Tension response in skinned Ca-activated skeletal muscle fibers of the frog to temperature jump following stepwise changes in the fiber length

Joulean temperature jump from 4-7 degrees to 20-25 degrees completed in 0.2 ms was applied to suspended in the air chemically skinned Ca-activated (pCa = 5.5-6) skeletal muscle fibres of the frog 2 ms after stepwise length changes (duration 0.3 ms, amplitudes --6. +3 nm per half sarcomere). The temperature jump induced a biphasic rise of tension, as was described earlier. Neither the time constant of the 2nd slow phase, nor maximum tension after the temperature jump were dependent on the length step amplitude. The amplitude and time constant of the 1st phase (1.2-0.28 ms) decreased after the fibre release. It shows that the 1st phase of the tension rise induced by the temperature jump is due to conformation in cross-bridges attached to thin filaments.     

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)     

Effect of joule temperature jump on tension and stiffness of skinned rabbit muscle fibers.

The effects of a temperature jump (T-jump) from 5-7 degrees C to 26-33 degrees C were studied on tension and stiffness of glycerol-extracted fibers from rabbit psoas muscle in rigor and during maximal Ca2+ activation. The T-jump was initiated by passing an alternating current pulse (30 kHz, up to 2.5 kV, duration 0.2 ms) through a fiber suspended in air. In rigor the T-jump induces a drop of both tension and stiffness. During maximal activation, the immediate stiffness dropped by (4.4 +/- 1.6) x 10(-3)/1 degree C (mean + SD) in response to the T-jump, and this was followed by a monoexponential stiffness rise by a factor of 1.59 +/- 0.14 with a rate constant ks = 174 +/- 42 s-1 (mean +/- SD, n = 8). The data show that the fiber stiffness, determined by the cross-bridge elasticity, in both rigor and maximal activation is not rubber-like. In the activated fibers the T-jump induced a biexponential tension rise by a factor of 3.45 +/- 0.76 (mean +/- SD, n = 8) with the rate constants 500-1,000 s-1 for the first exponent and 167 +/- 39 s-1 (mean +/- SD, n = 8) for the second exponent. The data are in accordance with the assumption that the first phase of the tension transient after the T-jump is due to a force-generating step in the attached cross-bridges, whereas the second one is related to detachment and reattachment of cross-bridges.     

Kinetic characteristics of cross bridges in the heart muscles by a temperature jump method]

Experiments were performed on rat skinned trabecular muscles dissected from the right ventricles. They were activated (pCa = 5.9) at 10 C, 2.3 nm sarcomere length. The temperature jump induced the biexponential tension rise. The rate constant of the fast tension rise (k1) was 3-4.5 ms and that of the slow tension rise (k2) was about 15 ms. These values were slower (3 times) comparing with those of a single skeletal fibre. The discrepancy can be explained by different kinetic properties of heart and skeletal myosin or presents a well developed connection tissue (series elastic element) in the heart muscle.     

Kinetics of the lamellar-inverse hexagonal phase transition determined by time-resolved X-ray diffraction.

The kinetics of the lamellar (L alpha)-inverse hexagonal (HII) phase transition in diacylphosphatidylethanolamine (PE)--water systems were probed with time-resolved X-ray diffraction. Transition kinetics in the fast time regime (approximately 100 ms) were studied by initiating large temperature jumps (up to 30 degrees C) with a 50-ms electrical current pulse passed through a lipid-salt water dispersion, resulting in ohmic heating of the sample. Diffraction with a time resolution to 10 ms was acquired at the National Synchrotron Light Source. The time constant for the phase transition for 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was on the order of 100 ms for the largest temperature jumps recorded. Faster transition behavior was found for a 1,2-dielaidoyl-sn-glycero-3-PE mixture. The HII lattice parameters for both systems were seen to swell from an initial value commensurate with the lamellar lattice to the final equilibrium value. The rate of swelling was seen to be independent of the magnitude of the temperature jump. For small temperature jumps (less than 10 degrees C), the phase transition kinetics slow dramatically, and transition studies can readily be performed on a conventional rotating anode X-ray source. At 4 degrees C, a DOPE sample was observed to slowly convert to the hexagonal phase over the course of a week, with the decay in the lamellar intensity fitting a power law behavior over four decades of time. This power law behavior is shown to have interesting consequences to the determination of the phase transition temperature of lipid-water dispersions by conventional methods such as calorimetry.     

Rapid charge translocation by the cardiac Na(+)-Ca2+ exchanger after a Ca2+ concentration jump.

