Ca2+-sensitive cross-bridge dissociation in the presence of magnesium pyrophosphate in skinned rabbit psoas fibers.

We find that at 6 degrees C in the presence of 4 mM MgPPi, at low or moderate ionic strength, skinned rabbit psoas fibers exhibit a stiffness and an equatorial x-ray diffraction pattern similar to that of rigor fibers. As the ionic strength is increased in the absence of Ca2+, both the stiffness and the equatorial x-ray diffraction pattern approach those of the relaxed state. This suggests that, as in solution, increasing ionic strength weakens the affinity of myosin cross-bridges for actin, which results in a decrease in the number of cross-bridges attached. The effect is Ca2+-sensitive. Assuming that stiffness is a measure of the number of cross-bridge heads attached, in the absence of Ca2+, the fraction of attached cross-bridge heads varies from approximately 75% to approximately 25% over an ionic strength range where ionic strength in solution weakens the binding constant for myosin subfragment-1 binding to unregulated actin by less than a factor of 3. Therefore, this phenomenon appears similar to the cooperative Ca2+-sensitive binding of S1 to regulated actin in solution (Greene, L. E., and E. Eisenberg, 1980, Proc. Natl. Acad. Sci. USA, 77:2616). By comparing the binding constants in solution and in the fiber under similar conditions, we find that the "effective actin concentration," that is, the concentration that gives the same fraction of S1 molecules bound to actin in solution as cross-bridge heads are bound to actin in a fiber, is in the millimolar range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnesium ion-dependent contraction of skinned frog muscle fibers in calcium-free solution

Skinned frog fibers were reversibly activated in Ca-free solutions containing 0 mM KCl, 23 microM free Mg, and having an ionic strength of approximately 50 mM. Contractile force was nearly maximal at 22 degrees - 25 degrees C and decreased at lower temperatures. Maximal force in Ca-free solution at 50 mM ionic strength was close to twice the calcium-activated force with pCa 5 and 190 mM ionic strength. The force in Ca-free solution could be reduced to zero by raising the concentration of free Mg from 23 microM to 1.0 mM at the same ionic strength (50 mM). On stretching the fiber from 2.0 to 3.2 micron the force decreased; this effect was similar to that seen with Ca-activated fiber and the data support the idea that Ca-free tension is made at the cross-bridge level. Isotonic contraction during Ca-free activation showed a velocity transient as in Ca-activated fiber at 190 mM ionic strength, but the transient in the present case was very much prolonged. This finding suggests that contraction mechanisms for force generation and for shortening are essentially the same in the two conditions, but that certain rate constants of cross-bridge turnover are slower for the Ca-free contraction. Also, the results indicate that, in low ionic strength, Ca binding to thin filaments is not essential for unmasking the cross-bridge attachment sites, which suggests that the steric blocking mechanism is modified under these conditions.     

Characterization of the myosin adenosine triphosphate (M.ATP) crossbridge in rabbit and frog skeletal muscle fibers.

In the presence of ATP and absence of Ca2+, muscle crossbridges have either MgATP or MgADP.Pi bound at the active site (S. B. Marston and R. T. Tregear, Nature [Lond.], 235:22:1972). The behavior of these myosin adenosine triphosphate (M.ATP) crossbridges, both in relaxed skinned rabbit psoas and frog semitendinosus fibers, was analyzed. At very low ionic strength, T = 5 degrees C, mu = 20 mM, these crossbridges spend a large fraction of the time attached to actin. In rabbit, the attachment rate constants at low salt are 10(4) - 10(5) s-1, and the detachment rate constants are approximately 10(4) s-1. When ionic strength is increased up to physiological values by addition of 140 mM potassium propionate, the major effect is a weakening of the crossbridge binding constant approximately 30-40-fold. This effect occurs because of a large decrease, approximately 100-fold, in the crossbridge attachment rate constants. The detachment rate constants decrease only 2-3-fold. The effect of ionic strength on crossbridge binding in the fiber is very similar to the effect of ionic strength on the binding of myosin subfragment-1 to unregulated actin in solution. Thus, the effect of increasing ionic strength in fibers appears to be a direct effect on crossbridge binding rather than an effect on troponin-tropomyosin. The finding that crossbridges with ATP bound at the active site can and do attach to actin over a wide range of ionic strengths strongly suggests that troponin-tropomyosin keeps a muscle relaxed by blocking a step subsequent to crossbridge attachment. Thus, rather than troponin-tropomyosin serving to keep a muscle relaxed by inhibiting attachment, it seems quite possible that the main way in which troponin-tropomyosin regulates muscle activity is by preventing the weakly-binding relaxed crossbridges from going on through the crossbridge cycle into more strongly-binding states.     

