Effect of temperature on Mg-ITP-induced superprecipitation of actomyosin

Superprecipitation of an actomyosin suspension was measured at various temperatures (2.5 degrees - 20 degrees) using Mg-ITP as substrate. Superprecipitation was induced by the addition of Mg-ITP at all temperatures, but decreased in extent with decrease in temperature. The predominant intermediate in the Mg-ITP hydrolysis of myosin depends on the temperature; at 20 degrees it is the myosin-IDP-Pi complex, while below 8 degrees it is the myosin-ITP complex (Hozumi, T. (1976) Eur. J. Biochem. 63, 241). Therefore, the occurrence of superprecipitation below 8 degrees is not compatible with muscle models in which formation of a myosin-product complex is the rate-limiting intermediate.     

Separation of myosin subfragment 1 into two fractions, one having the burst site and the other having the non-burst site.

During Mn(II)-ATP hydrolysis by myosin, the predominant intermediate formed at the burst site of the enzyme below 10 degrees is the myosin-ADP complex formed by adding ADP to myosin, while above 10 degrees it is the myosin -ADP-P1 complex generated by ATP hydroolysis (Yazawa, Morita, & Yagi (1973) J. Biochem. 74, 1107; Hozumi & Tawada (1975) Biochim. Biophys. Acta 376, 1; Tawada & Yoshida (1975) J. Biochem. 78, 293). It is suggested that the second (non-burst) site of myosin predominantly forms the myosin-ATP complex (Hozumi & Tawada, ibid.). From these findings, it is expected that (i) myosin subfragment 1 (S1) having the burst site is bound to actin in Mn(II)-ATP solution containing ADP below 10 degrees, because it forms the S1-ADP complex even in the presence of ATP; (ii) the other S1, i.e., that having the non-burst site, is dissociated from actin, because it forms the S1-ATP complex. These two expectations were confirmed by viscosity measurements of acto-S1 solutions, giving a basis for the separation of S1 into two fractions: one having the burst site and the other having the non-burst site. S1 having the non-burst site could be extracted from partially papain [EC 3.4.22.2]-digested myofibrils of rabbit skeletal muscle with a solution containing MnCl2, ATP, and ADP at 0 degrees. S1 having the burst site was extracted from myofibrils already used for the extraction of S1 having the non-burst site, with a solution containing MgCl2 and ATP at 20 degrees. The former S1 fraction had Mg-ATPase [EC 3.6.1.3] activity, but scarcely showed any initial burst of Pi liberation. The latter S1 showed a Pi burst of more than 0.5 (M/M). The steady state ATPase activity of the former S1 was slightly higher than that of the latter. The burst size of normal S1, i.e., that extracted from papain-digested myofibrils with Mg-PPi or Mg-ATP, was 0.5 (M/M). The ultraviolet absorption spectrum of the non-burst type S1 was not changed by ADP but was changed by ATP, though the difference spectrum was distinct from that of normal S1 and the difference molar extinction coefficient at 289 nm was only 20% of that of normal S1. No significant difference was seen in the compositions of these two S1's and normal S1, as determined by SDS gel electrophoresis.     

Temperature-dependent transitions of the myosin-product intermediate at 10 degrees during Mn(II)-ATP hydrolysis by myosin from rabbit psoas muscle.

The initial burst of Pi liberation during the hydrolysis of Mn(II)-ATP by heavy meromyosin from rabbit psoas muscle was investigated. Below 10 degrees, the initial burst of Pi liberation was inhibited by the pre-addition of ADP without any change in the steady-state activity, but it was not inhibited above 10 degrees. The burst size was about one mole per mole of heavy meromyosin. The initial burst of Pi liberation in Mg-ATP hydrolysis at 8 degrees, however, was not inhibited by the pre-addition of ADP. These results, obtained with psoas muscle heavy meromyosin, were almost the same as those obtained with heavy meromyosin from rabbit leg and back muscles (Hozumi and Tawada (1975) Biochim. Biophys. Acta 376, 1-12) and, therefore, indicate that in Mn-ATP above 10 degrees there is at the burst site a predominant myosin -product complex generated by ATP hydrolysis. Similarly, below 10 degrees there is a myosin-product complex identical with the one generated by adding ADP (and Pi) to myosin.     

