Paradoxical effects of endurance training and chronic hypoxia on myofibrillar ATPase activity.

This study aimed to determine the changes in soleus myofibrillar ATPase (m-ATPase) activity and myosin heavy chain (MHC) isoform expression after endurance training and/or chronic hypoxic exposure. Dark Agouti rats were randomly divided into four groups: control, normoxic sedentary (N; n = 14), normoxic endurance trained (NT; n = 14), hypoxic sedentary (H; n = 10), and hypoxic endurance trained (HT; n = 14). Rats lived and trained in normoxia at 760 mmHg (N and NT) or hypobaric hypoxia at 550 mmHg (approximately 2,800 m) (H and HT). m-ATPase activity was measured by rapid flow quench technique; myosin subunits were analyzed with mono- and two-dimensional gel electrophoresis. Endurance training significantly increased m-ATPase (P < 0.01), although an increase in MHC-I content occurred (P < 0.01). In spite of slow-to-fast transitions in MHC isoform distribution in chronic hypoxia (P < 0.05) no increase in m-ATPase was observed. The rate constants of m-ATPase were 0.0350 +/- 0.0023 s(-1) and 0.047 +/- 0.0050 s(-1) for N and NT and 0.033 +/- 0.0021 s(-1) and 0.038 +/- 0.0032 s(-1) for H and HT. Thus, dissociation between variations in m-ATPase and changes in MHC isoform expression was observed. Changes in fraction of active myosin heads, in myosin light chain isoform (MLC) distribution or in MLC phosphorylation, could not explain the variations in m-ATPase. Myosin posttranslational modifications or changes in other myofibrillar proteins may therefore be responsible for the observed variations in m-ATPase activity.

At physiological temperatures the ATPase rates of shortening soleus and psoas myofibrils are similar.

We obtained the temperature dependences of the adenosine triphosphatase (ATPase) activities (calcium-activated and relaxed) of myofibrils from a slow muscle, which we compared with those from a fast muscle. We chose rabbit soleus and psoas because their myosin heavy chains are almost pure: isoforms I and IIX, respectively. The Arrhenius plots of the ATPases are linear (4-35 degrees C) with energies of activation for soleus myofibrils 155 kJ mol(-1) (activated) and 78 kJ mol(-1) (relaxed). With psoas myofibrils, the energies of activation were 71 kJ mol(-1) (activated) and 60 kJ mol(-1) (relaxed). When extrapolated to 42 degrees C the ATPase rates of the two types of myofibril were identical: 50 s(-1) (activated) and 0.23 s(-1) (relaxed). Whereas with psoas myofibrils the K(m) for adenosine triphosphate (activated ATPase) is relatively insensitive to temperature, that for soleus myofibrils increased from 0.3 microM at 4 degrees C to 66.5 microM at 35 degrees C. Our results illustrate the importance of temperature when comparing the mechanochemical coupling in different types of muscle. We discuss the problem of how to reconcile the similarity of the myofibrillar ATPase rates at physiological temperatures with their different mechanical properties.

Kinetics of the initial steps of rabbit psoas myofibrillar ATPases studied by tryptophan and pyrene fluorescence stopped-flow and rapid flow-quench. Evidence that cross-bridge detachment is slower than ATP binding.