The substrate reactivity of mu-monothiopyrophosphate with pyrophosphate-dependent phosphofructokinase: evidence for a dissociative transition state in enzymatic phosphoryl group transfer.

mu-Monothiopyrophosphate (MTP), an analogue of pyrophosphate (PPi) with sulfur in place of oxygen in the bridge position, is a substrate for the enzyme pyrophosphate-dependent phosphofructokinase. At pH 9.4 and 6 degrees C, the maximal velocity for the phosphorylation of fructose 6-phosphate (F6P) by MgMTP is about 2.8% of that with MgPPi as the phosphoryl donor. The kinetic mechanism is equilibrium random with rate-limiting transformation of the substrate ternary complex to the product when either MgMTP or MgPPi is the phosphoryl donor. This is known from independent studies to be kinetic mechanism at pH 8.0 and 25 degrees C [Bertagnolli, B. L., & Cook, P. F. (1984) Biochemistry 23, 4101-4108]. The dissociation constant of MgPPi is 14 microM, that of MgMTP is 64 microM, and that of F6P from the enzyme is about 5 mM. The Km values for MgPPi and MgMTP are 14.5 and 173 microM, respectively. MgMTP competes with MgPPi for binding to the enzyme. The values of kcat are 3.4 s-1 and 140 s-1 for MgMTP and MgPPi, respectively, at pH 9.4 and 6 degrees C. The estimated rate enhancement factors are 3.6 x 10(5) and 1.4 x 10(14) for the reactions of MgMTP and MgPPi, respectively. Therefore, MgMTP is a reasonably good substrate for PPi-dependent PKF, on the basis of comparisons of kcat. However, the rate enhancement factors show that the enzyme is a poor catalyst for the reaction of MgMTP. Lesser enzymatic catalysis in the reaction of MgMTP compared with MgPPi is largely compensated for by the greater intrinsic reactivity of MgMTP. Thus, the larger substrate MgMTP is well accommodated in the active site, and the dissociative reaction of MgMTP is well accommodated in the transition state. The results are interpreted to indicate a dissociative transition state for phosphoryl group transfer by PPi-dependent PFK. A modified synthesis and purification of MTP are described, in which (trimethylsilyl)trifluoromethanesulfonate and tetra-N-butylammonium iodide are used in place of iodotrimethylsilane to dealkylate tetramethyl-MTP.     

Physical characteristics of hyaluronate binding to the surface of simian virus 40-transformed 3T3 cells

The binding of labeled hyaluronate to the surface of Simian virus 40-transformed 3T3 cells was studied as a function of 1) pH, 2) ionic strength, 3) temperature, and 4) molecular weight of the hyaluronate. Binding occurred over a wide range of pH values with optima at pH 7 and at less than pH 4. Binding at low pH was eliminated at high ionic strength whereas that at physiological pH was enhanced, with a maximum at 0.5 M NaCl. The enhancement of binding at pH 7 was reversible and independent of the particular salt used. Scatchard plot analysis showed that increasing the ionic strength resulted in both a decrease in the dissociation constant (Kd) and an increase in the amount bound at saturation (Bmax). Temperature also influenced the binding of hyaluronate to the cell surface. The amount bound at low temperatures (0 degrees C) was 3 to 5 times that bound at high temperatures (40 degrees C) with a sharp transition occurring at 18 degrees C, the temperature of phase transition of the plasma membrane. The temperature effect was primarily a change in the Bmax and was reversible. Finally the molecular weight of the ligand influenced the binding. High molecular weight preparations of hyaluronate had a higher binding affinity (lower Kd) and a lower Bmax than did smaller molecular weight preparations.     

