Biomedical subjects

M R Webb

Publications and source records attributed to M R Webb.

At least 37 records · Page 2Linked to original sources

PubMed1998

ATPase and shortening rates in frog fast skeletal myofibrils by time-resolved measurements of protein-bound and free Pi.

Shortening and ATPase rates were measured in Ca2+-activated myofibrils from frog fast muscles in unloaded conditions at 4 degrees C. ATPase rates were determined using the phosphate-binding protein method (free phosphate) and quench flow (total phosphate). Shortening rates at near zero load (V0) were estimated by quenching reaction mixtures 50 ms to 10 s old at pH 3.5 and measuring sarcomere lengths under the optical microscope. As with the rabbit psoas myofibrils (C. Lionne, F. Travers, and T. Barman, 1996, Biophys. J. 70:887-895), the ATPase progress curves had three phases: a transient Pi burst, a fast linear phase (kF), and a deceleration to a slow phase (kS). Evidence is given that kF is the ATPase rate of shortening myofibrils. V0 is in good agreement with mechanical measurements in myofibrils and fibers. Under the same conditions and at saturation in ATP, V0 and kF are 2.4 microm half-sarcomere(-1) s(-1) and 4.6 s(-1), and their Km values are 33 and 200 microM, respectively. These parameters are higher than found with rabbit psoas myofibrils. The myofibrillar kF is higher than the fiber ATPase rates obtained previously in frog fast muscles but considerably lower than obtained in skinned fibers by the phosphate-binding protein method (Z. H. He, R. K. Chillingworth, M. Brune, J. E. T. Corrie, D. R. Trentham, M. R. Webb, and M. R. Ferenczi, 1997, J. Physiol. 50:125-148). We show that, with frog as with rabbit myofibrillar ATPase, phosphate release is the rate-limiting step.

Adenosine Triphosphatases↗

PubMed1997

Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate.

We have measured the kinetics of inorganic phosphate (Pi) release during a single turnover of actomyosin nucleoside triphosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin-ATP and myosin-ADP-Pi to actin. Myosin subfragment 1 and a series of nucleoside triphosphates were mixed and incubated for approximately 1-10 s to allow NTP to bind to myosin and generate a steady state mixture of myosin-NTP and myosin-NDP-Pi. The steady state intermediates were then mixed with actin. The kinetics of Pi release were measured using a fluorescent probe for Pi, based on a phosphate binding protein [Brune et al. (1994) Biochemistry 33, 8262-8271]. These data are correlated with quenched-flow data, where the extent of the rapid burst of hydrolysis during the first turnover of ATP hydrolysis was followed by chemical quenching of the reaction mix at various times after rapidly mixing ATP and myosin subfragment 1. From the double-mixing actomyosin measurements, the kinetics of Pi release are biphasic. The fast phase corresponds to Pi release from the associated actomyosin-ADP-Pi complex. The slow phase measures the rate of the cleavage step on associated actomyosin. At saturating actin, there is a correlation between the amplitude of the fast phase and the size of the Pi burst observed by quenched flow in the absence of actin: the size of this phase corresponds to the amount of myosin-ADP-Pi formed during the first mix. For ATP at 20 degrees C the rate of the Pi release step is 75 (+/-5) s-1, 25-fold larger than the cleavage step, which is the rate-limiting step of actomyosin ATP hydrolysis at saturating actin. The rate constant of Pi release varies only slightly with nucleoside structure. The rate constant of the slow phase of the Pi release (measuring cleavage) is highly dependent upon the structure of the NTP substrate.

Actomyosin↗

PubMed1997

ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay.

1. The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM. This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release. Tension was at 67% of its isometric level by the time 150 microM Pi was released. ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C. The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1. 4. On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl-EGTA. At 5 degrees C the ATPase rate was 5.8 and 4.0 s-1 in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pi on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0.5 mM) decreased the initial ATPase rate by 23%. Pi (10 mM) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5 degrees C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s-1 during the first turnover (based on 180 microM myosin subfragment 1). Tension was 74% of its isometric level by the time 180 microM Pi was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached. Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.

Actin Cytoskeleton↗

PubMed1997

The crystal structure of human rac1, a member of the rho-family complexed with a GTP analogue.

The crystal structure of human rac1, a member of the rho family of small G-proteins, complexed with the non-hydrolysable GTP analogue, guanosine-5'-(beta gamma-imino)triphosphate (GMPPNP), has been determined by X-ray analysis at a resolution of 1.38 A. Comparison with the structure of H-ras indicates that rac1 has an extra alpha-helical domain that is characteristic of the rho G proteins, and may be involved in the signalling pathway of this family.