The kinetics of the tryptophan fluorescence enhancement that occurs when myofibrils (rabbit psoas) are mixed with Mg-ATP were studied by stopped-flow in different solvents (water, 40% ethylene glycol, 20% methanol) at 4 degrees C. Under relaxing conditions (low Ca(2+)) in water (mu = 0.16 M, pH 7.4) and at high ATP concentrations, the transient was biphasic, giving a k(fast)(max) of 230 s(-)(1) and a k(slow)(max) of 15 s(-)(1). The kinetics of the two phases were compared with those obtained by chemical sampling using [gamma-(32)P]ATP and quenching in acid (P(i) burst experiments: these give unambiguously the ATP cleavage kinetics), or cold Mg-ATP (cold ATP chase: ATP binding kinetics). k(slow) is due to ATP cleavage, as with S1. Interestingly, k(fast) is slower than the ATP binding kinetics. Instead, this constant appears to report ATP-induced cross-bridge detachment from actin because (1) it was identical to the fluorescence transient obtained on addition of ATP to pyrene-labeled myofibrils; (2) when the initial filament overlap in the myofibrils was decreased, the amplitude of the fast phase decreased; (3) there was no fluorescent enhancement upon the addition of ADP to myofibrils. This is different from the situation with S1 or actoS1 where there was also a fast fluorescent ATP-induced transient but whose kinetics were identical to those of the tight ATP binding. To increase the time resolution and to confirm our results, we also carried out transient kinetics in ethylene glycol and methanol. We interpret our results by a scheme in which a rapid equilibrium between attached (AM.ATP) and detached (M.ATP) states is modulated by the fraction of myosin heads in rigor (AM) during the time of experiment.

Cryoenzymic studies on an organized system: myofibrillar ATPases and shortening.

We have exploited cryoenzymology, first, to probe the product release steps of myofibrillar ATPase under relaxing conditions and, second, to define the conditions for studying the contractile process in slow motion. Cryoenzymology implies perturbation by temperature and by the antifreeze added to allow for work at subzero temperatures. Here, we studied myofibrillar shortening and ATPases by the rapid quench flow method over a wide temperature range (-15 to 30 degrees C) in two antifreezes, 40% ethylene glycol and 20% methanol. The choice of solvent and temperature was dictated by the purpose of the experiment. Ethylene glycol (40%) is suitable for investigating the kinetics of the products release steps which is difficult in water. In this cryosolvent, the myofibrillar ATPase is not activated by Ca2+ nor is there shortening, except under special conditions, i.e., Ca2+ plus strong rigor bridges [Stehle, R., Lionne, C., Travers, F., and Barman, T. (1998) J. Muscl. Res. Cell Motil. 19, 381-392]. By the use of the glycol, we show that at low Ca2+ the kinetics of the ADP release are much faster with myofibrils than with S1. On the other hand, the kinetics of the Pi release were very similar for the two materials. Therefore, we suggest that, upon Ca2+ activation, only the Pi release kinetics are accelerated. In 20% methanol, in the presence of Ca2+, myofibrils shortened at temperatures above -2 degrees C but not below. At a given temperature above -2 degrees C, both the shortening and ATPase rates were reduced by the methanol. The temperature dependences of the myofibrillar ATPases (+/-Ca2+) converged with a decrease in temperature: at 20 degrees C, Ca2+ activated 30-fold, but at -15 degrees C, only about 5-fold. We suggest that studies in methanol may open the way for an investigation of muscle contraction in slow motion and, further, to obtain thermodynamic information on the internal forces involved in the shortening process.

The subsites structure of bovine pancreatic ribonuclease A accounts for the abnormal kinetic behavior with cytidine 2',3'-cyclic phosphate.

The kinetics of the hydrolysis of cytidine 2',3'-cyclic phosphate (C>p) to 3'-CMP by ribonuclease A are multiphasic at high substrate concentrations. We have investigated these kinetics by determining 3'-CMP formation both spectrophotometrically and by a highly specific and quantitative chemical sampling method. With the use of RNase A derivatives that lack a functional p2 binding subsite, evidence is presented that the abnormal kinetics with the native enzyme are caused by the sequential binding of the substrate to the several subsites that make up the active site of ribonuclease. The evidence is based on the following points. 1) Some of the unusual features found with native RNase A and C>p as substrate disappear when the derivatives lacking a functional p2 binding subsite are used. 2) The kcat/Km values with oligocytidylic acids of increasing lengths (ending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: increase in going from the monomer to the trimer, a decrease from tetramer to hexamer, and then an increase in going to poly(C). 3) Adenosine increases the kcat obtained with a fixed concentration of C>p as substrate. 4) High concentrations of C>p protect the enzyme from digestion with subtilisin, which results in a more compact molecule, implying large substrate concentration-induced conformational changes. The data for the hydrolysis of C>p by RNase A can be fitted to a fifth order polynomial that has been derived from a kinetic scheme based on the sequential binding of several monomeric substrate molecules.