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.     

Properties of herpes simplex virus type 1 and type 2 DNA polymerase

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA polymerases were highly purified from infected HeLa BU cells by DEAE cellulose, phosphocellulose and DNA cellulose column chromatography. DNA exonuclease activity but not endonuclease activity was found associated with both types of DNA polymerase. Both DNA polymerase activities could be activated by salt in a similar fashion with the optimal activity in the range of ionic strength between 0.22 and 0.29 alpha. At an ionic strength of 0.14, spermidine and putrescine in the concentration range (0--5 mM) studied could mimic the action of KCI in stimulating DNA polymerase activity. Spermine, in the same concentration range, had a biphasic effect. At an ionic strength of 0.29 all three polyamines were inhibitory. HSV-1 and HSV-2 DNA polymerase are similar in their column chromatographic behavior, sedimentation rate in sucrose gradient centrifugation, and activation energy, but they differ in their heat stability at 45 degrees C with the HSV-2 enzyme more stable than the HSV-1 enzyme. Kinetic behavior of both enzymes is similar, with Km values for deoxyribonucleoside triphosphates in the range of 5 . 10(-7) to 1.8 . 10(-8) M. IdUTP and dUTP served as apparent competitive inhibitors with respect to dTTP, and AraATP acted as an apparent competitive inhibitor with respect to dATP. AraATP could not replace dATP in the DNA polymerization reaction; in contrast, IdUTP could replace TTP. Phosphonoformic acid behaved as an uncompetitive inhibitor with respect to DNA. The ID(50) value estimated was foind to be dependent on the purity of the DNA polymerase used and the ionic strength of the assay condition. Each DNA-polymerase associated DNA exonuclease had the same stability at 45 degrees C as its DNA polymerase. The associated DNAase activity was inhibited by phosphonoformic acid and high ionic strength of the assay condition.     

Stiffness of skinned rabbit psoas fibers in MgATP and MgPPi solution.

The stiffness of single skinned rabbit psoas fibers was measured during rapid length changes applied to one end of the fibers. Apparent fiber stiffness was taken as the initial slope when force was plotted vs. change in sarcomere length. In the presence of MgATP, apparent fiber stiffness increased with increasing speed of stretch. With the fastest possible stretches, the stiffness of relaxed fibers at an ionic strength of 20 mM reached more than 50% of the stiffness measured in rigor. However, it was not clear whether apparent fiber stiffness had reached a maximum, speed independent value. The same behavior was seen at several ionic strengths, with increasing ionic strength leading to a decrease in the apparent fiber stiffness measured at any speed of stretch. A speed dependence of apparent fiber stiffness was demonstrated even more clearly when stiffness was measured in the presence of 4 mM MgPPi. In this case, stiffness varied with speed of stretch over about four decades. This speed dependence of apparent fiber stiffness is likely due to cross-bridges detaching and reattaching during the stiffness measurement (Schoenberg, 1985. Biophys. J. 48:467). This means that obtaining an estimate of the maximum number of cross-bridges attached to actin in relaxed fibers at various ionic strengths is not straightforward.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature-dependent modes for the binding of the polyene antibiotic amphotericin B to human erythrocyte membranes. A circular dichroism study