Temperature-dependent change in rate-limiting step of the magnesium-stimulated ITPase of myosin.

The effects of temperature on Mg-ITPase activity of heavy meromyosin and myosin subfragment 1 were measured in 0.1 M KC1. The initial burst of Pi liberation was one mol per mol of heavy meromyosin or two mol of myosin subfragment 1, i.e. one mol per two mol of myosin active sites, at 20 degrees C. However, it was almost zero mol below 8degrees C. Effects of KC1 concentration and pH on ITPase activity of heavy meromyosin at 20 degrees C were different from those below 8 degrees C, suggesting that the rate-limiting step in the Mg-ITP hydrolysis of myosin depends on temperature. The effect of temperature on the actin activation of heavy meromyosin Mg-ITPase was analyzed by measuring the temperature dependence of double-reciprocal plots of ITPase activity against actin concentration. The extent of actin activation was larger at low temperture. The results presented in this paper might be explained by assuming the existence of two kinds of active sites on a myosin molecule.     

Preparation of a myosin-extracted "ghost" myofibril Sephadex conjugate column and its application to the separation of myosin subfragment-1 giving and not giving the initial burst of phosphate.

A "ghost" myofibril (myosin-extracted myofibril) Sephadex conjugate which specifically binds myosin, HMM and S-1 in the absence of Mg-ATP or Mg-PP can be prepared in a few days by conjugating "ghost" myofibrils to Sephadex beads. Binding ability is retained for over a month. It is used, therefore, for actin-affinity chromatography of myosin and its active fragments. It is under debate whether the two heads of the myosin molecule are functionally identical. Recently several reports have indicated that S-1 could be separated into two kinds of S-1, one giving the initial burst of phosphate and the other not, by assuming a difference in the affinity of the two kinds of S-1 to F-actin. Attempts are reported here to obtain these two components of S-1 separately by using the "ghost" myofibril Sephadex conjugate column. The method of S-1 separation reported by Shibata-Sekiya and Tonomura ((1976) J. Biochem, 80, 1371-1380), which used S-1 treated with CMB, was applied to the "ghost" myofibril Sephadex conjugate column. This resulted in the successful separation of S-1 modified with CMB giving no initial burst of phosphate and unmodified S-1 giving the initial burst of phosphate. A separation method based essentially on the principle employed by Taniguichi and Tawada ((1976) J. Biochem. 80, 853-860) gave an unsuccessful result.     

Temperature and ligand dependence of conformation and helical order in myosin filaments

Mammalian myosin filaments are helically ordered only at higher temperatures (>20 degrees C) and become progressively more disordered as the temperature is decreased. It had previously been suggested that this was a consequence of the dependence of the hydrolytic step of myosin ATPase on temperature and the requirement that hydrolysis products (e.g., ADP.P(i)) be bound at the active site. An alternative hypothesis is that temperature directly affects the conformation of the myosin heads and that they need to be in a particular conformation for helical order in the filament. To discriminate between these two hypotheses, we have studied the effect of temperature on the helical order of myosin heads in rabbit psoas muscle in the presence of nonhydrolyzable ligands. The muscle fibers were overstretched to nonoverlap such that myosin affinity for nucleotides was not influenced by the interaction of myosin with the thin filament. We show that with bound ADP.vanadate, which mimics the transition state between ATP and hydrolysis products, or with the ATP analogues AMP-PNP or ADP.BeF(x)() the myosin filaments are substantially ordered at higher temperatures but are reversibly disordered by cooling. These results reinforce recent studies in solution showing that temperature as well as ligand influence the equilibrium between multiple myosin conformations [Málnási-Csizmadia, A., Pearson, D. S., Kovács, M., Woolley, R. J., Geeves, M. A., and Bagshaw, C. R. (2001) Biochemistry 40, 12727-12737; Málnási-Csizmadia, A., Woolley, R. J., and Bagshaw, C. R. (2000) Biochemistry 39, 16135-16146; Urbanke, C., and Wray, J. (2001) Biochem. J. 358, 165-173] and indicate that helical order requires the myosin heads to be in the closed conformation. Our results suggest that most of the heads in the closed conformation are ordered, and that order is not produced in a separate step. Hence, helical order can be used as a signature of the closed conformation in relaxed muscle. Analysis of the dependence on temperature of helical order and myosin conformation shows that in the presence of these analogues one ordered (closed) conformation and two disordered conformations with distinct thermodynamic properties coexist. Low temperatures favor one disordered conformation, while high temperatures favor the ordered (closed) conformation together with a second disordered conformation.     

