Identical Kinetic Constants Research Articles

Kinetic models and phenomenological analysis of passive lipid translocation in single-file.

Passive movement of lipids through a membrane-embedded pore is analysed with kinetic equations of transport in single-file. The number of lipids arranged along the translocation coordinate in the pore is not limited in the calculations. The assumption is made that the energetic state of a pore is independent of the sequence of lipids contained in it. The results are valid for an arbitrary number of species with identical kinetic constants. It is shown that infinitely fast diffusion of one vacant site is equivalent to the filled pore approximation, which has been used here. We introduce the concept of non-strict single-file, which allows also for exchanges of neighbouring lipids inside the pore at specified rates. The model successfully simulates the redistribution of lipids between the monolayers of red blood cell plasma membranes under operation of an active aminophospholipid translocase. Kinetic equations are related to linear flux force relations. Phenomenological coefficients are expressed and analysed in terms of kinetic constants. Plausible kinetic pore model parameters are derived from comparison with a reference simulation of a thermodynamic model of the erythrocyte transmembrane lipid distribution. Mechanical forces due to differences in compressions of the lipid molecules between the monolayers are incorporated in kinetic rate constants. It is seen how the active inward transport of aminophospholipids causes an unsymmetrical passive redistribution of the other components due to mechanical effects and cross-coupling of fluxes.

Uptake of neutral α- and β-amino acids by human proximal tubular cells

The transport characteristics of amino acids in primary cell cultures from the proximal tubule of human adults (AHKE cells) were examined, using α-aminoisobutyric acid (AIB) and β-alanine as representatives of α- and β-amino acids, respectively. The Na +-gradient dependent influx of AIB occurred by a single, saturable transport system, whereas the Na +-gradient dependent uptake data for β-alanine could be described in terms of two-independent transport components as well as one-transport one-leak model with identical kinetic constants for the high-affinity system. Competition experiments revealed that all the neutral amino acids tested reduced the uptake of AIB, whereas there was no effect of taurine, l-aspartic acid, and l-arginine. By contrast, the influx of β-alanine was only drastically reduced by β-amino acids, whereas the inhibition by neutral α-amino acids was relatively low. Nor did l-arginine and l-aspartic acid affect the uptake of β-alanine into AHKE cells. Comparison with the results obtained for normal (NHKE) and immortalized (IHKE) embryonic cells suggested an unaltered expression of the types of transport carriers for neutral α- and β-amino acids in the embryonic and AHKE cells. However, the uptake capacity of the above-mentioned transport proteins was relatively smaller in the embryonic kidney compared with the adult human kidney, which may explain, at least partly, the phenomenon of physiologic amino aciduria in neonates.

Development and testing of a new computerized method for multicomponent kinetic determinations based on multiwavelength spectrophotometric detection

We developed a new kinetic multidetermination method based on multiwavelength spectrophotometric detection and used simulated processes to evaluate its performance. The method uses spectral differences between analytes and kinetic equations solved by means of a nonlinear regression procedure. It allows the simultaneous determination of rate constants and analyte concentrations, and takes account of potential matrix effects and changes in the rate constants arising from temperature oscillations. The results obtained by using simulated Gaussian spectra revealed that the method allows correct resolution of simultaneous kinetic processes, even if they involve strong spectral overlap and identical kinetic constants. The “standardized spectral difference”, concept introduced with this work, allows results to be extrapolated to non-Gaussian spectra.

Isolation of the gene for murine glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1A.

Glycogen storage disease (GSD) type 1a (von Gierke disease) is caused by a deficiency in glucose-6-phosphatase, the key enzyme in glucose homeostasis catalyzing the terminal step in gluconeogenesis and glycogenolysis. Despite its clinical importance, this membrane-bound enzyme has eluded molecular characterization. Here we report the cloning and characterization of a murine glucose-6-phosphatase cDNA by screening a mouse liver cDNA library differentially with mRNA populations representing the normal and the albino deletion mouse known to express markedly reduced glucose-6-phosphatase activity. Additionally, we identified the gene that consists of 5 exons. Biochemical analyses indicate that the in vitro expressed enzyme is indistinguishable from mouse liver microsomal glucose-6-phosphatase exhibiting essentially identical kinetic constants, latency, thermal lability, and vanadate sensitivity. The characterization of the murine glucose-6-phosphatase gene opens the way for studying the molecular basis of GSD type 1a in humans and its etiology in an animal model.