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.

Probing the coupling of Ca2+ and rigor activation of rabbit psoas myofibrillar ATPase with ethylene glycol.

We have exploited solvent perturbation to probe the coupling of Ca2+ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4 degrees C, the glycol had little effect on the myofibrillar ATPase at low [Ca2+], but at high [Ca2+] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca2+) was reduced only 3-4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca2+. Further experiments revealed that in 40% ethylene glycol, Ca2+ does initiate shortening but only with the aid of strong interactions and at temperatures above 15 degrees C. This confirms that in the organized and intact myofibril, Ca2+ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1.

Cryoenzymic studies on yeast 3-phosphoglycerate kinase. Attempt to obtain the kinetics of the hinge-bending motion.

This is a continuation of a study on the 3-phosphoglycerate kinase (PGK) reaction in the direction of 1,3-bisphosphoglycerate (bPG) formation: ATP + 3-phosphoglycerate (PG) <==> ADP + bPG [Schmidt, P. P., Travers, F., & Barman, T. (1995) Biochemistry 34, 824-832]. We showed that species containing bPG accumulate in the steady state, but their low concentrations and rapid kinetics of formation precluded a full study, even under cryoenzymic conditions in 40% ethylene glycol. Here we studied the PGK reaction in 30% methanol. The transient kinetics of bPG formation were obtained by chemical sampling: PGK was mixed with PG and [gamma-32P]ATP in a rapid flow quench apparatus, the mixture aged 4 ms up and quenched in acid, and the [1-(32)P]bPG was determined. The time course consisted of a rapid rise of bPG (kinetics k(obs)) and a steady state phase. In methanol, the amplitude of the rise was large (>50% of the PGK in the steady state), and k(obs) was measurable. Fluorescence stopped flow was used to study the formation of the binary E x PG and E x ATP. The affinities of PGK for ATP and PG were high in methanol (Kd = 102 and 1.5 microM, respectively), but the kinetics of the formation of E x PG and E x ATP were too rapid to be measured. From these and the chemical sampling experiments, we propose a reaction scheme for PGK: a rapid formation of the collision complex E x PG x ATP (K1), a slow isomerisation to E* x PG x ATP (k2,k(-2)), a rapid phosphorylation transfer step to E x bPG x ADP (K3), and a slow release of the products (k4). In our scheme, k(obs) is the reflection mainly of k2 and k(-2) and the steady state of k4. Using a computer simulation procedure, k2/K1 = 0.37 microM(-1) s(-1), k(-2) = 33 s(-1), K3 = 4, and k4 = 7.1 s(-1). We propose that k(obs) measures the kinetics of the putative hinge-bending motion of PGK, i.e., the conformational change that is necessary for the substrates to line up for phosphoryl transfer.

Mechanochemical coupling in muscle: attempts to measure simultaneously shortening and ATPase rates in myofibrils.

We studied the ATPase of shortening myofibrils at 4 degrees C by the rapid flow quench method. The progress curve has three phases: a P(i) burst, a fast linear phase kF of duration tB, and a deceleration to a slow kS. We propose that kF is the ATPase of myofibrils shortening under zero external load; at tB shortening and ATPase rates are reduced by passive resistance. The total ATP consumed during the rapid shortening is ATPc. Our purpose was to obtain information on the myofibrillar shortening velocity from their ATPase progress curves. We tested tB as an indicator of shortening velocity by determining the effects of different probes upon it and the other ATPase parameters. The dependence of tB upon the initial sarcomere length was linear, giving a shortening velocity close to that of muscle fibres (Vo). The Km of ATP was larger for tB than for kF, as found with fibers for Vo and their ATPase. ADP and 2,3-butanedione monoxime, but not P(i), inhibited tB to the same extent as Vo. The delta H for tB and Vo were similar. ATPc was independent of the sarcomere length, implying that the more the myofibrils shorten, the less ATP expended per myosin head per micron shortened. We propose that tB can be used as an indicator for myofibrillar shortening velocities.

