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F Travers

Publications and source records attributed to F Travers.

At least 55 records · Page 3Linked to original sources
PubMed1984

Transient kinetics of the binding of ATP to actomyosin subfragment 1: evidence that the dissociation of actomyosin subfragment 1 by ATP leads to a new conformation of subfragment 1.

The initial steps by which ATP dissociates and binds to actomyosin subfragment 1 (acto-SF-1) were studied. Two techniques were used: stopped-flow (for acto-SF-1 dissociation kinetics) and rapid-flow quench with ATP chase quenching (for ATP binding kinetics). The experiments were carried out in 40% ethylene glycol-5 mM KCI, pH 8, at 15 degrees C. Under these conditions, the binding of SF-1 to actin remains very tight. As with SF-1, the ATP chase technique could be used, first, to titrate active sites and, second, to study the kinetics of ATP binding to acto-SF-1. The kinetic constants obtained were compared with those of SF-1 alone and with the acto-SF-1 dissociation kinetics under identical conditions. The kinetics of the acto-SF-1 dissociation did not vary with the actin to SF-1 ratio, but the ATP binding kinetics did, and a maximum value was reached at a mole ratio of 2.5. At high ATP (100 microM), kdiss = 300 s-1, which compares with 49 s-1 and 13 s-1 for the ATP binding kinetics for acto-SF-1 (actin to SF-1 = 1:1) and SF-1, respectively. As with SF-1, the ATP binding to acto-SF-1 follows a hyperbolic law with the ATP concentration. This suggests a rapid equilibrium (K) followed by an essentially irreversible step (k).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1984

Cryoenzymic studies on myosin subfragment 1: perturbation of an enzyme reaction by temperature and solvent.

The effects of temperature and solvent on myosin subfragment 1 ATPase have been studied. Under all of the conditions used the data could be fitted to the Bagshaw - Trentham pathway: (formula; see text) Ethylene glycol (40%) was used as the cryosolvent ; this makes K1 and k+2 measurable and allows for temperature studies over an extensive temperature range (+35 to -20 degrees C) and thus to reasonably accurate thermodynamic parameters. The following techniques were used: ATP chase (for K1 and k+2); Pi burst (k+2 or k+3 + k-3); single-turnover Pi burst [k0 = k +4K3 /(1 + K3)] absorption stopped flow (k+2 or k+3 + k-3); steady state (k+6 or k0). Myosin provides examples of causes for nonlinear Arrhenius and van't Hoff plots. A temperature-induced structural change is exemplified by a "jump" in an Arrhenius plot of k+2 and "breaks" in van't Hoff plots of K1 and K3. A change in rate-limiting step is illustrated from stopped-flow experiments ( kobsd approximately k+2 at low and approximately k+3 + k-3 at high temperatures) and steady-state experiments (kcat approximately k+6 at low and approximately k0 at high temperatures). A third cause is illustrated by k0: an Arrhenius plot of k0 is nonlinear since there is a break in K3. These studies illustrate the use of temperature perturbation as a way of revealing reaction intermediates and of defining the conditions required for the isolation of a particular intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1983

A jump in an Arrhenius plot can be the consequence of a phase transition. The binding of ATP to myosin subfragment 1.

The temperature dependence of the kinetics of the binding of ATP to myosin subfragment-1 was studied by an ATP chase technique in a rapid-flow-quench apparatus: (formula; see text) A temperature range of 30 degrees C to -15 degrees C was obtained with ethylene glycol as antifreeze. The Arrhenius plot of k2 is discontinuous with a jump at 12 degrees C. Above the jump delta H+ = 9.5 kcal/mol, below delta H+ = 28.5 kcal/mol. Few such Arrhenius plots are recorded in the literature but they are predicted from theory. Thus, we explain our results as a phase change of the subfragment 1-ATP system at 12 degrees C. This is in agreement with certain structural studies.

Adenosine Triphosphate↗
PubMed1983

Evidence for the two-step binding of ATP to myosin subfragment 1 by the rapid-flow-quench method.

