A single order-disorder transition generates tension during the Huxley-Simmons phase 2 in muscle.

Increasing temperature was used to progressively interconvert non-force-generating into force-generating states in skinned rabbit psoas muscle fibers contracting isometrically. Laser temperature-jump and length-jump experiments were used to characterize tension generation in the time domain of the Huxley-Simmons phase 2. In our experiments, phase 2 is subdivisible into two kinetic steps each with quite different physical properties. The fast kinetic component has rate constant of 950 s-1 at 1 degrees C and a Q10 of approximately 1.2. Its rate is tension insensitive and its normalized amplitude declines with rising temperature--behavior that closely parallels the instantaneous stiffness of the cross-bridge. It is likely that this kinetic step is a manifestation of a damped elastic element/s in the fiber. The slow component of phase 2 is temperature-dependent with a Q10 of approximately 3.0. Its rate is sensitive to tension. Unlike the fast component, its amplitude remains in fixed proportion to isometric tension at different temperatures indicating direct participation in tension generation. Similar T-jump studies on frog fibers are also included. The combined results (frog and rabbit) suggest that tension generation occurs in a single endothermic (entropy driven) step in phase 2.     

Kinetic and thermodynamic studies of the cross-bridge cycle in rabbit psoas muscle fibers.

The effect of temperature on elementary steps of the cross-bridge cycle was investigated with sinusoidal analysis technique in skinned rabbit psoas fibers. We studied the effect of MgATP on exponential process (C) to characterize the MgATP binding step and cross-bridge detachment step at six different temperatures in the range 5-30 degrees C. Similarly, we studied the effect of MgADP on exponential process (C) to characterize the MgADP binding step. We also studied the effect of phosphate (Pi) on exponential process (B) to characterize the force generation step and Pi-release step. From the results of these studies, we deduced the temperature dependence of the kinetic constants of the elementary steps and their thermodynamic properties. We found that the MgADP association constant (K0) and the MgATP association constant (K1) significantly decreased when the temperature was increased from 5 to 20 degrees C, implying that nucleotide binding became weaker at higher temperatures. K0 and K1 did not change much in the 20-30 degree C range. The association constant of Pi to cross-bridges (K5) did not change much with temperature. We found that Q10 for the cross-bridge detachment step (k2) was 2.6, and for its reversal step (k-2) was 3.0. We found that Q10 for the force generation step (Pi-isomerization step, k4) was 6.8, and its reversal step (k-4) was 1.6. The equilibrium constant of the detachment step (K2) was not affected much by temperature, whereas the equilibrium constant of the force generation step (K4) increased significantly with temperature increase. Thus, the force generation step consists of an endothermic reaction. The rate constant of the rate-limiting step (k6) did not change much with temperature, whereas the ATP hydrolysis rate increased significantly with temperature increase. We found that the force generation step accompanies a large entropy increase and a small free energy change; hence, this step is an entropy-driven reaction. These observations are consistent with the hypothesis that the hydrophobic interaction between residues of actin and myosin underlies the mechanism of force generation. We conclude that the force generation step is the most temperature-sensitive step among elementary steps of the cross-bridge cycle, which explains increased isometric tension at high temperatures in rabbit psoas fibers.     

Kinetic effects of fiber type on the two subcomponents of the Huxley-Simmons phase 2 in muscle

The Huxley-Simmons phase 2 controls the kinetics of the first stages of tension recovery after a step-change in fiber length and is considered intimately associated with tension generation. It had been shown that phase 2 is comprised of two distinct unrelated phases. This is confirmed here by showing that the properties of phase 2(fast) are independent of fiber type, whereas those of phase 2(slow) are fiber type dependent. Phase 2(fast) has a rate of 1000-2000 s(-1) and is temperature insensitive (Q(10) approximately 1.16) in fast, medium, and slow speed fibers. Regardless of fiber type and temperature, the amplitude of phase 2(fast) is half (approximately 0.46) that of phase 1 (fiber instantaneous stiffness). Consequently, fiber compliance (cross-bridge and thick/thin filament) appears to be the common source of both phase 1 elasticity and phase 2(fast) viscoelasticity. In fast fibers, stiffness increases in direct proportion to tension from an extrapolated positive origin at zero tension. The simplest explanation is that tension generation can be approximated by two-state transition from attached preforce generating (moderate stiffness) to attached force generating (high stiffness) states. Phase 2(slow) is quite different, progressively slowing in concert with fiber type. An interesting interpretation of the amplitude and rate data is that reverse coupling of phase 2(slow) back to P(i) release and ATP hydrolysis appears absent in fast fibers, detectable in medium speed fibers, and marked in slow fibers contracting isometrically. Contracting slow and heart muscles stretched under load could employ this enhanced reversibility of the cross-bridge cycle as a mechanism to conserve energy.     