The effects of ionic conditions, temperature, and chemical modification on the fluorescence of myosin during the steady state of ATP hydrolysis. A comparison of the fluorescnece and electron spin resonance spectra of the spin-labeled enzyme.

The ATP-induced enhancement of the intrinsic fluorescence of myosin and heavy meromyosin (HMM) that persists during the steady state of hydrolysis has been investigated. To compare the substrate-induced changes in fluorescence with those in the electron spin resonance spectrum of the spin-labeled enzyme, we studied the influence of temperature, pH, and ionic strength, as well as the effect of chemical modification (spin labeling) of the SH-1 sulfhydryl groups. Changing the pH between 6 and 9 does not affect the enhancement of fluorescence of myosin or HMM; changing the ionic strength, which could be studied only with HMM, also has no effect; and decreasing the temperature from 20 to 5 degrees slightly diminishes the enhancement with both myosin and HMM. Chemical modification with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, which blocks the SH-1 thiol groups, reduces the enhancement of fluorescence, induces a strong dependence on ionic strength and pH, and substantially increases the dependence on temperature. The enhancement with labeled myosin or labeled HMM increases with increasing pH, ionic strength, and temperature, closely paralleling the effects of these parameters on the electron spin resonance spectrum of spin-labeled myosin (SEIDEL, J.C. and GERGELY, J. (1973) Arch. Biochem. Biophys. 158, 853), suggesting that the same molecular change, induced by ATP and associated with formation of the MADP-P1 complex, underlies both the change in fluorescence and the change in ESR spectrum. Those analogues of ATP that produce the maximal enhancement of fluorescence (WERBER, M., SZENT-GYORGYL, A.G., and FASMAN, G. (1972) Biochemistry 11, 2872) also produce the maximal change in the ESR spectra. Both an amino group at position 6 of the substrate and an unmodified triphosphate chain are required for maximal change in either fluorescence or ESR spectra. The smaller enhancement of fluorescence produced by spin labeling the SH-1 groups persists after the nitroxide has been chemically changed to a diamagnetic species. Thus the small enhancement cannot be attributed to paramagnetic quenching of tryptophan fluorescence by the spin label. An initial burst of phosphate liberation accompanies the hydrolysis of ATP, cytidine 5'-triphosphate, uridine 5'-triphosphate, guanosine 5'-tryphosphate, iosine 5'-triphosphate, 2'-deoxyadenosine 5'-tryphosphate, adenosine 5'-tetraphosphate, and tripolyphosphate. The presence or absence of the burst does not correlate with the extent of the spectral change.     

Magnesium ion dependent adenosine triphosphatase activity of heavy meromyosin as a function of temperature between +20 and -15 degrees C.