The kinetics of Na(+)-Ca2+ exchange current after a cytoplasmic Ca2+ concentration jump (achieved by photolysis of DM-nitrophen) was measured in excised giant membrane patches from guinea pig or rat heart. Increasing the cytoplasmic Ca2+ concentration from 0.5 microM in the presence of 100 mM extracellular Na+ elicits an inward current that rises with a time constant tau 1 < 50 microseconds and decays to a plateau with a time constant tau 2 = 0.65 +/- 0.18 ms (n = 101) at 21 degrees C. These current signals are suppressed by Ni2+ and dichlorobenzamil. No stationary current, but a transient inward current that rises with tau 1 < 50 microseconds and decays with tau 2 = 0.28 +/- 0.06 ms (n = 53, T = 21 degrees C) is observed if the Ca2+ concentration jump is performed under conditions that promote Ca(2+)-Ca2+ exchange (i.e., no extracellular Na+, 5 mM extracellular Ca2+). The transient and stationary inward current is not observed in the absence of extracellular Ca2+ and Na+. The application of alpha-chymotrypsin reveals the influence of the cytoplasmic regulatory Ca2+ binding site on Ca(2+)-Ca2+ and forward Na(+)-Ca2+ exchange and shows that this site regulates both the transient and stationary current. The temperature dependence of the stationary current exhibits an activation energy of 70 kj/mol for temperatures between 21 degrees C and 38 degrees C, and 138 kj/mol between 10 degrees C and 21 degrees C. For the decay time constant an activation energy of 70 kj/mol is observed in the Na(+)-Ca2+ and the Ca(2+)-Ca2+ exchange mode between 13 degrees C and 35 degrees C. The data indicate that partial reactions of the Na(+)-Ca2+ exchanger associated with Ca2+ binding and translocation are very fast at 35 degrees C, with relaxation time constants of about 6700 s-1 in the forward Na(+)-Ca2+ exchange and about 12,500 s-1 in the Ca(2+)-Ca2+ exchange mode and that net negative charge is moved during Ca2+ translocation. According to model calculations, the turnover number, however, has to be at least 2-4 times smaller than the decay rate of the transient current, and Na+ inward translocation appears to be slower than Ca2+ outward movement.     

温度对菊小长管蚜生长发育的影响

在15、19、22、25、28、31和35℃及RH80％的组合条件下,测定了菊小长管蚜的发育起点温度及有效积温,分析了温度与菊小长管蚜发育速率的关系．结果表明,菊小长管蚜各龄若虫的发育时间随温度的增加而缩短,4龄若虫的发育历时最长,平均为前三龄若虫历期的1．4倍．模拟分析表明,菊小长管蚜发育速率随温度的升高而加快,2、3龄若虫较1、4龄若虫对极端温度的忍耐性强,发育最适温度高．2、3龄若虫的极端温度忍耐值δ为6．70,最适发育温度为21．76℃,最高发育温度为40．97℃;1、4龄若虫极端温度忍耐值δ分别为4．70和4．50,最适发育温度分别23．76℃和22．49℃,最高发育温度为39．97℃和40．56℃;龄期越高,发育起点温度相对较低,发育所需的有效积温则越高．1～4龄若蚜的发育起点温度分别为6．93、5．02、4．58和4．46℃,有效积温分别为26．88、33．41、33．63和48．49℃·d^-1。     

Thermal stress and Ca-independent contractile activation in mammalian skeletal muscle fibers at high temperatures.

Temperature dependence of the isometric tension was examined in chemically skinned, glycerinated, rabbit Psoas, muscle fibers immersed in relaxing solution (pH approximately 7.1 at 20 degrees C, pCa approximately 8, ionic strength 200 mM); the average rate of heating/cooling was 0.5-1 degree C/s. The resting tension increased reversibly with temperature (5-42 degrees C); the tension increase was slight in warming to approximately 25 degrees C (a linear thermal contraction, -alpha, of approximately 0.1%/degree C) but became more pronounced above approximately 30 degrees C (similar behavior was seen in intact rat muscle fibers). The extra tension rise at the high temperatures was depressed in acidic pH and in the presence of 10 mM inorganic phosphate; it was absent in rigor fibers in which the tension decreased with heating (a linear thermal expansion, alpha, of approximately 4 x 10(-5)/degree C). Below approximately 20 degrees C, the tension response after a approximately 1% length increase (complete < 0.5 ms) consisted of a fast decay (approximately 150.s-1 at 20 degrees C) and a slow decay (approximately 10.s-1) of tension. The rate of fast decay increased with temperature (Q10 approximately 2.4); at 35-40 degrees C, it was approximately 800.s-1, and it was followed by a delayed tension rise (stretch-activation) at 30-40.s-1. The linear rise of passive tension in warming to approximately 25 degrees C may be due to increase of thermal stress in titin (connectin)-myosin composite filament, whereas the extra tension above approximately 30 degrees C may arise from cycling cross-bridges; based on previous findings from regulated actomyosin in solution (Fuchs, 1975), it is suggested that heating reversibly inactivates the troponin-tropomyosin control mechanism and leads to Ca-independent thin filament activation at high temperatures. Additionally, we propose that the heating-induced increase of endo-sarcomeric stress within titin-myosin composite filament makes the cross-bridge mechanism stretch-sensitive at high temperatures.     