Amino Acid Sequence↗

PubMed1995

Kinetics of inorganic phosphate release during the interaction of p21ras with the GTPase-activating proteins, p120-GAP and neurofibromin.

The rate of GTP hydrolysis on p21ras is accelerated by approximately 10(5) times by the catalytic domains of GTPase-activating proteins (GAPs), p120-GAP (GAP-344) or neurofibromin (NF1-334). The kinetic mechanism of this activation has been investigated by following the release of inorganic phosphate (Pi), using a fluorescent probe that is sensitive to Pi [Brune, M., Hunter, J., Corrie, J. E. T., & Webb, M. R. (1994) Biochemistry 33, 8262-8271]. Measurements were made in real time with a stopped-flow apparatus, in which the p21ras complex with the 2',3'-methanthraniloyl analogue of GTP (mantGTP) was mixed with the GAP in the presence of this Pi probe. The results show that Pi release is fast and that the overall hydrolysis is controlled by the cleavage itself or a conformational change preceding the cleavage. The time courses were single exponentials over a range of [GAP-344] and were modeled to show that a single step controlled Pi release. The maximum rate constant was 15 s-1 (all data at 30 degrees C, pH 7.6, low ionic strength) in experiments in which GAP-344 underwent a single turnover, compared with 5 s-1 for multiple-turnover experiments, and possible causes of this discrepancy were investigated and discussed. With NF1-334 the time courses were more complex, showing a lag prior to rapid release of Pi. The results were consistent with a Kd of 0.04 microM for NF1-344 affinity is some 3 orders of magnitude tighter than that of GAP-344.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗

PubMed1995

Time resolved measurements show that phosphate release is the rate limiting step on myofibrillar ATPases.

The myofibril is a good model to study the ATPase of the muscle fibre. When myofibrillar ATPase reaction mixtures are quenched in acid, there is a burst of Pi formation, due to AM.ADP.Pi or Pi, as shown in the scheme: AM+ATP<-->A.M.ATP<-->AM.ADP.Pi<-->AM.ADP+Pi<-->AM+ADP. Therefore, in the steady state, either AM.ADP.Pi or AM.ADP or both predominate. To determine which, we studied the reaction using a Pi binding protein (from E. coli) labeled with a fluorophore such that it is specific and sensitive to free Pi [Brune, M. et al. (1994) Biochemistry 33, 8262-8271]. We show that the Pi bursts with myofibrillar ATPases (calcium-activated or not, or crosslinked) are due entirely to protein bound Pi. Thus, with myofibrillar ATPases the AM.ADP.Pi state predominates.

Actins↗

PubMed1995

Pathway of processive ATP hydrolysis by kinesin.

Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin-ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin.

Adenosine Diphosphate↗

PubMed1995

A new method for the time-resolved measurement of phosphate release in permeabilized muscle fibers.

A new method for the measurement of phosphate release in contracting and relaxed permeabilized muscle fibers is described. The assay is based on a genetically engineered phosphate-binding protein labeled with a coumarin fluorescent probe, which binds inorganic phosphate tightly and shows a fourfold increase in fluorescence upon binding. Measurements of Pi release on the millisecond time scale with sensitivity in the 10 microM range are obtained that provide new information about the relationship between ATP hydrolysis and force production.

Adenosine Triphosphate↗

PubMed1994

Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.

A probe has been developed that can rapidly measure micromolar concentrations of inorganic phosphate (Pi), in particular to follow the release of Pi in real time from enzymes such as phosphatases. Its application is described to investigate the mechanism of actomyosin subfragment 1 ATPase. The probe uses the A197C mutant of Escherichia coli phosphate binding protein (PBP), generated by oligonucleotide-directed mutagenesis. A new fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), was attached to the single cysteine to produce the reporter molecule that was purified free of unlabeled protein and unattached MDCC. The labeled protein has an excitation maximum at 425 nm and emission maximum at 474 nm in the absence of Pi, shifting to 464 nm with a 5.2-fold increase in fluorescence (lambda max/lambda max) when complexed with Pi at pH 7.0, low ionic strength, 22 degrees C. The fluorescence increase is not much altered by change to pH 8 or by increase in ionic strength to 1 M. Pi binds tightly (Kd approximately 0.1 microM) and rapidly (1.36 x 10(8) M-1 s-1) and the dissociation rate constant is 21 s-1, at pH 7.0, low ionic strength, 22 degrees C. A variety of phosphate esters were tested to investigate the specificity of the MDCC-PBP and none gave a significant fluorescence increase at 100 microM or higher concentration. ATP weakly inhibited the Pi-induced fluorescence change, indicating that it binds at least 3000-fold weaker than Pi. Because Pi is a widespread contaminant, the probe is used in conjunction with a "Pi mop", consisting of 7-methylguanosine and purine nucleoside phosphorylase, to remove free Pi from solutions by its conversion to ribose 1-phosphate. Because the equilibrium constant of this reaction is > 100, free Pi can be reduced below 0.1 microM. The probe was used to measure the rate of Pi release during a single turnover of ATP hydrolysis with actomyosin subfragment 1 from rabbit skeletal muscle, to determine to what extent Pi release contributes to the rate limitation of this ATPase. Using a stopped-flow apparatus, a small lag prior to rapid Pi release was detected at pH 7.0, low ionic strength, between 5 and 22 degrees C at both high and low [ATP]. For measurements of a single turnover at low [ATP], the observed rate increased with [actin], showing saturation with a Km with respect to actin of 26 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1994