1. The initial steps on the myosin ATPase (EC 3.6.1.3) pathway are taken to be: (formula; see text) A two-step binding for ATP is assumed, but the evidence for it is unconvincing; because of the rapidity of the process unambiguous values for K1 and K2 are not available. 2. We investigated the myosin mechanism by the chemical flow-quench technique. Reaction mixtures containing [gamma-32P]ATP plus myosin subfragment 1 were quenched in unlabelled ATP (ATP chase) or acid (Pi burst). 3. We show that the ATP-chase method can lead directly to unambiguous values for K1 and k+2. 4. The binding process was slowed down by 40% ethylene glycol. It was studied as a function of the ATP concentration. A limiting plateau resulted, showing a two-step binding for ATP, and values for K1 and k+2 were obtained. 5. K1 and k+2 are rather sensitive to the experimental conditions. Ethylene glycol and lowering of the pH decrease both constants, but an increase in KCl concentration increases them. This suggests that the binding of ATP to myosin is of an electrostatic nature. 6. The Pi-burst method can lead directly to k+3 + k-3, but under certain conditions the kinetics are governed by K1 and k+2. This uncertainty of the interpretation of Pi-burst experiments is discussed.

Adenosine Triphosphate↗
PubMed1982

Influence of the structure of water on the hydrolysis of cytidine 2',3'-phosphate catalysed by bovine pancreatic ribonuclease A.

The study of the temperature dependence of the hydrolysis of cytidine 2',3'-phosphate by bovine pancreatic ribonuclease A (EC 3.1.27.5) at pH 7.0 by using the pH-stat method showed a transition at 4 degrees C [J. A. Biosca and C. M. Cuchillo (1980) Biochem. J. 189, 655-657]. The breaks found in the Van't Hoff and Arrhenius plots at pH 7.0 were confirmed in the present work by following the reaction spectrophotometrically with a stopped-flow spectrophotometer adapted to the use of sub-zero temperatures. Similar results were found at pH 5.5. In addition it was found that the discontinuity disappears when 40% ethyleneglycol is present in the reaction mixture. However, in this latter instance a discontinuity around 0 degrees C appears in the Arrhenius plot. To explore the possibility that all these effects were due to a conformational transition in the protein, thermal perturbation experiments were carried out with the enzyme. A change in the slope of the plot of delta A290 as a function of temperature was found around 6 degrees C at pH 7.0 but not at pH 5.5. The results reported here can be interpreted as due to a change in the protein structure induced by the change of the structure of water. The studies carried out in the presence of ethyleneglycol also open the way to the cryoenzymological experimentation on ribonuclease.

Animals↗
PubMed1981

The first step in the polymerisation of actin.

In the presence of certain cations (e.g. K+ or Mg2+) actin polymerizes. Below a certain concentration (the critical concentration) the monomer G-actin does not polymerize on the addition of K+ or Mg2+. However, the proteolysis experiments of Rich and Estes [J. Mol. Biol. 104, 777--792 (1976)] strongly suggest that cations induce a change in conformation of G-actin leading to a novel form of actin, G*-actin. This conformational change may be the first step in the polymerization of actin. We have studied G*-actin induced by K+, by difference spectroscopy. We show that G*-actin is a monomer and we confirm that the bound ATP is not cleaved. We also studied the G-actin in equilibrium with G*-actin equilibrium at 4 degrees C as a function of K+ or Mg2+ concentration. With KCl, the transformation can be accounted for as a screening effect. The effect of Mg2+ is more specific and the change in conformation of the G-actin could result from the binding of two or three Mg2+ ions/molecule. We suggest that the G-actin in equilibrium with G*-actin transformation results from the neutralization of a polyanionic region on the actin surface and that this region could be the highly negatively charged N terminus.

Actins↗
PubMed1981

Lack of evidence for a tetrahedral intermediate in the hydrolysis of nitroanilide substrates by serine proteinases. Subzero-temperature stopped-flow experiments.

We have used a stopped-flow apparatus to reinvestigate reports, based on the observation of "burst" kinetics, of an intermediate prior to the acyl-enzyme complex in hydrolysis reactions of anilides catalyzed by trypsin and elastase [M. W. Hunkapiller, M. D. Forgac and J. H. Richards (1976) Biochemistry 15, 5581-5588; D. D. Petkov (1978) Biochim. Biophys. Acta, 523, 538-541; A. L. Fink and P. Meehan (1979) Proc. Natl Acad. Sci. USA, 76, 1566-1569; P. Compton and A. L. Fink (1980) Biochem. Biophys. Res. Commun. 93, 427-431]. We studied the hydrolysis of several anilide substrates by bovine and porcine trypsin and porcine elastase between -30 degrees C and 20 degrees C. In no case did we record true "burst" kinetics. We show that confusion spectral changes can arise from incomplete mixing, thermal gradients, or heterogeneity of the substrate. We conclude that there is no solid spectroscopic evidence at present for the existence of a tetrahedral intermediate in the hydrolysis of amides by serine proteinases. The substrate N-acetyl-L-alanyl-L-prolyl-L-alanine 4-nitroanilide is a mixture of two isomers trans and cis about the L-alanyl-L-propyl peptide bond. It appears that elastase hydrolysis the cis isomer more rapidly than the trans isomer and this could lead to false "burst" kinetics. We describe the construction of the stopped-flow apparatus designed for cryoenzymology used for this work that has novel features and is adaptable to a variety of spectrophotometers. Solutions can be handled under anaerobic conditions. A window allows the drive syringes to be observed or exposed to light for photochemical experiments. The apparatus operates over the temperature range -35 degrees C to + 25 degrees C. The dead time is under 5 ms. A recording system is described that permits one to follow reactions over a wide time scale covering half-time of the order of several milliseconds to hours.