PMN elastases: A comparison of the specificity of human isozymes and the enzyme from other species toward substrates and inhibitors

The human elastases isolated from polymorphonuclear neutrophils (PMN) and purulent sputum displayed identical kinetic constants toward substrates and inhibitors. The elastases from the two sources yield identical N-terminal sequences and were recognized by antiserum prepared against human sputum elastase (HSE) isozyme-4 (I-4). The data support the proposal put forth by Twumasi and Liener (1977, J. Biol. Chem. 252, 1917–1926) that the human elastase from sputum is of PMN origin. PMN elastases from other species displayed kinetic constants toward both substrates and inhibitors significantly different from the human enzyme. Therefore, extrapolation of inhibitor profiles from these elastases to the human source should be avoided. Four groups of isozymes were resolved from HSE by FPLC. Only the most basic isozyme (I-4) was obtained as a single species. The isozymes displayed identical macroscopic kinetic constants toward several substrates and two classes of inhibitors. The similar partition ratios observed with a cephalosporin-derived inhibitor suggest that the microscopic rate constants are also identical. The data support the proposal suggested by Baugh and Travis (1976, Biochemistry 15, 836–841) that HLE isozymes differ only in carbohydrate content. Whatever the source of human PMN elastase heterogeneity, it does not result in heterogeneous catalytic properties. In addition, a new protein was identified in elastase preparations derived from human sputum. This protein displayed homology to serine proteases and properties suggesting that it is identical to azurocidin.

Inactivation of monoamine oxidase by 3,3-dimethyl analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-2,3-dihydropyridinium ion: Dramatic effect of β-mercaptoethanol on substrate turnover and enzyme inactivation

It was previously shown (Sayre, L. M., Arora, P. K., Feke, S. C., and Urbach, F. L. (1986) J. Am. Chem. Soc. 108, 2464-2466) that 1,3,3-trimethyl-4-phenyl-2,3-dihydropyridinium salt (the 3,3-dimethyl analogue of 1-methyl-4-phenyl-2,3-dihydropyridinium ion or MPDP+) is a good model for MPDP+ on the basis of its redox potential and was used to show that MPDP+ is unlikely to possess reactivity characteristics which could contribute to the neurotoxicity observed with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). 3,3-Dimethyl-MPTP and 3,3-dimethyl-MPDP+ are now shown to interact with monoamine oxidase similar to MPTP and MPDP+, but only in the presence of beta-mercaptoethanol (beta-ME). In the absence of beta-ME, mixed competitive-noncompetitive inhibition kinetics are observed for 3,3-dimethyl-MPTP and 3,3-dimethyl-MPDP+, whereas competitive inhibition kinetics are exhibited by MPTP. In the presence of beta-ME, however, 3,3-dimethyl-MPTP also is a competitive inhibitor. 3,3-Dimethyl-MPTP and 3,3-dimethyl-MPDP+ also are time-dependent inactivators of monoamine oxidase, having identical kinetic constants, as is the case with MPTP and MPDP+. In the presence of beta-ME, but not glutathione, the rate of inactivation increases dramatically. When [beta-ME] and [3,3-dimethyl-MPTP] or [3,3-dimethyl-MPDP+] are varied, there is an optimal concentration of 1.0 mM for all three at which maximal inactivation rates are obtained. Another dramatic effect of the beta-ME is to lower the partition ratio for inactivation from greater than 50 to about one. This suggests that the effect of the beta-ME toward inactivation may be to induce a conformational change in the enzyme, which reorients an active site nucleophile for attack on the activated species. Support for involvement of an active site nucleophile is the finding that inactivation does not lead to a flavin adduct. Three possible mechanisms for inactivation of monoamine oxidase by MPTP and MPDP+ are suggested.