Activation of NADPH oxidase involves the dissociation of p21rac from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane.

Activation of the NADPH oxidase of phagocytes involves the small GTP-binding protein p21rac. In this paper we report that neutrophil cytosol contains predominantly p21rac2 rather than p21rac1, and that the P21rac2 is almost entirely complexed with rhoGDI (GDP dissociation inhibitor) to form a heterodimer with a molecular mass of 45-50 kDa. Activation of superoxide production by phorbol 12-myristate 13-acetate or formylmethionyl-leucyl-phenylalanine in whole cells, and by SDS in the cell-free assay, led to the dissociation of some of the p21rac2 from rhoGDI and its movement to the plasma membrane together with p47phox and p67phox. The appearance of these proteins at the plasma membrane was related to the dose of the agonist and to the rate of superoxide generation. The nucleotide bound to p21rac2 in this complex following isolation was almost exclusively GDP, with less than 2% GTP, and the complex was active in the cell-free assay. Although the rac/GDI complex could activate the NADPH oxidase in the absence of exogenous GTP, the rate of superoxide production was increased 3-fold by the addition of GTP and was almost completely inhibited by GDP. Our findings confirm that rhoGDI serves as GDP dissociation inhibitor and that the release of p21rac2 from this inhibitor is an important step in activation of the NADPH oxidase.

Amino Acid Sequence↗

PubMed1994

Behavioral responses and self-reported pain in postoperative patients.

Pain has been difficult to assess because of its multidimensional nature. The primary purpose of this study was to investigate the relationship between the nurse's assessment of behavioral cues to pain and self-reports of pain made by patients using patient controlled analgesia (PCA). This descriptive-correlational study used the PACU Behavioral Pain Rating Scale (BPRS), patient's self-report, and hospital's PCA pain-rating scale to investigate pain measurement. The convenience sample consisted of 36 postoperative, gynecological surgery patients. Pain assessments for this study were completed during the first 6 postoperative hours on patients who did not have complications. Five pain assessments were collected for each patient. Pain scores were highest during the immediate postoperative period and continued over 2 hours. Significant relationships (rs = 0.56 to 0.80; P < 0.05) were found between the BPRS scores and the self-reports of pain. The relationship between the hospital's PCA pain rating scores and self-reported pain was significant only during the second assessment (rs = 0.45; P < 0.05). The BPRS consistently showed a moderate to high relationship with the patient's self-report of pain and had a stronger relationship with the patient's self-reported pain than with the hospital's pain scale.

Adult↗

PubMed1993

Mechanism of GTP hydrolysis by p21N-ras catalyzed by GAP: studies with a fluorescent GTP analogue.

The mechanism of the hydrolysis of GTP by p21N-ras and its activation by the catalytic domain of p120 GTPase activating protein (GAP) have been studied using a combination of chemical and fluorescence measurements with the fluorescent GTP analogue, 2'(3')-O-(N-methylanthraniloyl)GTP (mantGTP). Since the concentration of active p21 is important in these measurements, various assays for both total protein and active p21 were investigated. All assays gave good agreement except the filter binding assay of [3H]-GDP bound to p21, which gave values of 35-40% compared to the other methods. Concentrations of p21 were thus based on the absorbance of the mant-chromophore of the p21-mant-nucleotide complexes. The rate constants of the elementary steps of the p21 intrinsic GTPase activity and the GAP activated activity were similar between GTP and mantGTP. Incubation of a stoichiometric complex of p21.mantGTP results in a biphasic decrease in fluorescence. The second phase occurs with the same rate constant as the cleavage step and is accelerated by GAP. No other steps of the mechanism are affected by GAP. Incubation of a stoichiometric complex of p21.mantGpp[NH]p also results in a biphasic decrease in fluorescence even though cleavage does not occur. This is interpreted that the cleavage step of p21.GTP is preceded by and controlled by an isomerization of the p21.GTP complex. GAP accelerates the rate constant of the second fluorescence phase occurring with p21.mantGpp[NH]p. This result shows that GAP accelerates the proposed isomerization which limits GTP cleavage rather than the cleavage step itself.