Determination of membrane lipid phase transition temperature from C-NMR intensities

A new and simple approach for the determination of the temperature of gel-to-liquid crystalline phase transitions (T_C) of biological (chloroplast) membrane lipids from ¹³C-NMR resonance intensities is proposed. The variation of intensity of a temperature-sensitive NMR resonance is monitored by recording the spectra of the sample at a range of temperatures. From such a series of spectra recorded at different temperatures, a temperature-insensitive resonance is located. Then the ratio of the intensity of the temperature-sensitive to the intensity of the temperature-insensitive resonance is calculated from each spectrum to even out the procedural error, if any. The values of this ratio at different temperatures, when plotted against sample temperature, shows a break at T_C as confirmed by spin label ESR studies.     

T-jump infrared study of the folding mechanism of coiled-coil GCN4-p1

Partially folded intermediates have been frequently observed in equilibrium and kinetic protein folding studies. However, folding intermediates that exist at the native side of the rate-limiting step are rather difficult to study because they often evade detection by conventional folding kinetic methods. Here, we demonstrated that a laser-induced temperature-jump method can potentially be used to identify the existence of such post-transition or hidden intermediates. Specifically, we studied two cross-linked variants of GCN4-p1 coiled-coil. The GCN4 leucine zipper has been studied extensively and most of these studies have regarded it as a two-state folder. Our static circular dichroism and infrared data also indicate that the thermal unfolding of these two monomeric coiled-coils can be adequately described by an apparent two-state model. However, their temperature-jump-induced relaxation kinetics exhibit non-monoexponential behavior, dependent upon sequence and temperature. Taken together, our results support a folding mechanism wherein at least one folding intermediate populates behind the main rate-limiting step.     

Kinetic effects of myosin regulatory light chain phosphorylation on skeletal muscle contraction

Kinetic analysis of contracting fast and slow rabbit muscle fibers in the presence of the tension inhibitor 2,3-butanedione monoxime suggests that regulatory light chain (RLC) phosphorylation up-regulates the flux of weakly attached cross-bridges entering the contractile cycle by increasing the actin-catalyzed release of phosphate from myosin. This step appears to be separate from earlier Ca(2+) regulated steps. Small step-stretches of single skinned fibers were used to study the effect of phosphorylation on fiber mechanics. Subdivision of the resultant tension transients into the Huxley-Simmons phases 1, 2(fast), 2(slow), 3, and 4 reveals that phosphorylation reduces the normalized amplitude of the delayed rise in tension (stretch activation response) by decreasing the amplitudes of phase 3 and, to a lesser extent, phase 2(slow). In slow fibers, the RLC P1 isoform phosphorylates at least 4-fold faster than the P2 isoform, complicating the role of RLC phosphorylation in heart and slow muscle. We discuss the functional relevance of the regulation of stretch activation by RLC phosphorylation for cardiac and other oscillating muscles and speculate how the interaction of the two heads of myosin could account for the inverse effect of Ca(2+) levels on isometric tension and rate of force redevelopment (k(TR)).     

Temperature differentially affects encounter and docking thermodynamics of antibody--antigen association

Using BIACORE SPR, we have examined the mechanism of temperature effects on the binding kinetics of two closely related antibody Fabs (H10 and H26) which recognize coincident epitopes on hen egg-white lysozyme (HEL), and whose association and dissociation kinetics are best described by the two-step conformational change model which we interpret as molecular encounter and docking. Time-course series data obtained at a series of six temperatures (6, 10, 15, 25, 30 and 37 degrees C) showed that temperature differentially affects the rate constants of the encounter and docking steps. Docking is more temperature-sensitive than the encounter step, and energetically less favorable at higher temperatures. At elevated temperatures, the time required for docking is longer and the apparent increase in off-rate reflects the greater proportion of the molecules failing to dock and remaining in the less stable encounter state. As a consequence, distribution of free energy change between the encounter and docking steps is altered. At physiological temperature (37 degrees C) the docking step of the H26 complex is energetically unfavorable and most complexes essentially do not dock. There is a significant decrease in total free energy change of the H26 complex at higher temperatures. Elevated temperature changes the rate-limiting step of H26--HEL association from the encounter to the docking step, but not that of H10--HEL. Our results indicate that the mechanism by which elevated temperature reduces the affinities of antigen--antibody complexes is to decrease the net docking rate, and/or stability of the docked complex; at higher temperatures, a smaller proportion of the complexes actually anneal to a more stable docked state. This mechanism may have broad applicability to other receptor--ligand complexes.     