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.

Kinetics of the interaction of myosin subfragment-1 with G-actin. Effect of nucleotides and DNaseI.

The kinetics of interaction of monomeric pyrenyl-labeled G-actin with myosin subfragment-1 (S1 (A1) and S1(A2) isomers) has been examined in the stopped-flow at low ionic strength. The data confirm the previously reported existence of binary GS and ternary G2S complexes. The increase in pyrenyl-actin fluorescence which monitors the G-actin-S1 interactions is linked to the isomerization of these complexes following rapid equilibrium binding steps. The rates of isomerization are approximately 200 s-1 for GS and approximately 50 s-1 for G2S at 4 degrees C and in the absence of ATP. DNaseI and S1 bind G-actin essentially in a mutually exclusive fashion. Both GS and G2S are dissociated by MgATP and MgADP. The kinetics and mechanism of ATP-induced dissociation of G2S are quantitatively close to the ATP-induced dissociation of F-actin-S1, which indicates the G2S is a good model for the F-actin-S1 interface. GS and G2S display different kinetic behaviors in response to nucleotides, GS being less efficiently dissociated than G2S by MgATP. This result suggests that different mechanical properties of the cross-bridge might correlate with different orientations of the myosin head and different actin/myosin binding ratios.

Transient and equilibrium kinetic studies on yeast 3-phosphoglycerate kinase. Evidence that an intermediate containing 1,3-bisphosphoglycerate accumulates in the steady state.

The structure of the key glycolytic enzyme 3-phosphoglycerate kinase (PGK) is known in detail, but there is little information on its reaction pathway. We have studied its equilibrium and transient kinetics in the direction of 1,3-bisphosphoglycerate (1,3-bis-P-glycerate) production: ATP + 3-P-glycerate<==>ADP + 1,3-bis-P-glycerate. We devised a sensitive method for following this production. PGK is mixed with 3-P-glycerate and [gamma-32P]ATP in a rapid flow quench apparatus. The reaction mixtures are aged for 4 ms or more and then quenched in acid in which any [1-32P]-1,3-P-glycerate decomposes to 3-P-glycerate and 32Pi, which is determined specifically. The Pi reflects accurately the 1,3-bis-P-glycerate in the original reaction mixture, and the kcat obtained is identical to that obtained by the conventional linked assay method with glyceraldehyde-3-phosphate dehydrogenase. This does not support the postulate of a rapid direct transfer of the 1,3-bis-P-glycerate between the kinase and the dehydrogenase [Srivastava, D. K., & Bernhard, S. A. (1986) Science 234, 1081-1086]. We fitted our data to a simple scheme with the formation of binary complexes, the interconversion of substrates to products via ternary complexes, and the release of products. Because of the high turnover of PGK, the work was carried out under cryoenzymic conditions with 40% ethylene glycol in the buffer. The glycol decreased kcat from 80 to 8.5 s-1 (pH 7.5, 4 degrees C), but the Km for 3-P-glycerate and ATP and the equilibrium constants in the scheme were little affected. We carried out two types of experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryoenzymology: how to practice kinetic and structural studies.

For a full understanding of an enzyme reaction pathway, one must identify the reaction intermediates and obtain their structures and rates of interconversion. It is impossible to obtain all this information under normal conditions. An approach is to work suboptimally, in particular at subzero temperatures. This is cryoenzymology, an approach that implies both kinetic and structural measurements on enzyme systems below 0 degrees C. To work below 0 degrees C one must add an antifreeze so cryoenzymology means perturbation by two agents: temperature and antifreeze, usually an organic solvent. Certain precautions are needed with these agents, which we will discuss here. In particular, we discuss the importance of choosing the right solvent: this requires extensive exploratory studies but it is the key for the successful practice of cryoenzymology. Each system has its particularities and its own 'good' solvent. Cryoenzymology is not only a way of reducing reaction rates, it is also a way of perturbing one's system. Thus, it is a method that allows for the accumulation of intermediates that cannot be observed under normal conditions by slowing down their kinetics of formation, by changes in rate limiting steps or by shifts in equilibria. We illustrate the usefulness of cryoenzymology by myosin and actomyosin ATPases and by creatine, arginine and 3-phosphoglycerate kinases. We also discuss recent results obtained by X-ray crystallography.

