the respiration between some steps and displacement of the ATP/ADP carrier from equilibrium. (2) The overshoot characteristics of the delta microH+ time-course after reoxygenation can be explained without using the lag-phase kinetics of ATP-synthetase. (3) A 'hot region' (sharp changes of many parameters) can be distinguished when the oxygen concentration approaches zero; only cytochrome oxidase is clearly sensitive on oxygen concentration in all its range. (4) Control over oxidative phosphorylation is shared mainly between inputs of the system (ATP utilization and substrate dehydrogenation) and the proton leak.     

Thermodynamic and kinetic analysis of an RNA kissing interaction and its resolution into an extended duplex

Kissing hairpin interactions form when the loop residues of two hairpins have Watson-Crick complementarity. In a unimolecular context, kissing interactions are important for tertiary folding and pseudoknot formation, whereas in a bimolecular context, they provide a basis for molecular recognition. In some cases, kissing complexes can be a prelude to strand displacement reactions where the two hairpins resolve to form a stable extended intermolecular duplex. The kinetics and thermodynamics of kissing-complex formation and their subsequent strand-displacement reactions are poorly understood. Here, biophysical techniques including isothermal titration calorimetry, surface plasmon resonance, and single-molecule fluorescence have been employed to probe the factors that govern the stability of kissing complexes and their subsequent structural rearrangements. We show that the general understanding of RNA duplex formation can be extended to kissing complexes but that kissing complexes display an unusual level of stability relative to simple duplexes of the same sequence. These interactions form and break many times at room temperature before becoming committed to a slow, irreversible forward transition to the strand-displaced form. Furthermore, using smFRET we show that the primary difference between stable and labile kissing complexes is based almost completely on their off rates. Both stable and labile complexes form at the same rate within error, but less stable species dissociate rapidly, allowing us to understand how these complexes can help generate specificity along a folding pathway or during a gene regulation event.     

Multi-enzyme kinetic analysis of glycolipid biosynthesis.

Gangliosides are acidic glycosphingolipids synthesized sequentially by a series of glycosyltransferases acting in parallel biosynthetic pathways. While most glycosyltransferases are highly specific, some, however, may catalyze equivalent steps in each pathway using different gangliosides as substrates (e.g. N-acetylgalactosaminyltransferase, sialyltransferase-IV). A multi-enzyme kinetic analysis was developed on the condition that serial enzymatic reactions operate below substrate saturation. A multi-enzyme kinetic analysis enabled a simultaneous calculation of the Vmax/Km value of each enzyme derived from the equilibrium concentration of the respective substrate. Substrate concentrations [S] were determined by radioactive labelling of gangliosides in intact cells with the precursor sugars [14C]galactose and [14C]glucosamine, followed by high-performance thin-layer chromatography and autoradiography of the radiolabelled glycolipids. On the basis of Michaelis-Menten kinetics, Vmax/Km values were derived from [S] by a system of linear equations. The procedure was used to analyze the development of the glycolipid composition during differentiation of rat gliomaxmurine neuroblastoma (NG108-15) cells. The Vmax/Km values calculated by multi-enzyme kinetic analysis were consistent with the kinetic data obtained with solubilized enzymes. Application of multi-enzyme kinetic analysis to published data on the correlation of enzyme activities with ganglioside levels in various cell lines and tissues indicated the validity of this method for analysis of the glycolipid biosynthesis, in particular, of its initial steps. On the basis of the kinetic analysis, it is suggested that the cell lines can be divided into two groups with respect to the substrate pools of GM3 used by sialyltransferase-II and N-acetylgalactosaminyltransferase-I. The first group encompasses the majority of the neuroblastoma cell lines and the embryonic rat brain where the two enzymes share a common pool of GM3. In the second group, the two enzymes do not compete for the same pool of GM3, indicating a different subcellular localization of CMP-NeuAc:GM3 alpha2-8-sialyltransferase and UDP-N-acetylgalactosaminyl:GM3 N-acetylgalactosaminyltransferase. In this study, the theory of a multi-enzyme kinetic analysis is discussed and its application to analysis of the glycolipid biosynthesis in neuroblastoma cells is demonstrated. A multi-enzyme kinetic analysis can be applied to other biosynthetic pathways and provides the advantage of analyzing kinetic data with intact cells or tissue samples.