The amino terminus regulates binding to and activation of cGMP‐dependent protein kinase

The allosteric regulation of binding to and the activation of cGMP-dependent protein kinase (cGMP kinase) was studied under identical conditions at 30 degrees C using three forms of cGMP-kinase which differed in the amino-terminal segment, e.g. native cGMP kinase, phosphorylated cGMP kinase which contained 1.4 +/- 0.4 mol phosphate/subunit and constitutively active cGMP kinase which lacked the amino-terminal dimerization domain. These three enzyme forms have identical kinetic constants, e.g. number of cGMP-binding sites, Km values for MgATP and the heptapeptide kemptide, and Vmax values. In the native enzyme, MgATP decreases the affinity for binding site 1. This effect is abolished by 1 M NaCl. In contrast, high concentrations of Kemptide increase the affinity of binding site 2 about fivefold. Under the latter conditions, identical Kd values of 0.2 microM were obtained for sites 1 and 2. Salt, MgATP and Kemptide do not affect the binding kinetics of the phosphorylated or the constitutively active enzyme, suggesting that allosteric regulation depends solely on the presence of a native amino-terminal segment. Cyclic GMP activates the native enzyme at Ka values which are identical with the Kd values for both binding sites. The activation of cGMP-dependent protein kinase is noncooperative but the Ka value depends on the substrate peptide concentration. These results show that the activity of cGMP kinase is primarily regulated by conformational changes within the amino-terminal domain.

Pathway of the microtubule-dynein ATPase and the structure of dynein: a comparison with actomyosin.

Dynein and myosin show several important similarities in design as well as some interesting differences in detail. Both ATPases function as crossbridges that undergo microscopic movements to drive the sliding of filaments, which results in macroscopic movements. They share a common design employing globular heads attached to flexible strands. Each head contains one ATP-binding site and one filament-binding site, and the binding of ATP induces an extremely rapid dissociation of the crossbridge-filament "rigor" complex. Following ATP hydrolysis, which is readily reversible, the crossbridge reassociates with the filament and returns to its original state with the release of products. Thus, the nucleotide-induced changes in conformation are effectively used to couple the hydrolysis of ATP to the dissociation and reassociation of the crossbridge in order to produce a force for net movement according to the Lymn-Taylor-Eisenberg model. The utilization of nucleotide-binding energy to induce a change in conformation can be rationalized in terms of our understanding of enzyme catalysis in general, whereby substrate binding energy is used to induce a change in conformation that stabilizes the transition state for catalysis. In these crossbridge ATPases, the substrate-induced change in conformation also serves to weaken the crossbridge-filament interaction. The pathway is symmetrical, with a return to the tight (filament) binding state coupled to product release. The ball on a string design may provide a reasonable basis to explain how a unidirectional force is obtained from a symmetrical cycle; opposite changes in conformation with the binding and release of the nucleotide produce a significant force only when pulling on the flexible strand. Moreover, the very rapid dissociation of the crossbridge following ATP binding limits the time that a negative force is in effect and also prevents a rigor crossbridge from retarding the sliding movements generated by other crossbridges. Myosin and dynein exhibit nearly identical kinetic constants governing ATP binding and the ATP-induced dissociation of the crossbridge. These appear as invariant steps that may reflect the basic principles of enzyme catalysis as applied to the mechanochemical cycle. The rates of ATP hydrolysis and synthesis by myosin and dynein differ slightly, but in each case the reactions are readily reversible with an equilibrium constant less than one. Steps involving the loss and rebinding of products occur at rates two to three orders of magnitude faster for dynein than for myosin.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics of interaction of N epsilon-fluorescein isothiocyanate-lysine-23-cobra alpha-toxin with the acetylcholine receptor

We have studied the kinetics of N epsilon-fluorescein isothiocyanate-lysine-23 cobra alpha-toxin (FITC-toxin) binding to the membrane-associated acetylcholine receptor from the Torpedo californica electric organ. The fluorescent toxin not only enabled us to monitor the binding reaction continuously but also to examine simultaneously the enhancement of ligand fluorescence and the increase in steady state polarization of fluorescence associated with binding of the alpha-toxin. Over the range of concentrations employed, both parameters yielded identical kinetic constants, suggesting that the enhancement of fluorescence of fluorescein and its immobilization are occurring in the same time frame. Both an initial rate analysis and the integrated rate expression showed association to be a simple, reversible bimolecular process. The apparent second-order association rate constant derived from the integrated rate analysis was constant within a factor of 2 over a 40-fold concentration range (6.7 +/- 1.7 X 10(3) M-1 S-1). The unimolecular dissociation rate constant was found to be 3.3 +/- 0.5 X 10(-5) S-1.

Seven mammalian aminoacyl-tRNA synthetases associated within the same complex are functionally independent.