Physicochemical properties of carbons prepared from pecan shell by phosphoric acid activation

Activated carbons were prepared from pecan shell by phosphoric acid activation. The pore structure and acidic surface groups of these carbons were characterized by nitrogen adsorption, Boehm titration and transmittance Fourier infrared spectroscopy (FTIR) techniques. The characterization results demonstrated that the development of pore structure was apparent at temperatures 250 degrees C, and reached 1130m(2)/g and 0.34cm(3)/g, respectively, at 500 degrees C. Impregnation ratio and soaking time at activation temperature also affected the pore development and pore size distribution of final carbon products. At an impregnation ratio of 1.5, activated carbon with BET surface area and micropore volume as high as 861m(2)/g and 0.289cm(3)/g was obtained at 400 degrees C. Microporous activated carbons were obtained in this study. Low impregnation ratio (less than 1.5) and activation temperature (less than 300 degrees C) are favorable to the formation of acidic surface functional groups, which consist of temperature-sensitive (unstable at high temperature) and temperature-insensitive (stable at high temperature) two parts. The disappearance of temperature-sensitive groups was significant at temperature 300 degrees C; while the temperature-insensitive groups are stable even at 500 degrees C. FTIR results showed that the temperature-insensitive part was mostly phosphorus-containing groups as well as some carbonyl-containing groups, while carbonyl-containing groups were the main contributor of temperature-sensitive part.     

The Rates of Ca2+ Dissociation and Cross-bridge Detachment from Ventricular Myofibrils as Reported by a Fluorescent Cardiac Troponin C

The rate-limiting step of cardiac muscle relaxation has been proposed to reside in the myofilament. Both the rates of cross-bridge detachment and Ca(2+) dissociation from troponin C (TnC) have been hypothesized to rate-limit myofilament inactivation. In this study we used a fluorescent TnC to measure both the rate of Ca(2+) dissociation from TnC and the rate of cross-bridge detachment from several different species of ventricular myofibrils. The fluorescently labeled TnC was sensitive to both Ca(2+) dissociation and cross-bridge detachment at low Ca(2+) (presence of EGTA), allowing for a direct comparison between the two proposed rates of myofilament inactivation. Unlike Ca(2+) dissociation from TnC, cross-bridge detachment varied in myofibrils from different species and was rate-limited by ADP release. At subphysiological temperatures (<20 °C), the rate of Ca(2+) dissociation from TnC was faster than the rate of cross-bridge detachment in the presence of ADP. These results support the hypothesis that cross-bridge detachment rate-limits relaxation. However, Ca(2+) dissociation from TnC was not as temperature-sensitive as cross-bridge detachment. At a near physiological temperature (35 °C) and ADP, the rate of cross-bridge detachment may actually be faster than the rate of Ca(2+) dissociation. This provides evidence that there may not be a simple, single rate-limiting step of myofilament inactivation.     

Elementary processes in the interaction of serine protease with a possible transition state analog. Subtillisin-benzeneboronic acid system.

The interaction of benzeneboronic acid(BBA), a possible transition state analog, with subtilisin BPN' [EC 3.4.21.14] was studied by the temperature-jump method at various pH's, temperatures and in D2O as well as H2O. From analysis of the concentration dependence of the relaxation times, it was suggested that the subtillsin-BBA interactions consist of at least two elementary steps, a fast bimolecular association followed by a slow unimolecular process. Similar concentration dependence was observed at pH 6.1-6.7 at 25degrees. However, in D2O the reciprocal relaxation times generally decreased compared to those in H2O and became concentration-independent below pD 6.5. The relaxation times were influenced considerably by the temperature. From these results, the slow unimolecular process was assigned to the trigonal-tetrahedral interconversion of BBA at the active site of the enzyme.     

Heat changes during transient tension responses to small releases in active frog muscle.