The interaction of amphotericin B with isolated human erythrocyte ghosts was monitored by circular dichroism at 37 degrees C and 15 degrees C. Although different, these spectra were not concentration dependent over a concentration range covering the inducement of K+ leakage and hemolysis, which suggests the existence of only one bound amphotericin B species. At 15 degrees C, the spectra indicate that amphotericin B is complexed with membrane cholesterol; the complex formation is saturable but not cooperative. At 37 degrees C new spectra are observed, and their existence is conditioned by the presence of membrane proteins. The binding is cooperative but not saturable. The amphotericin B right side-out vesicles complexation is temperature as well as ionic strength dependent: at high ionic strength it is the same as with ghosts, with the same temperature dependence. At low ionic strength it is characteristic of an interaction with cholesterol, regardless of temperature. In the large unilamellar vesicles reconstituted from the total lipid extracts of erythrocyte membranes, amphotericin B is complexed with cholesterol, regardless of temperature and ionic strength. These results indicate that there are two different modes of amphotericin B complexation with erythrocyte membranes, reversible one in the other, depending on the molecular organization of the membrane and the presence of membrane proteins.     

Golgi β-glucuronidase of androgen-stimulated mouse kidney

The equilibrium constant of the phosphoglyceromutase reaction was determined over a range of pH (5.4-7.9), in solutions of different ionic strength (0.06-0.3) and in the presence of Mg(2+), at 30 degrees C and at 20 degrees C. The values obtained (8.65-11.65) differ substantially from previously published values. The third acid dissociation constants were redetermined for 2- and 3-phosphoglycerate, and in contrast with previous reports the pK values (7.03 and 6.97 respectively at zero ionic strength) were closely similar. The Mg(2+)-binding constants were measured spectrophotometrically and the values, 286mm(-1) and 255mm(-1) for 2- and 3-phosphoglycerate at pH7 and ionic strength 0.02, were also very similar. From the relative lack of effect of temperature, pH and ionic strength it is concluded that the equilibrium constant differs from unity largely because of entropic factors. At low ionic strength, in the neutral region, the pH-dependence can be attributed to the small difference in the acid dissociation constants, but the difference in dissociation constants does not explain the pH-dependence in the acid region or at high ionic strength. Within physiological ranges of pH, Mg(2+) concentration and ionic strength there will be little variation in equilibrium constant.     

Comparison of mammalian mitochondrial ribosomal ribonucleic acid from different species

Mitochondrial ribosomal RNA species from mouse L cells, rat liver, rat hepatoma, hamster BHK-21 cells and human KB cells were examined by electrophoresis on polyacrylamide-agarose gels and sedimentation in sucrose density gradients. The S(E) (electrophoretic mobility) and S values of mitochondrial rRNA of all species were highly dependent on temperature and ionic strength of the medium; the S(E) values increased and the S values decreased with an increase in temperature at a low ionic strength. At an ionic strength of 0.3 at 23-25 degrees C or an ionic strength of 0.01 at 3-4 degrees C the S and S(E) values were almost the same being about 16.2-18.0 and 12.3-13.6 for human and mouse mitochondrial rRNA. The molecular weights under these conditions were calculated to be 3.8x10(5)-4.3x10(5) and 5.9x10(5)-6.8x10(5), depending on the technique used. At 25 degrees C in buffers of low ionic strength mouse mitochondrial rRNA species had a lower electrophoretic mobility than those of human and hamster. Under these conditions the smaller mitochondrial rRNA species of hamster had a lower electrophoretic mobility than that of human but the larger component had an identical mobility. Mouse and rat mitochondrial rRNA species had identical electrophoretic mobilities. Complex differences between human and mouse mitochondrial rRNA species were observed on sedimentation in sucrose density gradients under various conditions of temperature and ionic strength. Mouse L-cell mitochondrial rRNA was eluted after cytoplasmic rRNA on a column of methylated albumin-kieselguhr.     

Characterization of an adenosine 3'':5''-cyclic monophosphate phosphodiesterase from baker''s yeast. Its binding to subcellular particles, catalytic properties and gel-filtration behaviour.