A heterotypic multienzyme complex from sheep liver containing seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, glutamine, glutamic acid, lysine and arginine was subjected to kinetic analyses to examine the possibility that association of these enzymes may impart kinetic properties which differ from those of their unassociated counterparts. The evidence obtained by two different approaches leads to the conclusion that the associated enzymes are functionally independent. Firstly, the kinetic constants of the methionyl-tRNA and lysyl-tRNA synthetase components of the complex do not differ significantly from those of their unassociated counterparts obtained after controlled proteolysis of the complex. Secondly, the methionyl-tRNA synthetase component of the complex displays identical kinetic constants, whether assayed in the presence of [14C]methionine, ATP and highly enriched tRNAMet alone, or in the additional presence of the substrates required for unlabeled aminoacyl-tRNA formation by each of the other six enzymes. Similarly, the initial rates of [14C]aminoacyl-tRNA formation catalyzed by any of the six other enzymes was unaffected by the concomitant functioning of the other aminoacyl-tRNA synthetases. The sedimentation behaviour of the aminoacyl-tRNA synthetase components of the complex under conditions prevailing in the tRNA aminoacylation assay indicates that they remain associated under these conditions. The implications of these findings on the structural organization of the enzymes within the complex are discussed.

Glucose-initiated germination of mucor racemosus sporangiospores.

Treatments leading to the initiation of germination of Mucor racemosus sporangiospores were examined. The results support the hypothesis that glucose is a specific trigger molecule for the initiation of Mucor racemosus sporangiospores. Glucose and some of the glucose analogues tested could initiate germination, mannose, 3-O-methyl-D-glucose, 5-thio-D-glucose and 6-deoxy-D-glucose being the most effective. The initiation event appeared to depend on the concentration of the initiator, with glucose and 3-O-methyl-D-glucose exhibiting nearly identical kinetic constants. Spores accumulated not only glucose and 3-O-methyl-D-glucose, but also the 1-O-methyl-D-glucose analogue, which did not initiate germination. The accumulated 3-O-methyl-D-glucose was not metabolized. The initiation sequence appeared to require the continued presence of the initiator as well as protein synthesis.

Purification of a soluble and a wall-bound form of beta-glucosidase from Mucor racemosus.

beta-Glucosidase activity in crude extracts of Mucor racemosus exists in a soluble form and in a wall-bound form which sediments at 3,500 x g. The soluble form and a wall-bound form were purified to homogeneity by ammonium sulfate fractionation. DEAE-Sephadex chromatography, and SP-Sephadex chromatography. Both forms were identical in all parameters measured. Each enzyme is a glycoprotein of 91,000 daltons, with an identical amino acid composition and N-terminal amino acid of lysine; both contain about 10% carbohydrate. Both forms catalyze the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside with identical kinetic constants.

Properties of a Dicarboxylic Amino Acid Transport-deficient Mutant of Streptococcus faecalis

A mutant of Streptococcus faecalis has been isolated which has a 20-fold higher growth requirement for l -glutamic acid than the parent strain. The ability to utilize l -glutamine for growth is not affected. The mutant also has a greater dependence than the parent strain on an external supply of vitamin B6. The rate of glutamic acid transport at low extracellular concentrations (0.05 m m ) was greatly reduced in the mutant but was only moderately affected at higher concentrations (5 m m ). In contrast to the parent, in which kinetic studies revealed the operation of a high affinity and a low affinity system for glutamic acid transport, the mutant possessed a single catalytic component with kinetic constants equal to those of the low affinity system. These findings and the greatly elevated growth requirement for glutamic acid indicate that the high affinity system is inactive in the mutant. The transport of aspartic acid and d -glutamic acid by the mutant were similarly adversely affected, whereas essentially identical kinetic constants for glutamine, α-aminoisobutyric acid, and cycloleucine transport were obtained for the mutant and parent strains. The absence of a functional high affinity dicarboxylic amino acid transport system in the mutant allowed a study of the specific properties of the low affinity system. In contrast to the parent strain, the initial rate of uptake of isotopically labeled glutamate in the mutant was not stimulated by glucose at any concentration. However, the accumulation of large amounts of this amino acid on extended incubation was dependent on an energy source. Glutamine competitively inhibited glutamic acid uptake in the mutant. There was no evidence that the mutant produced an inhibitor of the high affinity transport component.