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.     

Kinetics and mechanism of the barotropic lamellar gel/lamellar liquid crystal phase transition in fully hydrated dihexadecylphosphatidylethanolamine: a time-resolved x-ray diffraction study using pressure jump.

The kinetics and mechanism of the barotropic lamellar gel (L beta')/lamellar liquid crystal (L alpha) phase transition in fully hydrated 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) has been studied using time-resolved x-ray diffraction (TRXRD). The phase transition was induced by pressure jumps of varying amplitudes in both the pressurization and depressurization directions at controlled temperature (78 degrees C). Both low- and wide-angle diffracted x rays were recorded simultaneously in live time using an x-ray-sensitive image intensifier coupled to a CCD camera and Super-VHS videotape recorder. Such an arrangement allowed for the direct and quantitative characterization of the long- (lamellar repeat spacing) and short-range order (chain packing) during a kinetic experiment. The image-processed live-time x-ray diffraction data were fitted using a nonlinear least-squares model, and the parameters of the fits were monitored continuously throughout the transition. The pressure-induced transitions from the L alpha to the L beta' phase and from the L beta' to the L alpha phase was two-state (no formation of intermediates apparent during the transition) to within the sensitivity limits of the method. The corresponding transit time (the time during which both phases coexist) associated with the long- and short-range order of the pressurization-induced L alpha-to-L beta' phase transition decreased to a limiting value of approximately 50 ms with increasing pressure jump amplitude. This limiting value was close to the response time of the detector/recording system. Thus, the intrinsic transit time of this transition in fully hydrated DHPE at 78 degrees C was less than or equal to 50 ms. In contrast, the depressurization-induced L beta'-to-L alpha phase transition was slower, taking approximately 1 s to complete, and occurred with no obvious dependence of the transit time on pressure jump amplitude. In the depressurization jump experiment, the lipid responded rapidly to the pressure jump in the L beta' phase up to the rate-determining L beta'-to-L alpha transition. Such behavior was examined carefully, as it could complicate the interpretation of phase transition kinetic measurements.     

Voltage clamp study of fast excitatory synaptic currents in bullfrog sympathetic ganglion cells

Excitatory postsynaptic currents (EPSCs) have been studied in voltage- clamped bullfrog sympathetic ganglion B cells. The EPSC was small, rose to a peak within 1-3 ms, and then decayed exponentially over most of its time-course. For 36 cells at --50 mV (21-23 degrees C), peak EPSC size was --6.5 +/- 3.5 nA (mean +/- SD), and the mean decay time constant tau was 5.3 +/- 0.9 ms. tau showed a small negative voltage dependence, which appeared independent of temperature, over the range -- 90 to --30 mV; the coefficient of voltage dependence was --0.0039 +/- 0.0014 mV-1 (n = 29). The peak current-voltage relationship was linear between --120 and --30 mV but often deviated from linearity at more positive potentials. The reversal potential determined by interpolation was approximately --5 mV. EPSC decay tau had a Q10 = 3. The commonly used cholinesterase inhibitors, neostigmine and physostigmine, exhibited complex actions at the ganglia. Neostigmine (1 X 10(-5)M) produced a time-dependent slowing of EPSC decay without consistent change in EPSC size. In addition, the decay phase often deviated from a single exponential function, although it retained its negative voltage dependence. With 1 x 10(-6) M physostigmine, EPSC decay was slowed by the decay phase remained exponential. At higher concentrations of physostigmine, EPSC decay was markedly prolonged and was composed of at least two decay components. High concentrations of atropine (10(-5) to 10(-4) M) produced complex alterations in EPSC decay, creating two or more exponential components; one decay component was faster and the other was slower than that observed in untreated cells. These results suggest that the time-course of ganglionic EPSC decay is primarily determined by the kinetics of the receptor-channel complex rather than hydrolysis or diffusion of transmitter away from the postsynaptic receptors.     