Tension and heat production were measured in frog sartorius muscles in response to small shortening ramps (releases) at high and moderate speed. Transient tension responses to fast releases (0.1 to 0.4 mm in 1 or 4 ms) were similar to the tension transients length-clamped single fibers. Tension time courses during releases at 25 mm/s were like fiber responses calculated from the first two phases of the step responses (Ford et al., 1977). We conclude that similar crossbridge transitions produce tension transients observed in whole muscles and single fibers. Heat was absorbed during rapid tension recovery after fast releases and during the later part of releases at 25 mm/s. Variation of heat absorption with release size was compared with that of crossbridge movement predicted by the Huxley-Simmons hypothesis of force generation (Huxley and Simmons, 1971). Agreement between the two supports the conclusion that heat is absorbed by the crossbridge transitions responsible for rapid tension recovery after release. The results indicate that the entropy change of these transitions is positive.     

Flat revertants of temperature-insensitive transformants induced by simian virus 40 tsA mutants lose their ability to express T-antigen.

Temperature-insensitive transformants that contained simian virus 40 sequences at only one or a few sites in the rat chromosome and that were induced by a temperature-sensitive A gene mutant of simian virus 40 were used to select flat revertants (revertants that had lost the transformed phenotype). The isolation was performed at the nonpermissive temperature so as not to select against temperature-sensitive transformants. Nonetheless, all of the revertants examined had lost their ability to express the T-antigen at both temperatures, and all contained rearrangements of the integrated simian virus 40 sequences. These results are most compatible with the hypothesis that the T-antigen of simian virus 40 is required for the maintenance of the transformed state even in temperature-insensitive cell lines.     

The rate of changes in tension within fused tetani of single motor units in rat medial gastrocnemius muscle.

The time course of fused tetani of three main types of motor units: slow (S), fast resistant (FR) and fast fatigable (FF) was studied in the rat medial gastrocnemius. The rate of tension generation and of the relaxation within a tetanus was measured under isometric conditions. These measurements were performed at three points during both the contraction and relaxation: the beginning, the middle and the end of the phase of changes in tension. Significant differences were found in the rate of tension changes between fast and slow units. Comparison of FF and FR units showed less pronounced differences in their rates of the contraction and the relaxation. Moreover, slow units showed significantly greater slowing of both the contraction and relaxation within a tetanus in relation to the speed of their twitch when compared to fast motor units. The rate of changes in tetanic tension correlated to twitch time parameters and to tension generated during twitch or tetanus. The results point out that the well known difference in the speed of twitch contraction between fast and slow units is also visible in their fused tetani.     

Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium

Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzyme kinetics above denaturation temperature: a temperature-jump/stopped-flow apparatus

We constructed a "temperature-jump/stopped-flow" apparatus that allows us to study fast enzyme reactions at extremely high temperatures. This apparatus is a redesigned stopped-flow which is capable of mixing the reactants on a submillisecond timescale concomitant with a temperature-jump even as large as 60 degrees C. We show that enzyme reactions that are faster than the denaturation process can be investigated above denaturation temperatures. In addition, the temperature-jump/stopped-flow enables us to investigate at physiological temperature the mechanisms of many human enzymes, which was impossible until now because of their heat instability. Furthermore, this technique is extremely useful in studying the progress of heat-induced protein unfolding. The temperature-jump/stopped-flow method combined with the application of structure-specific fluorescence signals provides novel opportunities to study the stability of certain regions of enzymes and identify the unfolding-initiating regions of proteins. The temperature-jump/stopped-flow technique may become a breakthrough in exploring new features of enzymes and the mechanism of unfolding processes.     

Conformational equilibria accompanying the electron transfer between cytochrome c (P551) and azurin from Pseudomonas aeruginosa.

The redox reaction between cytochrome c (Cyt c) (P-551) and the blue copper protein azurin, both from Pseudomonas aeruginosa, was studied using the temperature-jump technique. Two relaxation times were observed in a mechanism assumed to involve three equilibria. The fast relaxation time (0.4 less than tau less than 8 ms) was ascribed to the electron exchange step. The slow relaxation time (tau congruent to 37 ms) was assigned to a conformational equilibrium of the reduced azurin that was coupled through the electron exchange step to a faster conformational equilibrium of the oxidized Cyt c (P551). But because the Cyt c (P551) isomerization, being very rapid, was uncoupled from the two slower equilibria, and was assumed to involve no spectral change, the amplitude of its relaxation time (tau congruent to 0.1 ms) would be zero. At 25 degrees C and pH 7.0 the rate constants for the oxidation and reduction of Cyt c (P551) by azurin were 6.1 X 10(6) and 7.8 X 10(6) M-1 s-1, respectively; for the formation and disappearance of the reactive conformational isomer of azurin they were 12 and 17 s-1, respectively. The rates for the Cyt c (P551) isomerization could only be estimated at approximately 10(4) s-1. The thermodynamic parameters of each reaction step were evaluated from the amplitudes of the relaxations and from Eyring plots of the rate constants. Measurements of the overall equilibrium constant showed it to be temperature independent (5-35 degrees C), i.e. deltaHtot = 0. This zero enthalpy change was found to be compatible with the enthalpies calculated for the individual steps. In the electron exchange equilibrium, the values of the activation enthalpies were two to three times higher than the values published for various low molecular weight reagents in their electron exchange with copper proteins, yet the rate of exchange between Cyt c (P551) and azurin was some hundreds of times faster. This was explained in terms of the measured positive or zero entropies of activation that could result from a high level of specificity between the proteins particularly in areas of complementary charges. The mechanism of electron transfer was considered as essentially an outer sphere reaction, of which the rate could be approximated by the Marcus theory.     

