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S Highsmith

Publications and source records attributed to S Highsmith.

At least 37 records · Page 2Linked to original sources
PubMed1992

Nucleotide- and temperature-induced changes in myosin subfragment-1 structure.

The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5 degrees C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25 degrees C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15 degrees C was observed for S1, S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25 degrees C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15 degrees C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented.

Adenine Nucleotides↗
PubMed1992

Electrostatic changes at the actomyosin-subfragment 1 interface during force-generating reactions.

The ionic strength dependence of the binding of rabbit skeletal muscle myosin subfragment 1, S1, to F-actin in the presence of saturating concentrations of MgATP or MgADP was analyzed in order to determine the association constants at zero ionic strength [K(0)] and the products of the net effective electric charges (magnitude of zMzA) at the binding interfaces. K(0) and magnitude of zM A were 1 x 10(6) M-1 and 17 esu2 for S1-MgADP,P, and 5 x 10(7) M-1 and 7 esu2 for S1-MgADP, respectively, for binding to F-actin at 25 degrees C. At ionic strengths near physiological, the increase in affinity is close to 10(4)-fold for this transition that may correspond to force generation in muscle fibers. The large, from 17 to 7 esu2, decrease in the electrostatic contribution to binding appears to be correlated with a much larger increase in nonelectrostatic interactions, unlike the simpler transition of actin-bound S1-MgADP to S1, which appears to be due entirely to electrostatic changes [Highsmith, S. (1990) Biochemistry 29, 10690-10694]. These results for acto-S1-MgADP,P to acto-S1-MgADP suggest that a substantial transformation of the actin binding site on S1 occurs even if there is a translocation to a new interface.

Actins↗
PubMed1990

Electrostatic contributions to the binding of myosin and myosin-MgADP to F-actin in solution.

The ionic strength dependence of skeletal myosin subfragment 1 (S1) binding to unregulated F-actin was measured in solutions containing from 0 to 0.50 M added lithium acetate (LiOAc) in the absence and presence of MgADP. The data were analyzed by using a theory based on an ion interaction model that is rigorous for high ionic strength solutions [Pitzer, K. S. (1973) J. Phys. Chem. 77, 268-277] in order to obtain values for K, the equilibrium association constant when the ionic strength is zero, and for [zMzA[, the absolute value of the product of the net electric charges of the actin binding site on myosin (zM) and the myosin binding site on actin (zA). The presence of MgADP reduced K by a factor of 10, as expected, and reduced [zMzA[ by about 1 esu2. Because the presence of MgADP is not likely to change the net charge of the myosin binding site on actin, these data are consistent with a model in which MgADP binding to S1 reduces its affinity for actin by a mechanism that reduces the net electric charge of the acting binding site on S1. The value of [zMzA[ in the absence of ADP was 8.1 +/- 0.9 esu2, which, if one uses integer values, suggests that zM and zA are in the 8+ to 1+ esu and 1- to 8- esu ranges, respectively. ADP binding then reduces zM to the 7+ to 0.88+ esu range.

Actins↗
PubMed1990

Ligand-induced myosin subfragment 1 global conformational change.

The effects of selected ligands on the structure of myosin subfragment 1 (S1) were compared by using transient electrical birefringence techniques. With pairs of dilute solutions of S1 at 3.5 degrees C in low ionic strength (mu = 0.020 M) buffers that had matched electrical impedances, S1 with Mg2+, MgADP, or MgADP.Vi bound was subjected to 6-7-microseconds external electrical fields in the Kerr law range. Specific Kerr constants and the rates of rotational Brownian motion after the electric field was removed were measured. Neither Mg2+ nor MgADP had a measurable effect on either observable, but when orthovanadate (Vi) bound S1.MgADP it decreased the rotational correlation coefficient from 267 +/- 6 to 244 +/- 10 ns. Parallel measurements of MgATPase activity indicated that S1.MgADP.Vi was greater than 95% inhibited. These results confirm the conclusion of Aguirre et al. [(1989) Biochemistry 28, 799] that Vi binding to S1.MgADP increases its rate of rotational Brownian motion and provide data that are more quantitatively correlated with S1 structure. The Vi-induced change in the rotational correlation coefficient is consistent with S1 becoming more flexible or more compact when Vi binds. Assuming that S1.MgADP.Vi is an analogue for S1.MgADP.Pi, the structural changes observed for S1-ligand complexes in solution are discussed in relation to possible structural changes of intermediates on the kinetic pathway of ATPase hydrolysis. A new model of force generation by S1 in muscle is hypothesized.

Adenosine Triphosphate↗
PubMed1990

On the mechanism of detergent modification of myosin structure and function.