The hydrolysis of Mg2+-adenosine 5'-triphosphate (ATP) by heavy meromyosin has been studied between +20 and -15 degrees C, especially in the low-temperature range, in a medium containing 30% (v/v) ethylene glycol by fluorometric, spectrophotometric, and potentiometric measurements. The time course of the fluorescence changes of the enzyme during the reaction depends markedly on the temperature in consequence of large differences between the activation energies of the various steps. The observed kinetics have been analyzed according to the simplified scheme of Bagshaw & Trentham [Bagshaw, C. R., & Trentham, D. R. (1974) Biochem. J. 141, 331-349]. The following results have been obtained. (1) The rate-limiting step of the reaction changes in this temperature range; at 20 degrees C M**.ADP.Pi is the predominant steady-state complex, and M*.ADP predominates at -15 degrees C, with a half-life of approximately 10 min. (2) As expected, on the basis that it is the dissociation of the M*.ADP complex which becomes rate limiting at low temperature, one observes, in the pre-steady-state below 0 degrees C, both a proton burst and a lag phase in ADP release. (3) At low temperature, the equilibrium M*.ATP in equilibrium M**.ADP.Pi is displaced to the left All the kinetic data obtained in this study are compatible with a simple pathway for the Mg2+-ATP hydrolysis by myosin and with sequential release of the reaction products.     

Interaction of a spin-labeled analog of ATP with myosin

By means of spin labeled analogs of ATP we have shown that conformational changes in myosin molecule induced by variation of temperature take place in the region of the active centre. In case of Mg-ATP and unmodified myosin conformation of the active centre changes monotonously with the change in temperature but after the modification of S1 thiol groups by N-ethylmaleimide on the temperature dependence curve of rotational mobility of the spin label a discontinuous is observed at 14-16 degrees C. It is also observed in case of K+-EDTA-ATP, or Ca2+-ATP and unmodified myosin. It is shown that the chemical analogs of Mg2+-paramagnetic ion Mn2+ are directly connected with the myosin active centre in the presence of ATP(ADP), i. e. a triple complex enzyme-bivalent cation-substrate is formed.     

Temperature-dependence of isometric tension and cross-bridge kinetics of cardiac muscle fibers reconstituted with a tropomyosin internal deletion mutant

The effect of temperature on isometric tension and cross-bridge kinetics was studied with a tropomyosin (Tm) internal deletion mutant AS-Delta23Tm (Ala-Ser-Tm Delta(47-123)) in bovine cardiac muscle fibers by using the thin filament extraction and reconstitution technique. The results are compared with those from actin reconstituted alone, cardiac muscle-derived control acetyl-Tm, and recombinant control AS-Tm. In all four reconstituted muscle groups, isometric tension and stiffness increased linearly with temperature in the range 5-40 degrees C for fibers activated in the presence of saturating ATP and Ca(2+). The slopes of the temperature-tension plots of the two controls were very similar, whereas the slope derived from fibers with actin alone had approximately 40% the control value, and the slope from mutant Tm had approximately 36% the control value. Sinusoidal analysis was performed to study the temperature dependence of cross-bridge kinetics. All three exponential processes A, B, and C were identified in the high temperature range (30-40 degrees C); only processes B and C were identified in the mid-temperature range (15-25 degrees C), and only process C was identified in the low temperature range (5-10 degrees C). At a given temperature, similar apparent rate constants (2pia, 2pib, 2pic) were observed in all four muscle groups, whereas their magnitudes were markedly less in the order of AS-Delta23Tm < Actin < AS-Tm approximately Acetyl-Tm groups. Our observations are consistent with the hypothesis that Tm enhances hydrophobic and stereospecific interactions (positive allosteric effect) between actin and myosin, but Delta23Tm decreases these interactions (negative allosteric effect). Our observations further indicate that tension/cross-bridge is increased by Tm, but is diminished by Delta23Tm. We conclude that Tm affects the conformation of actin so as to increase the area of hydrophobic interaction between actin and myosin molecules.     

Extraction and functional reformation of thick filaments in chemically skinned molluscan catch muscle fibers.