Chromatography, Gel↗

PubMed1993

Interaction of GTPase activating proteins (GAPs) with p21ras measured by a novel fluorescence anisotropy method. Essential role of Arg-903 of GAP in activation of GTP hydrolysis on p21ras.

Ras GTPase activating proteins (GAPs) contain an invariant motif, -FLR-, within the most conserved region of their catalytic domains. Certain mutations in this motif have greatly reduced activity (Skinner, R. H., Bradley, S., Brown, A. L., Johnson, N. J., Rhodes, S., Stammers, D. K., and Lowe, P. N. (1991) J. Biol. Chem. 266, 14163-14166), but it was not determined whether the reduced activity was due to loss of binding or impaired catalysis. In order to address this question, we have developed a simple physical method to study formation of GAP.p21ras complexes. This utilizes the increase of fluorescence anisotropy upon binding of GAP to p21ras complexed with 2'(3')-O-(N-methylanthraniloyl) (mant) derivatives of guanine nucleotides. Dissociation constants obtained for the catalytic domains of either p120-GAP (GAP-344) or neurofibromin (NF1-GRD) with normal and Leu-61 p21ras proteins are comparable with those obtained by kinetic methods. In the course of these studies, we found, in contrast to previous observations, that both GAP and NF1-GRD can weakly activate the GTPase of Leu-61 mutant p21, showing that Gln-61 is not absolutely required for the stimulation of GTPase activity by GAPs. The fluorescence anisotropy method allowed us to show that mutation of Arg-903, within the FLR motif of GAP, can result in protein defective in catalysis but not in binding to p21ras. These data suggest a direct role for this residue in catalyzing GTP hydrolysis on p21ras, possibly by contributing a catalytic group to the p21 active site. This method is independent of the catalytic activity of the proteins, and so it could be extended generally to the measurement of binding of effector molecules, exchange factors, or other macromolecules to guanine nucleotide-binding proteins.

Amino Acid Sequence↗

PubMed1992

Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release.

The mechanism of GTPase-activating protein (GAP) activation of p21ras GTP hydrolysis has been investigated by measuring the kinetics of release of Pi during the hydrolysis. The measurement uses a continuous spectroscopic assay for Pi, based on a guanosine analogue, 2-amino-6-mercapto-7-methylpurine ribonucleoside, as substrate for purine nucleoside phosphorylase [Webb, M.R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4884-4887]. This phosphorolysis gives an absorbance increase at 360 nm, so that when the reaction is coupled to GTP hydrolysis, the change in absorbance gives the total amount of Pi released from the p21ras. The rate of the absorbance increase gives the GTPase activity. This provides a non-radioactive method of determining p21ras concentration and GAP activity. It was used to determine the interaction of GAP with wild-type p21ras and two mutants (Leu-61/Ser-186 and Asp-12), all in the GTP (or guanosine 5'-[ beta gamma-imido]triphosphate) form. The Leu-61/Ser-186 mutant binds 10-fold tighter than does the wild-type protein. The Asp-12 mutant binds to GAP with the same affinity as the wild-type protein. A novel GTPase activity was characterized whereby the EDTA-induced nucleotide release and GAP-activated cleavage of bound GTP leads to steady-state turnover of GTP hydrolysis. An assay for GAP is described based on this activity.

Ammonium Sulfate↗

PubMed1992

A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems.

A spectrophotometric method for the measurement of inorganic phosphate (P(i)) has been developed by using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1). This substrate gives an absorbance increase at 360 nm on phosphorolysis at pH 6.5-8.5, and at pH 7.6 the change in extinction coefficient is 11,000 M-1.cm-1. The Michaelis-Menten constants of the two substrates with the enzyme are 70 microM for the nucleoside and 26 microM for P(i); the kcat is 40 s-1 (25 degrees C). The assay was shown to quantitate P(i) in solution at concentrations at least down to 2 microM. It can be used to measure the kinetics of P(i) release from phosphatases, such as GTPases and ATPases, by coupling the two enzymic reactions. The utility of this assay was shown by three test systems: glycerol kinase plus D-glyceraldehyde acting as an ATPase and actin-activated myosin ATPase, and myosin subfragment 1, hydrolyzing a single turnover of ATP, releasing P(i) with a rate constant the same as the steady-state ATPase activity.