Evidence for the existence of three or more slow phases in the refolding of ribonuclease A and some characteristics of the phases

The slow refolding kinetics of RNase A have been analyzed, by using a nonlinear least-squares program for deconvoluting the kinetic phases and applying statistical tests for quality of fit. It is found that a minimum of three slow phases are required to fit the kinetic data properly, and this is true whether the method of detection is absorbance of fluorescence. Since the number of phases and the relaxation times for each phase are independent of the method of detection, it is concluded that the same three rate-limiting processes are seen by absorbance and fluorescence. These phases correspond to the XY, CT, and ct phases described in our earlier studies. The fact that fluorescence-detected kinetics are somewhat slower than absorbance-detected kinetics is a trivial effect due not to differences in relaxation times but to the fact that the amplitude of the CT phase is enhanced in fluorescence measurements, at the expense of the faster XY phase, because of intrinsic fluorescence changes associated with the isomerization of proline-93. By use of a new double-jump technique [Schmid, F.X., Grafl, R., Wrba, A., & Beintema, J.J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 872], it is shown that proline-93 isomerizes as the rate-limiting step in only one of the three phases, the CT phase, and that this phase involves only 25-30% of the RNase molecules. There is still no indication as to the molecular events that occur in the large, ammonium sulfate dependent XY phase, which is the pathway for formation of the nativelike intermediate.     

Calcium diffusion in uterine smooth muscle sheets

The potassium contracture in the longitudinal muscle of estrogen- treated rat uterus was kinetically investigated. The rates of tension development after Ca addition and relaxation after Ca removal were measured under the high-potassium depolarization. Both rates decreased with an increase in preparation thickness. The relaxation rate had only a slight dependence on temperature. On the contrary, both relaxation and contraction rates in a contraction induced by an electrical stimulation strongly depended on temperature, but not on preparation size. These results suggest that the Ca diffusion process in the extracellular space is the rate-limiting step in relaxation of Ca- dependent contracture under potassium depolarization. The diffusion model, in which the effect of the unstirred layer was considered, could quantitatively explain the experimental results. The apparent diffusion coefficient in the muscle sheet was estimated to be approximately 3 x 10(-7) cm2/s. The difference from that in aqueous solution is discussed.     

Effect of single amino acid replacements on the folding and stability of dihydrofolate reductase from Escherichia coli

The role of the secondary structure in the folding mechanism of dihydrofolate reductase from Escherichia coli was probed by studying the effects of amino acid replacements in two alpha helices and two strands of the central beta sheet on the folding and stability. The effects on stability could be qualitatively understood in terms of the X-ray structure for the wild-type protein by invoking electrostatic, hydrophobic, or hydrogen-bonding interactions. Kinetic studies focused on the two slow reactions that are thought to reflect the unfolding/refolding of two stable native conformers to/from their respective folding intermediates [Touchette, N. A., Perry, K. M., & Matthews, C. R. (1986) Biochemistry 25, 5445-5452]. Replacements at three different positions in helix alpha B selectively alter the relaxation time for unfolding while a single replacement in helix alpha C selectively alters the relaxation time for refolding. This behavior is characteristic of mutations that change the stability of the protein but do not affect the rate-limiting step. In striking contrast, replacements in strands beta F and beta G can affect both unfolding and refolding relaxation times. This behavior shows that these mutations alter the rate-limiting step in these native-to-intermediate folding reactions. It is proposed that the intermediates have an incorrectly formed beta sheet whose maturation to the structure found in the native conformation is one of the slow steps in folding.