A method for the almost complete extraction of myosin from smooth muscle fibers of the anterior byssal retractor muscle (ABRM) of Mytilus edulis was developed, and functional reformation of thick filaments in the fibers was achieved. Complete removal of myosin from the glycerol-extracted ABRM fibers with a solution containing 600 mM KCl, 5 mM MgCl2, and 5 mM ATP was difficult. However, successive treatments of the ABRM fibers with glycerol and saponin made the plasma membrane permeable to Mg-ATP and myosin. The extraction of myosin completely eliminated the tension induced by the addition of Mg-ATP. Partial recovery of tension development was observed by irrigation of myosin into fibers from which myosin had been extracted. Similar results were obtained using rabbit myosin instead of ABRM myosin. Addition of heavy meromyosin, on the other hand, had a suppressive effect on the tension development, as is the case in glycerinated rabbit psoas muscle fibers.     

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

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

Oxygen exchange reaction during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments

The oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two Pi species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20 degrees C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KCl. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KCl at 20 degrees C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissociation of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

The penicillin-binding site in the exocellular DD-carboxypeptidase-transpeptidase of Actinomadura R39.

Heat denaturation and Pronase degradation of the complex previously formed between benzylpenicillin and the exocellular DD-carboxypeptidase-transpeptidase of Actinomadura R39 yields a heptapeptide H-Leu-Pro-Ala-Ser-Asn-Gly-Val-OH, where the benzylpenicilloyl group is ester-linked to the serine residue. This linkage is very labile and its hydrolysis causes the release of benzylpenicilloate. In contrast, the native benzylpenicilloyl-enzyme complex is very stable (half-life 70 h at 37 degrees C) and its breakdown proceeds via fragmentation of the bound benzylpenicilloyl group [Fuad, Frère, Ghuysen, Duez & Iwatsubo (1976) Biochem. J. 155, 623-629].     

Transient phase of adenosine triphosphate hydrolysis by myosin, heavy meromyosin, and subfragment 1.

The transient phase of adenosine triphosphate (ATP) hydrolysis (early burst) was investigated for myosin, heavy meromyosin (HMM), and subfragment 1 (S-1) over a range of temperatures and pH's. The burst size at pH 8,20 degrees C, is 0.8-0.85, based on steady-state and transient measurements. The equilibrium constant for the enzyme-substrate to enzyme-product transition is 0.85 +/- 0.05. It is concluded that both myosin heads undergo the rapid hydrolysis step and that there are no significant differences for S-1 vs. HMM or myosin. The transient data are fitted reasonably well by a single rate process, but available evidence is consistent with some heterogeneity and a range of rate constants differing by a factor of two. At pH 6.9 and 3 degrees C, the burst size is 0.5 and the hydrolysis is slower than the configuration change measured by fluorescence. The results are consistent with the kinetic scheme (see article). The lower burst at low temperature and pH can be partly explained by a reduction in the equilibrium constant, K3, and ATP can be synthesized on the enzyme by a pH-temperature jump.     

ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition

Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects of varying load and shortening velocity during contraction were investigated. The myosin isoform composition was determined in each fiber by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At 12 degrees C large variations (three- to fourfold) were found between slow and fast (2A and 2A-2B) fibers in their maximum shortening velocity, peak power output, velocity at which peak power is produced, isometric ATPase activity, and tension cost. Isometric tension was similar in all fiber groups. The ATP consumption rate increased during shortening in proportion to shortening velocity. At 12 degrees C the maximum efficiency was similar (0.21-0.27) for all fiber types and was reached at a higher speed of shortening for the faster fibers. In all fibers, peak efficiency increased to approximately 0.4 when the temperature was raised from 12 degrees C to 20 degrees C. The results were simulated with a kinetic scheme describing the ATPase cycle, in which the rate constant controlling ADP release is sensitive to the load on the muscle. The main difference between slow and fast fibers was reproduced by increasing the rate constant for the hydrolysis step, which was rate limiting at low loads. Simulation of the effect of increasing temperature required an increase in the force per cross-bridge and an acceleration of the rate constants in the reaction pathway.     