Rapid relaxation of single frog skeletal muscle fibres following laser flash photolysis of the caged calcium chelator, diazo-2

Diazo-2 is a calcium chelator based on BAPTA [(1989) J. Biol. Chem., in press], whose electron withdrawing diazoacetyl group may be rapidly (2000 s-1) converted photochemically to an electron donating carboxymethyl group by exposure to near ultraviolet light, producing an increase in its calcium affinity (Kd changes from 2.2 microM to 0.073 microM) without steric modification of the metal binding site. Photolysis of a 2 mM solution of this compound with a brief flash of light from a frequency-doubled ruby laser (347 nm) caused single skinned muscle fibres from the semitendinosus muscle of the frog Rana temporaria to relax with a mean half-time of 60.4 +/- 5 ms (range 30-100 ms, n = 15) at 12 degrees C, which is faster than the relaxation observed in intact muscles (half-time 133 ms at 14 degrees C [(1986) J. Mol. Biol. 188, 325-342]) and similar to the rate of the fast phase of tension decay in intact single fibres (20 s-1 at 10 degrees C [(1982) J. Physiol. 329, 1-20]).     

Nanosecond temperature jump and time-resolved Raman study of thermal unfolding of ribonuclease A

A nanosecond temperature jump (T-jump) apparatus was constructed and combined with time-resolved Raman measurements to investigate thermal unfolding of a protein for the first time. The 1.56-microm heat pulse with 9 ns width at 10 Hz was obtained through the two-step stimulated Raman scattering in D(2) gas involving seeding and amplification. To achieve uniform temperature rise, the counter-propagation geometry was adopted for the heat pulse. The temperature rise was determined by anti-Stokes to Stokes intensity ratios of the 317 and 897 cm(-1) bands of MoO(4)(2-) ions in an aqueous solution. The T-jump as large as 9 degrees C in 10 ns was attained. The unfolding of bovine pancreatic ribonuclease A was monitored with time-resolved Raman spectra excited at 532 nm. The C-S stretching band of Met residues exhibited 10% change of that expected from the stationary state temperature-difference spectra in the initial 200 ns following T-jump and another 10% in 5 ms. The Raman intensity of SO(4)(2-) ions around 980 cm(-1) increased at 100 micros, presumably due to some conformational changes of the protein around the active site. The S-S stretches and tyrosine doublet displayed little changes within 5 ms. Thus, the conformational changes in the initial step of unfolding are not always concerted.     

Rapid reversible formation of a metastable subgel phase in saturated diacylphosphatidylcholines.

Formation of well ordered lamellar subgel (SGII) phase in aqueous dispersions of L-dipalmitoylphosphatidylcholine upon cooling from the lamellar gel phase, without low-temperature equilibration, is observed in real time using synchrotron x-ray diffraction. It has the same lamellar repeat period as the gel phase from which it was formed but differs in its wide-angle diffraction pattern. The SGII phase forms at about 7 degrees C upon cooling at 2 degrees C/min. In temperature jump experiments at 1 degree C/s from 50 to -5 degrees C, the relaxation time of the lamellar gel-SGII transition is found to be approximately 15 s. The conversion between the lamellar gel and SGII phase is cooperative and rapidly reversible. Upon heating, it coincides in temperature with an endothermic event with a calorimetric enthalpy of 0.35 kcal/mol, the so-called sub-subtransition. Similar sub-subtransitions are also observed calorimetrically at temperatures approximately 10 degrees C below the subtransition, without low-temperature storage, in aqueous dispersions of L-dimyristoylphosphatidylcholine and L-distearoylphosphatidylcholine, but not in racemic DL-dipalmitoylphosphatidylcholine. The formation of the equilibrium lamellar crystalline Lc phase appears to take place only from within the SGII phase.     

Aerobic power as a factor in women's response to work in hot environments.

Twelve young women, athletes (n = 6) and nonathletes (n = 6), walked on a treadmill at loads equivalent to approximately 30% Vo2 max for two 50-min periods in three environments: 1) 28 degrees C, 45% rh, 2) 35 degrees C, 65% rh, and 3) 48 degrees C, 10% rh. There were no differences between groups in rectal temperature, heart rate, evaporative heat loss, or mean skin temperature at 28 or 35 degrees C or during the first work period in the 48 degrees C environment. However, a significantly lower cardiac output (Q) and stroke volume (SV) observed for nonathletes by the 46th min of work at 48 degrees C may explain why no nonathletes were able to complete a 2nd h of work while four of six athletes successfully finished the period. It appears that in conditions of severe heat stress (48 degrees C) athletes were able to maintain a cardiac output sufficient to meet the metabolic requirements and the large increase in peripheral blood flow for a longer period of time than nonathletes.     