Anilides↗
PubMed1980

Characterization of actin microfilaments at the apical pole of thyroid cells.

In thyroid cells (rat or hog), actin has been detected by immunofluorescence with an antiactin antibody and, in electron microscopy by decoration "in situ" with heavy meromyosin. The antibody as the heavy meromyosin method have shown that actin microfilaments are especially localized at the apical pole of the cells, in a region where thin filaments are usually observed by conventional methods of electron microscopy. These microfilaments are attached to the apical membrane at the ends of the microvilli and form dense bundles at their cores. They are polarized towards the interior of the cell. Decorated filaments are also organized in a clear network, parallel to the apical membrane; they are associated with microvillar bundles, but also with small apical vesicles and lateral membranes, in tight or gap junctions.

Actins↗
PubMed1980

A flow-quench apparatus for cryoenzymic studies. Application to the creatine kinase reaction.

The flow-quench method was adapted to sub-zero conditions. Two apparatus were constructed: a rapid flow-quench apparatus (which take samples in the 5--300-ms time range) and a time delay flow-quench apparatus (0.5 s and longer). The apparatus were constructed so that the reagents are only in contact with chemically inert materials : glass, poly(tetrafluoroethylene) and poly(trifluorochloroethylene). The modified flow-quench apparatus were used to study the initial formation of creatine phosphate by creatine kinase at -15 degrees C in the time range 5 ms to 10 s. As at +4 degrees C [Travers, F., Barman, T. E. and Bertrand, R. (1979) Eur. J. Biochem. 100, 149--155], the time course of product formation was complex and consisted of three phase: a lag phase, a burst phase and the steady-state phase. The apparatus were also used to test chemically for reaction intermediates involving labile phosphate and phospho-enzyme complexes on the creatine kinase reaction pathway at -15 degrees C. Since neither type of intermediate could be detected down to 5 ms, this enzyme probably proceeds via a direct in-line type of mechanism.

Animals↗
PubMed1980

Cryoenzymic studies on the transition-state analog complex creatine kinase . ADPMg . nitrate . creatine.

Concomitant with the formation of the transition state analog complex creatine kinase . ADPMg . nitrate . creatine [Milner-White, E. J. and Watts, D. C. (1971) Biochem. J. 122, 727--740] there results a large difference spectrum. The shape of the spectrum was characteristic of tryptophan exposure. The kinetics of formation and final amplitude of the spectrum were studied at -15 degrees C using a stopped-flow apparatus. The results obtained allowed for a plausible reaction pathway for nitrate fixation: the ordered addition of nitrate and creatine to the binary enzyme-ADPMg complex, a slow protein isomerization and the final addition of a molecule each of nitrate and creatine. The kinetic parameters for the formation of the transition state analog complex were compared with those already known for the overall reaction obtained under the same conditions [Barman, T. E., Brun, A. and Travers, F. (1980) Dur. J. Biochem. 110, 397--403]. This comparison revealed a striking analogy between the two processes. This suggests that the conformation of creatine kinase in the analog complex is very similar to that in the transition state complex on the catalytic pathway.

Adenosine Diphosphate↗
PubMed1979

[Identification of actin microfilaments in the neurosecretory axons of rat neural lobe].

Incubation of rat neural lobes with heavy meromyosin (HMM) after prolonged glycerination, induced characteristic arrowhead decoration of a number of microfilaments at different levels of the neurosecretory axons. In non terminal sections of axons the labelled microfilaments showed preferential relationships with microtubules in addition to occasional contacts with the axolemma and various axonal organelles. In axonal endings, they were mainly associated to microvesicles and appeared to be anchored on the axolemma facing the perivascular space at the level of membranous densifications.