Kinetics and thermodynamics of the assembly of the parallel- and antiparallel-packed sections of synthetic thick filaments of skeletal myosin: a pressure-jump study

Earlier work on the length regulation mechanism of synthetic myosin filaments generated at pH 8.2 showed the process to be mediated through the dissociation rate constant which had an increasing and apparently monophasic exponential dependence on filament length and an association rate constant that was length independent, filament growth ceasing at the point of equilibrium [Davis, J.S. (1981) Biochem. J. 197, 309-314]. In this work, the exponential dependence of the dissociation rate constant on thick filament length was shown to be more complex than originally thought. Two phases were resolved, one of which correlated with the dissociation of parallel-packed myosin and the other with that of antiparallel-packed material. The pressure dependence of the dissociation reaction for the parallel-packed material showed that the activation volume decreased linearly with length while the Gibbs energy increased. This was interpreted as indicating that the weakening of the interaction between dimer and filament with length was accompanied by a decrease in the extent of ionic bonding. The case in the antiparallel-packed region was quite different, with the activation volume and the Gibbs energy both increasing linearly. The contribution from ionic bonding thus rises counter to the change in Gibbs energy, presumably at the expense of other noncovalent interactions. The relationship between the synthetic thick filaments and their in vivo counterparts is also considered in some detail.     

Production of nitrite ions from trinitrophenyl myosin and from trinitrophenyl subfragment-1

When trinitrophenyl (TNP) myosin of either chicken breast muscle or porcine cardiac muscle was left to stand in an alkaline medium at 20 degrees C for several hours, nitrite ions were found to be gradually produced. The nitrite production from myosin trinitrophenylated in the presence of PPi occurred at the same rate and to the same extent as that from myosin trinitrophenylated in the absence of PPi. The nitrite production was significantly reduced when thiols of myosin were modified with 2-nitro-5-thiocyanobenzoate. Four different preparations of TNP subfragment-1, S1(Aa), S1(Ab), S1(Ba), and S1(Bb), were obtained from chymotryptic digest of chicken breast myosin trinitrophenylated in the absence of PPi. When these preparations of TNP-S1 were left to stand at alkaline pH, a significant amount of nitrite was produced from S1(Ab) and S1(Bb), but very little from S1(Aa) and S1(Ba). In our previous report (J. Biochem. 97, 965-968, 1985), S1(Aa) and S1(Ba) were suggested to correspond to "non-burst" heads of myosin, and S1(Ab) and S1(Bb) to "burst" heads of the myosin molecule (Inoue et al. (1980) Adv. Biophys. 13, 1-194). Therefore, the present findings described above strongly suggest that the nitrite production involves some interaction of TNP groups with thiols, and that it occurs at the "burst" heads.     

A transient-kinetic study of the nitrogenase of Klebsiella pneumoniae by stopped-flow calorimetry. Comparison with the myosin ATPase.

The pre-steady-state kinetics of MgATP hydrolysis by nitrogenase from Klebsiella pneumoniae were studied by stopped-flow calorimetry at 6 degrees C and at pH 7.0. An endothermic reaction (delta Hobs. = +36 kJ.mol of ATP-1; kobs. = 9.4 s-1) in which 0.5 proton.mol of ATP-1 was released, has been assigned to the on-enzyme cleavage of MgATP to yield bound MgADP + Pi. The assignment is based on the similarity of these parameters to those of the corresponding reaction that occurs with rabbit muscle myosin subfragment-1 (delta Hobs. = +32 kJ.mol of ATP-1; kobs. = 7.1 s-1; 0.2 proton released.mol of ATP-1) [Millar, Howarth & Gutfreund (1987) Biochem. J. 248, 683-690]. MgATP-dependent electron transfer from the nitrogenase Fe-protein to the MoFe-protein was monitored by stopped-flow spectrophotometry at 430 nm and occurred with kobs. value of 3.0 s-1 at 6 degrees C. Thus, under these conditions, hydrolysis of MgATP precedes electron transfer within the protein complex. Evidence is presented that suggests that MgATP cleavage and subsequent electron transfer are reversible at 6 degrees C with an overall equilibrium constant close to unity, but that, at 23 degrees C, the reactions are essentially irreversible, with an overall equilibrium constant greater than or equal to 10.