Inhibition of ATP binding to myofibrils and acto-myosin subfragment 1 by caged ATP.

The inhibitory effect of P3-[1-(2-nitrophenyl)ethyl]adenosine 5'-triphosphate (caged ATP) on the binding of Mg2+-ATP to myofibrils was investigated. The most sensitive method was found to be the monitoring of single turnovers of [gamma-32P] ATP hydrolysis using the quench flow technique. The method was tested using ADP, which was found to have an inhibition constant of 145 microM, in agreement with previous reports. Caged ATP behaved as a simple competitive inhibitor of ATP binding with an inhibition constant of 1.6 mM. The inhibitory effect of these ligands on the binding of ATP to acto-myosin subfragment 1 was investigated using the same method. The inhibition constants of caged ATP and ADP were found to be 0.35 mM and 50 microM, respectively. This inhibitory effect of caged ATP on ATP binding accounts for the lower rate of ATP binding to fibers, deduced from caged ATP [(0.5-1) x 10(6) M-1 s-1], than that reported for acto-S1 (3.5 x 10(6) M-1 s-1) [Goldman, Y. E., Hibberd, M. G., & Trentham, D. R. (1984) J. Physiol. (London) 354, 577].

Correlation of ActoS1, myofibrillar, and muscle fiber ATPases.

Our objective was to determine a good in vitro model for muscle fiber ATPase, and we compared the kinetics of Ca(2+)-activated myofibrils and cross-linked actoS1 in a buffer of physiological ionic strength. The myofibrils were cross-linked chemically to mimic the isometric condition of fibers or were un-cross-linked (the isotonic condition), and temperature perturbation was used to probe their ATPase mechanisms. At 4 degrees C, we have already shown that the kinetics of cross-linked actoS1 and myofibrils (cross-linked or not) are similar: there were large P(i) bursts and kcat values of about 1 s-1, close to that obtained with fibers [Herrmann, C., Sleep, J., Chaussepied, P., Travers, F. & Barman, T. (1993) Biochemistry 32, 7255-7263]. So, at 4 degrees C cross-linked actoS1 and myofibrils are equally good as models for fiber ATPase. At 20 degrees C, this similarity vanishes: progress curves with the myofibrils (cross-linked or not) had large P(i) bursts, but with cross-linked actoS1, bursts could not be discerned. This shows that at 20 degrees C the predominant steady-state intermediates are ATP complexes with actoS1 but are products complexes with the myofibrils, as with fibers [Ferenczi, M.A. (1986) Biophys. J. 50, 471-477]. Further, the kcat values were different: 15.5 s-1 with cross-linked actoS1, 8.3 s-1 for myofibrils, and 3.5 s-1 for cross-linked myofibrils. With fibers, kcat = 3.3 s-1. These results show that cross-linked myofibrillar ATPase is a good model for muscle fibers contracting isometrically.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of 2',3'-cyclic phosphodiesters in the bovine pancreatic ribonuclease A catalysed cleavage of RNA: intermediates or products?