Actins↗
PubMed1979

Transient-phase studies on the creatine kinase reaction. The analysis of a reaction pathway with three intermediates.

1. The initial formation of creatine phosphate by creatine kinase was studied in the millisecond range and the effect of temperature on the transient and steady-state phases exploited. 2. At 25 degrees C and 35 degrees C there was no transient phase. This is in agreement with the results of Gutfreund [Engelborghs, Y., Marsh, A., and Gutfreund, H. (1975) Biochem. J. 151, 47--50]. 3. At 4 degrees C the time course of creatine phosphate formation was complex and consisted of three transient phases: a lag phase, a burst phase and a steady-state phase. Based on this result a reaction scheme for creatine kinase which includes three intermediates was proposed. Despite the completeness of the time course, the extraction of estimates for the rate constants was difficult and computer simulation and iterative methods had to be resorted to. 4. Attempts were made to provide evidence for the complex enzyme.ADP.metaphosphate.creatine on the creatine kinase reaction pathway [cf. Milner-White, E.J. and Watts, D.C. (1971) Biochem. J. 122, 727--740]. Under the conditions used these attempts were unsuccessful at times down to 2.5 ms, at 4 degrees C or 35 degrees C.

Animals↗
PubMed1978

Transient-phase studies on the arginine kinase reaction.

1. The initial formation of arginine phosphate by arginine kinase was studied in the time range 2.8--50 ms by the quenched-flow method. 2. A transient burst phase of product formation was obtained, the amplitude of which was temperature-dependent. At 35 degrees C it was 0.64 mol arginine phosphate/mol arginine kinase and at 12 degrees C, 0.25 mol/mol. 3. These results show that for the reaction pathway of arginine kinase the rate-limiting step follows the formation of arginine phosphate on the enzyme. This is in contrast to the creatine kinase reaction where no transient phase was observed [Engelborghs, Y., Marsh, A. & Gutfreund, H. (1975) Biochem. J. 151, 47--50]. 4. The rate-limiting step on the arginine kinase reaction pathway is only slightly affected by temperature: the change in Kcat with temperature is due to a change of an equilibrium constant pertaining to at least two previous steps.

Animals↗
PubMed1978

Cryoenzymologic studies on arginine kinase: solvent, temperature and pH effects on the overall reaction.

The overall reaction catalyzed by the phosphotransferase arginine kinase was studied at normal and subzero temperatures. Ethylene glycol was used as the antifreeze and its effects on the Km values of substances, kcat and pH profiles were investigated in detail. a) The Km values for the substrate (2 mM for ATP and 0.6 mM for arginine) were little affected by the solvent composition or temperature of the reaction mixture. b) At concentration of ethylene glycol higher than 40% there was a sharp drop of enzyme activity. c) Ethylene glycol induces a large shift in the enzymic pK D) At -5 degrees C in 40% of solvent there was a break in the Arrhenius plot suggesting a change of the rate-limiting step. The relevance of these results to the reaction pathway of arginine kinase is discussed. In addition, controlled perturbations induced by cosolvent and temperature appear as useful tools for further kinetic investigations.

Animals↗
PubMed1976

Electrostatic potential modulations on polynucleotides as a function of ionic content: potentiometric determinations.

pK values of ionisation of cytidine residues, in poly C and poly I. poly C, have been determined by potentiometric titration as a function of either ionic strength (Na+) or Mg2+ concentration. The results are interpreted as a consequence of the existence of a local pH, prevailing in the vicinity of the polynucleotides, smaller than that of the bulk solution. This local pH is dependent upon the electrostatic potential psi due to the negatively charged phosphate groups of the polynucleotide. Moreover psi can be modulated through variation of either ionic strength or Mg2+ concentration. At a given pH Mg2+ addition leads to the release of protons which, in the case of complex systems of limited stability, might be detected by the use of the pH-stat method. Possible functional implications for genetic translation systems are examined.

Binding Sites↗
PubMed1975

Ternary solvents to investigate proteins at zub-zero temperatures.

Mixtures of water, ethylene glycol and methanol in different volume ratios have been selected to carry out kinetics of enzyme reactions at sub-zero temperatures with the intention to reduce maximally the viscosity. Density, viscosity and dielectric constant values of these mixtures as a function of temperature are reported, as well as values of the protonic activity of several buffers under such conditions. A procedure to avoid or delay the eventual damaging effect of methanol on proteins is described.

Acetates↗