1. The 3':5'-cyclic AMP phosphodiesterase in the microsomal fraction of baker's yeast is highly specific for cyclic AMP, and not inhibited by cyclic GMP, cyclic IMP or cyclic UMP. Catalytic activity is abolished by 30 micrometer-EDTA. At 30 degrees C and pH8.1, the Km is 0.17 micrometer, and theophylline is a simple competitive inhibitor with Ki 0.7 micrometer. The pH optimum is about 7.8 at 0.25 micrometer-cyclic AMP, so that over the physiological range of pH in yeast the activity changes in the opposite direction to that of adenylate cyclase [PH optimum about 6.2; Londesborough & Nurminen (1972) Acta Chem. Scand. 26, 3396-3398].2. At pH 7.2, dissociation of the enzyme from dilute microsomal suspensions increased with ionic strength and was almost complete at 0.3 M-KCl. MgCl2 caused more dissociation than did KCl or NaCl at the same ionic strength, but at low KCl concentrations binding required small amounts of free bivalent metal ions. In 0.1 M-KCl the binding decreased between pH 4.7 and 9.3. At pH 7.2 the binding was independent of temperature between 5 and 20 degrees C. These observations suggest that the binding is electrostatic rather than hydrophobic. 3. The proportion of bound activity increased with the concentration of the microsomal fraction, and at 22 mg of protein/ml and pH 7.2 was 70% at I0.18, and 35% at I0.26. Presumably a substantial amount of the enzyme is particle-bound in vivo. 4. At 5 degrees C in 10 mM-potassium phosphate, pH 7.2, the apparent molecular weight of KCl-solubilized enzyme decreased with enzyme concentration from about 200 000 to 40 000. In the presence of 0.5M-KCl, a constant mol.wt. of about 55 000 was observed over a 20-fold range of enzyme concentrations.     

X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle.

X-ray diffraction patterns were obtained from skinned rabbit psoas muscle under relaxing and rigor conditions over a wide range of ionic strengths (50-170 mM) and temperatures (1 degree C-30 degrees C). For the first time, an intensification of the first actin-based layer line is observed in the relaxed muscle. The intensification, which increases with decreasing ionic strength at various temperatures, including 30 degrees C, parallels the formation of weakly attached cross-bridges in the relaxed muscle. However, the overall intensities of the actin-based layer lines are low. Furthermore, the level of diffuse scattering, presumably a measure of disorder among the cross-bridges, is little affected by changing ionic strength at a given temperature. The results suggest that the intensification of the first actin layer line is most likely due to the cross-bridges weakly bound to actin, and that the orientations of the weakly attached cross-bridges are hardly distinguishable from the detached cross-bridges. This suggests that the orientations of the weakly attached cross-bridges are not precisely defined with respect to the actin helix, i.e., nonstereospecific. Intensities of the myosin-based layer lines are only marginally affected by changing ionic strength, but markedly by temperature. The results could be explained if in a relaxed muscle the cross-bridges are distributed between a helically ordered and a disordered population with respect to myosin filament structure. Within the disordered population, some are weakly attached to actin and others are detached. The fraction of cross-bridges in the helically ordered assembly is primarily a function of temperature, while the distribution between the weakly attached and the detached within the disordered population is mainly affected by ionic strength. Some other notable features in the diffraction patterns include a approximately 1% decrease in the pitch of the myosin helix as the temperature is raised from 4 degrees C to 20 degrees C.     

Effects of magnesium pyrophosphate on mechanical properties of skinned smooth muscle from the guinea pig taenia coli.