There is a considerable degree of ambiguity in the literature regarding the role of the 2',3'-cyclic phosphodiesters formed during the reaction of RNA cleavage catalysed by ribonuclease. Usually the reaction is considered to take place in two steps: in the first step there is a transphosphorylation of the RNA 3',5'-phosphodiester bond broken yielding a 2',3'-cyclic phosphodiester which in the second step is hydrolysed to a 3'-nucleotide. Although in many occasions, either explicitly or implicitly, the reaction is treated as taking place sequentially, this is not the case as it has been shown that the 2',3'-phosphodiesters are actually released to the medium as true products of the reaction and that no hydrolysis of these cyclic compounds takes place until all the susceptible 3',5'-phosphodiester bonds have been cyclised. Comparison of the hydrolysis and alcoholysis of the 2',3'-phosphodiesters catalysed by RNase A indicates that the hydrolysis reaction has to be considered formally as a special case of the transphosphorylation back reaction in which the R group of the R-OH substrate is just H. It is thus concluded that the 2',3'-cyclic phosphodiesters formed in the ribonuclease A reaction are true products of the transphosphorylation reaction and not intermediates as usually considered.

A structural and kinetic study on myofibrils prevented from shortening by chemical cross-linking.

In previous work, we studied the early steps of the Mg(2+)-ATPase activity of Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4 degrees C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the Pi burst method. In Pi burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the kcat of Ca(2+)-activated myofibrils was reduced from 1.7 to 0.8 s-1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPases. An example of an uncompetitive inhibitor.

2,3-Butanedione monoxime (BDM) reversibly inhibits force production in muscle. At least part of its action appears to be directly on the contractile apparatus. To understand better its mechanism of action, we studied the effect of BDM on the steps of myosin subfragment 1 Mg(2+)-ATPase in 0.1 M potassium acetate, pH 7.4. Because of the rapidity of certain processes, we experimented at 4 degrees C and our main technique was the rapid flow quench method. By varying the experimental conditions (relative concentrations of reagents, time scale, quenching agent), it was possible to study selectively the different steps of the S1 Mg(2+)-ATPase: [formula: see text] At saturation (20 mM), BDM had two major effects on the ATPase. First, it increased the equilibrium constant of the cleavage step (K3) from 2 to > 10. Second, it slowed the kinetics of the release of Pi by an order of magnitude (k4; from 0.054 to 0.004 s-1). By contrast, the kinetics of the binding of ATP (k) and the release of ADP (k6) were little affected by BDM. Thus, the oxime appears to interact specifically with M**.ADP.Pi, and it is a rare example of an uncompetitive inhibitor. Its effect is to reduce the steady-state concentration of the "strong" actin binding state M*.ADP and to increase that of the "weak" binding state, M**.ADP.Pi. The effect of BDM on the initial ATPase of Ca2+ activated myofibrils was very similar to that on S1 ATPase. Thus, with myofibrils too BDM seems to exert its main effect subsequent to the initial binding and cleavage steps.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of engineered proteins with internal tryptophan reporter groups and pertubation techniques to probe the mechanism of ligand-protein interactions: investigation of the mechanism of calcium binding to calmodulin.

Stopped-flow kinetic and fluorescence spectroscopic analyses, including solvent and temperature perturbations, of five isofunctional structural mutants of calmodulin indicate that calcium binding to calmodulin follows the order site III, site IV, site I, site II, with dissociation occurring in the reverse order. Each of the isofunctional structural mutants contains a single tryptophan residue, introduced by site-specific mutagenesis, as an internal spectroscopic reporter group that was used as a probe of local conformational change. Calcium binding was studied by using flow dialysis or by using fluorescence spectroscopy and monitoring the change in the single tryptophan residue in each calcium-binding site. Calcium removal was examined by using EDTA and monitoring tryptophan fluorescence or by using Quin 2 and monitoring the change in the chromophoric chelator. Computational analysis of the data suggests a rate-limiting step for dissociation between calcium removal from sites I/II and sites III/IV. Unexpected results with the site IV isofunctional mutant (Q135W-CaM) indicated cross-talk between the amino and carboxyl terminal halves of CaM during the calcium-binding mechanism. Studies with ethylene glycol provided empirical data that suggest the functional importance of the electrostatic potential of CaM, or the molarity of water, in the calcium-binding process. Altogether, the data allowed a kinetic extension of the sequential, cooperative model for calcium binding to calmodulin and provided values for additional parameters in the model of calcium binding to CaM, a prototypical member of the family of proteins required for calcium signal transduction in eukaryotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)