Guanosine↗

PubMed1992

The role of nucleoside triphosphate hydrolysis in transducing systems: p21ras and muscle.

A variety of systems use nucleoside triphosphate hydrolysis to control or provide energy for biological processes, mediated through protein-protein interactions. The nature of this coupling may vary, but often there is a degree of similarity. In this paper, two systems are compared: actomyosin in muscle and p21ras in a signal transduction pathway as yet undefined. The mechanism of the nucleotide triphosphate hydrolysis and the consequent changes in the protein-nucleotide complex have been investigated, to understand how the coupling to biological function is achieved. The basal nucleoside triphosphatase mechanisms are compared and the roles of proteins that activate the hydrolysis, actin and GAP, are discussed. The cleavage process was probed by stereochemical techniques to determine the basic mechanism, of either a phosphorylated enzyme intermediate or direct displacement of nucleoside diphosphate by water. Phosphate-water oxygen exchange probes were used to investigate nucleoside triphosphate and inorganic phosphate release steps. A new method of probing the kinetics of inorganic phosphate release directly has been developed. In muscle, this process seems likely to be related directly to force generation. In the GAP-ras system, measurement of phosphate release is allowing the mechanism of the GAP-p21ras interaction to be probed.

Animals↗

PubMed1991

Nitrogenase of Klebsiella pneumoniae. Reversibility of the reductant-independent MgATP-cleavage reaction is shown by MgADP-catalysed phosphate/water oxygen exchange.

The steady-state kinetics of reductant-independent ATP hydrolysis by Klebsiella pneumoniae nitrogenase at 23 degrees C at pH 7.4 were determined as a function of component protein ratio (optimal at an oxidized Fe protein/MoFe protein ratio of 3:1) and MgATP concentration (Km 400 microM). Competitive inhibition was observed for MgADP (Ki 145 microM), [beta gamma-methylene]ATP (Mgp[CH2]ppA) (Ki 115 microM), [beta gamma-monofluoromethylene]ATP (Mgp[CHF]ppA) (Ki 53 microM) and [beta gamma-difluoromethylene]ATP (Mgp[CF2]ppA) (Ki 160 microM). The tighter binding of MgADP to free oxidized Fe protein (KD less than 10 microM) than to the oxidized Fe protein-MoFe protein complex (Ki 145 microM) is proposed as the driving force that induces rate-limiting protein dissociation in the catalytic cycle of nitrogenase. The reversible nature of the reductant-independent MgATP-cleavage reaction was demonstrated by an MgADP-induced enhancement of the rate of the phosphate/water oxygen exchange reaction with 18O-labelled phosphate ion. This enhancement, like the reductant-independent ATPase reaction, only occurred with the complex formed by oxidized Fe protein and MoFe protein and not with the individual proteins. The results are discussed in terms of the mechanism of ATP hydrolysis by nitrogenase and other systems involving protein-protein interactions.

Adenosine Diphosphate↗

PubMed1991

Kinetics of ATP release and Pi binding during the ATPase cycle of lethocerus flight muscle fibres, using phosphate-water oxygen exchange.

Rate constants have been obtained using oxygen isotope exchange techniques for steps controlling ATP release and Pi binding in the ATPase cycle of insect flight muscle fibres from the giant waterbug Lethecerus. The new exchange data for Pi binding and ATP release are compatible with a model developed previously in which only the rate constants controlling Pi and ATP release change during fibre activation. Phosphate-water oxygen exchange occurs into ATP remaining after partial hydrolysis by chemically skinned fibres in (18O) water. For fully activated fibres, the results are compatible with a single set of rate constants controlling this exchange and give a rate constant for ATP release of 1 s-1 (21 degrees C, pH 7.0 I = 120 mM). Oxygen exchange also occurs between (18O4)Pi in the medium and water during ATP hydrolysis. There is a strong correlation between the measured rate constant of exchange and the value of keat for the ATPase activity at different levels of activation. For fibres fully activated by oscillation or strain, the rate constant for Pi binding to an actomyosin. ADP state is greater than 960 M-1 s-1.

Adenosine Diphosphate↗