Effects of the non-hydrolyzable nucleotide analogue magnesium pyrophosphate (MgPPi) on cross-bridge properties were investigated in skinned smooth muscle of the guinea pig Taenia coli. A "high" rigor state was obtained by removing MgATP at the plateau of an active contraction. Rigor force decayed slowly towards an apparent plateau of approximately 25-35% of maximal active force. MgPPi markedly increased the rate of force decay. The initial rate of the force decay depended on [MgPPi] and could be described by the Michaelis-Menten equation with a dissociation constant of 1.6 mM. The decay was irreversible amounting to approximately 50% of the rigor force. Stiffness decreased by 20%, suggesting that the major part of the cross-bridges were still attached. The results can be interpreted as "slippage" of PPi-cross-bridges to positions of lower strain. The initial rate of MgPPi-induced force decay decreased with decreasing ionic strength in the range 45-150 mM and was approximately 25% lower in thiophosphorylated fibers. MgADP inhibited the MgPPi-induced force decay with an apparent Ki of 2 microM. The apparent Km of MgATP for the maximal shortening velocity in thiophosphorylated fibers was 32 microM. This low Km of MgATP suggests that steps other than MgATP-induced detachment are responsible for the low shortening velocity in smooth muscle. No effects were observed of 4 mM MgPPi on the force-velocity relation, suggesting that cross-bridges with bound MgPPi do not constitute an internal load or that binding of MgPPi is weaker in negatively strained cross-bridges during shortening.     

X-ray diffraction testing for weak-binding crossbridges in relaxed bony fish muscle fibres at low ionic strength

Equatorial X-ray diffraction patterns from single skinned fibres from bony fish muscle (turbot) were obtained with the fibres at 6 degrees C bathed in relaxing solutions of 170 down to 26 mM ionic strength. Diffraction patterns from rigor fibres were also obtained as controls. Unlike fibres from rabbit muscle, which show very clear evidence of substantial crossbridge formation at low ionic strength in what is mechanically a rapid equilibrium ("weak-binding") state (Brenner et al., 1982), diffraction patterns from bony fish fibres showed only a small change in relative peak intensities at low ionic strength (26 mM) compared with normal (170 mM) ionic strength. However, there was a slight ordering of the filament lattice at low ionic strength. The specimen temperature used (about 6 degrees C) was not far from the normal physiological temperature of the fish. Likewise, only a small change was seen by Xu et al. (1987) in patterns from frog fibres at low ionic strength at 2 to 6 degrees C. (Rabbit fibres previously studied, where large changes were seen at temperatures of 5 to 20 degrees C, were about 17 to 32 degrees C below physiological.) The I11/I10 ratio for fish fibres at 26 mM ionic strength was actually lower than that for rabbit even at normal ionic strength. This may be associated with an intrinsic structural difference between these muscles or alternatively with the disordering of the crossbridge helix in rabbit muscle found at low temperature by Wray (1987), and could support the view that rabbit fibres at 5 degrees C and normal ionic strength may already have a significant population of weak-binding crossbridges.     

Activation of the rat liver androgen-receptor complex

Activation of androgen receptor in rat liver cytosol was studied in vitro. The state of activation was judged by binding of [3H] R1881-receptor complex to chromatin. High ionic strength (0.4 M KCl as a final concentration) provoked the binding of [3H] R1881-receptor complex to chromatin at 0 degrees C. At low ionic strength, activation was very slow at 0 degrees C, but was very rapid at 25 degrees C and reached the maximum at 15 min of heating.     

Isotonic contraction of skinned muscle fibers on a slow time base: effects of ionic strength and calcium

The force development by calcium-activated skinned frog skeletal muscle fibers and the motion on a slow time base after a quick decrease in load were studied at 0-1 degrees C as a function of the ionic strength and the degree of activation. The ionic strength was varied between 50 and 190 mM by adding appropriate concentrations of KCl to the bathing solution. Under these conditions, the fibers could be maximally activated for several cycles at low ionic strength without developing residual tension. We found that the steady isometric force in fully activated fibers linearly decreased when the KCl concentration was increased from 0 to 140 mM. The steady isotonic motion at a given relative load in fully activated fibers was almost the same at KCl concentration greater than or equal to 50 mM. In 0 and 20 mM KCl, the isotonic velocity decreased continuously for more than 300 ms. At a given relative load, the initial velocity of the motion in 0 and 20 mM KCl was about 0.6 and 0.9 times, respectively, that in 140 mM KCl. The initial velocity decreased further when residual tension developed; this observation provides additional evidence that residual tension may reflect the presence of an internal load. The effect of calcium on the motion was examined at 70 mM KCl. In this solution, the motion during the velocity transient at a given relative load appeared to be the same at different levels of activation. The speed of the subsequent motion was almost steady at high calcium levels but decreased continuously in low calcium levels. These results support the idea that at low ionic strength the response of the fiber to calcium is switch-like, but that other factors also affect the contraction mechanism under these conditions.     