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.     

Control of sweating during the human menstrual cycle

Thermoregulatory responses were studied in seven women during two separate experimental protocols in the follicular (F, days 4-7) phase and during the luteal (L, days 19-22) phase of the menstrual cycle. Continuous measurements of esophageal temperature (Tes), mean skin temperature (Tsk), oxygen uptake and forearm sweating (ms) were made during all experiments. Protocol I involved both passive heat exposure (3 h) and cycle exercise at approximately 80% VO2 peak during which the environmental chamber was controlled at Ta = 50.0 degrees C, rh = 14% (Pw = 1.7 kPa). In protocol II subjects were tested during thirty-five minutes of exercise at approximately 85% VO2 peak at Ta = 35 degrees C and rh = 25% (Pw = 1.4 kPa). The normal L increase in resting Tes (approximately 0.3 degrees C) occurred in all seven subjects. Tsk was higher during L than F in all experiments conducted at 50 degrees C. During exercise and passive heat exposure, the Tes threshold for sweating was higher in L, with no change in the thermosensitivity (slope) of ms to Tes between menstrual cycle phases. This rightward or upward shift in Tes threshold for initiation of sweating averaged 0.5 degrees C for all experiments. The data indicate the luteal phase modulation in the control of sweating in healthy women is also apparent during severe exercise and/or heat stress.     

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.     

Time-resolved changes in equatorial x-ray diffraction and stiffness during rise of tetanic tension in intact length-clamped single muscle fibers.

We report the first time-resolved x-ray diffraction studies on tetanized intact single muscle fibers of the frog. The 10, 11, 20, 21, 30, and Z equatorial reflections were clearly resolved in the relaxed fiber. The preparation readily withstood 100 1-s duration (0.4-s beam exposure) tetani at 4 degrees C (less than 4% decline of force and no deterioration in the 10, 11 equatorial intensity ratio at rest or during activation). Equatorial intensity changes (10 and 11) and fiber stiffness led tension (t1/2 lead 20 ms at 4 degrees C) during the tetanus rise and lagged during the isometric phase of relaxation. These findings support the existence of a low force cross-bridge state during the rise of tetanic tension and isometric relaxation that is not evident at the tetanus plateau. In "fixed end" tetani lattice expansion occurred with a time course similar to stiffness during the tetanus rise. During relaxation, lattice spacing increased slightly, while the sarcomere length remained isometric, but underwent large changes after the "shoulder" of tension. Under length clamp control, lattice expansion during the tetanus rise was reduced or abolished, and compression (2%) of the lattice was observed. A lattice compression is predicted by certain cross-bridge models of force generation (Schoenberg, M. 1980. Biophys. J. 30:51-68; Schoenberg, M. 1980. Biophys. J. 30:69-78).     

Multiple folding pathways for the P4-P6 RNA domain

We recently described site-specific pyrene labeling of RNA to monitor Mg(2+)-dependent equilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescence with approximately 1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allows high-yield automated synthesis of oligoribonucleotides with pyrene incorporated at a specific 2'-amino-2'-deoxyuridine residue. P4-P6 forms its higher-order tertiary structure rapidly, with k(obs) = 15-31 s(-1) (t(1/2) approximately 20-50 ms) at 35 degrees C and [Mg(2+)] approximately 10 mM in Tris-borate (TB) buffer. The folding rate increases strongly with temperature from 4 to 45 degrees C, demonstrating a large activation enthalpy DeltaH(double dagger) approximately 26 kcal/mol; the activation entropy DeltaS(double dagger) is large and positive. In low ionic strength 10 mM sodium cacodylate buffer at 35 degrees C, a slow (t(1/2) approximately 1 s) folding component is also observed. The folding kinetics are both ionic strength- and temperature-dependent; the slow phase vanishes upon increasing [Na(+)] in the cacodylate buffer, and the kinetics switch completely from fast at 30 degrees C to slow at 40 degrees C. Using synchrotron hydroxyl radical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radical cleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionic strength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiary interactions are present even without Mg(2+) in the initial state. The fast kinetic phase reflects folding that is facilitated by these interactions, whereas the slow kinetics are observed when these interactions are disrupted at lower ionic strength and higher temperature.