Thermodynamics of the arginine kinase reaction.

The effect of temperature, pH, free [Mg(2+)], and ionic strength on the apparent equilibrium constant of arginine kinase (EC 2.7.3.3) was determined. At equilibrium, the apparent K' was defined as [see text] where each reactant represents the sum of all the ionic and metal complex species. The K' at pH 7.0, 1.0 mM free [Mg(2+)], and 0. 25 M ionic strength was 29.91 +/- 0.59, 33.44 +/- 0.46, 35.44 +/- 0. 71, 39.64 +/- 0.74, and 45.19 +/- 0.65 (n = 8) at 40, 33, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy (DeltaH degrees') is -8.19 kJ mol(-1), and the corresponding standard apparent entropy of the reaction (DeltaS degrees') is + 2. 2 J K(-1)mol(-1) in the direction of ATP formation at pH 7.0, free [Mg(2+)] =1.0 mM, ionic strength (I) =0.25 M at 25 degrees C. We further show that the magnitude of transformed Gibbs energy (DeltaG degrees ') of -8.89 kJ mol(-1) is mostly comprised of the enthalpy of the reaction, with 7.4% coming from the entropy TDeltaS degrees' term (+0.66 kJ mol(-1)). Our results are discussed in relation to the thermodynamic properties of its evolutionary successor, creatine kinase.     

Effects of neutral salts and temperature on skeletal muscle sarcolemmal Ca-TPase]

The temperature dependence and effects of sodium and potassium chloride on purified preparations of sarcolemmal Ca2+-activated ATPase were investigated. It was shown that within the concentration range of 0,1--1,0 M both salts have the same effect on the enzyme activity. A low ionic strength and concentration of the salts of 0,1 M the temperature maximum was 45 degrees and the shapes of temperature curves were the same. The Arrhenius plots showed a break at 16--19 degrees. The apparent activation energies were 27,3 kcal/mole below and 17,1 kcal/mole above the break point. At high ionic strength (0,5 M) the temperature maximum was observed at 40 degrees and the apparent activation energies decreased down to 18,0 kcal/mole below and 11,5 kcal/mole above the break point.     

Interaction of talin with actin: sensitive modulation of filament crosslinking activity.

Talin is an adhesion plaque protein believed important in linking actin filaments to the plasma membrane. The nature of a direct talin-actin interaction, however, is complex and has remained unclear. We have systematically characterized the effects of pH, ionic strength, temperature, and protein molar ratio on the interaction between highly purified talin and actin. The ability of talin to increase viscosity of F-actin at 25 degrees C and low ionic strength increased with decreasing pH from 7.3 to 6.4 and increasing molar ratio of talin to actin. At pH 6.4 and low ionic strength, talin could extensively crosslink actin filaments into ordered bundles as shown by negative staining and could cosediment with F-actin at molar ratios as high as one talin to two actin monomers. Talin crosslinked prepolymerized actin filaments to a similar extent as actin filaments polymerized in its presence. The 190-kDa calpain-generated proteolytic fragment of talin bound poorly to actin under conditions favorable for intact talin, but was able to crosslink actin filaments at a lower pH. Increasing the ionic strength within a relatively narrow range significantly decreased ability of talin to bind to actin, regardless of pH. The effects of pH and ionic strength on the talin-actin interaction were rapid and reversible. Low-shear-viscosity studies revealed a strong temperature dependence in the talin-actin interaction with significant crosslinking activity at physiological-like ionic conditions and temperature (37 degrees C). Our results consistently demonstrated that talin crosslinks actin filaments and that this direct interaction is highly sensitive to, and dependent upon, ionic conditions and temperature.