The concentration dependences of the activation of myosin subfragment-1 (S1) Mg-ATPase by the detergents CHAPS and C12E8 were determined at 23 degrees C in 25 mM Tris (pH 7.0), 250 microM EDTA, 5 mM MgCl2, and 100 microM ATP. At detergent concentrations expected to bind hydrophobic S1 surface areas equally, C12E8 caused an 8.5-fold greater increase in activity than CHAPS, which suggests that detergent binding to the surface of S1 is not the mechanism of activation. At detergent concentrations above their critical micelle concentrations, C12E8 was also much more effective than CHAPS, suggesting that micelles are not involved. A series of n-alcohols (which do not form micelles) with from 3 to 10 carbons all increased S1 Mg-ATPase activity as much or more than C12E8. The largest increase (5.7-fold) was caused by n-hexanol. The more hydrophobic alcohols activated S1 at lower concentrations. A linear plot of the alcohol concentration that caused 50% of maximum activity versus the number of carbons in the alcohol, indicated the apparent free energy of binding per CH2-group was -0.60 +/- 0.03 kcal/mol. There were two indications that alcohol binding caused an S1 conformational change. The intrinsic fluorescence increase of S1 during steady-state activity was reduced from 17.5 to 12.8%, and the apparent hydrodynamic rotational mobility of fluorescently labeled S1 was decreased 25% by the present of n-hexanol. The data suggest that S1 activation by C12E8 and by n-alcohols is due to hydrophobic binding to S1 at non-surface sites, which causes an S1 structural change.

Animals↗
PubMed1989

Detergent modification of myosin function and structure in solution.

Rabbit skeletal muscle myosin and myosin subfragment 1 (S1) MgATPase activities were increased 2-3-fold by the addition of a variety of molecules that contained single straight saturated 12-16-carbon chains. The nonionic detergent dodecyl nonaoxyethylene ether (C12E9) increased the activity of S1 to 50% of maximum at a free C12E9 concentration of 27 +/- 9 microM. The activation was reversible and was not due to chemical modification of S1 amino acid side chains. The Vmax for actin-activated S1 MgATPase activity was increased 3-fold by C12E9. The apparent association constant for S1 binding to pure F-actin was reduced 3-fold by C12E9. The [C12E9] dependencies of the increase in S1 and acto-S1 MgATPase activities and of the decrease in acto-S1 binding were equal, within experimental uncertainty, suggesting that a single detergent-induced S1 conformational change is sufficient to explain the results. The stoichiometry of C12E9 bound to S1 in the S1-C12E9 complex was estimated, by the S1 concentration dependence of the C12E9 activation midpoint and by the light-scattering increase when S1 and detergent were mixed, to be 7 and 57 C12E9 molecules per S1, respectively. The results are discussed in relation to possible structural aspects of the mechanism of action for S1 and acto-S1 MgATPase activities.

Animals↗
PubMed1987

Limited trypsinolysis changes in structural dynamics of myosin subfragment 1.

The effects of limited trypsinolysis of myosin subfragment 1 (S1) on its structural dynamics were investigated by using the method of transient electric birefringence. Conversion of S1 by trypsin to produce S1 (T) did not change the specific Kerr constant [(8.1 +/- 0.3) X 10(-7) and (8.0 +/- 0.3) X 10(-7) cm2/statvolt2 for S1(T) and S1, respectively] or the degree of alignment in a weak electric field, suggesting that the size of S1 and its permanent electric dipole moment are not modified by trypsin. On the other hand, the relaxation time for the field-free rotation, after achieving a steady-state birefringence signal, was reduced from 316 ns for S1 to 269 ns for S1(T), at 3.7 degrees C, suggesting that trypsinolysis increases the flexibility of the connections between S1 segments or introduces additional segmental motions. For both S1 and S1(T), the rate of decay for a steady-state signal was independent of the field strength, between 3.34 and 20.3 statvolt/cm. Shortening the duration of the weak electric field pulses to 0.35 microseconds, so that steady-state signals were not achieved, decreased the relaxation times for S1 and S1(T) to 240 and 210 ns, respectively, which is consistent with the segmented flexible S1 structure proposed earlier [Highsmith, S., & Eden, D. (1986) Biochemistry 25, 2237]. When the strength of the electric field was increased to above 10 statvolt/cm, in order to make the interaction energy for the S1(T) electric dipole moment in the electric field greater than the thermal energy, the relaxation time after a 0.35-microseconds pulse decreased from 210 to 170 ns as the field was increased from 7 to 20 statvolt/cm. (ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Spatial organization of CaATPase molecules in sarcoplasmic reticulum vesicles.

Fluorescence intensity, polarization, and (Ca2+-Mg2+)-ATPase (CaATPase) activity were measured for sarcoplasmic reticulum (SR) CaATPase with varying amounts of fluorescein isothiocyanate (FITC) attached at a specific site at or near the ATP binding site. The stoichiometry of attached FITC was proportional to the inhibition of ATPase activity, consistent with the independent labeling of one FITC site per CaATPase molecule. Polarization measurements on vesicular CaATPase indicated the occurrence of energy-transfer depolarization that increased as the fraction of binding sites labeled by FITC increased. Addition of the nonionic detergent dodecyl nonaoxyethylene alcohol (C12E9) eliminated the energy-transfer depolarization for all degrees of labeling with little direct effect on the attached FITC molecule. Fluorescence polarization measurements on sizing-column-purified FITC-labeled CaATPase in the presence of 30 mM C12E9 indicated that the sample consisted of homogeneous monomeric CaATPase. The attached FITC molecule was not sensitive to the bulk viscosity for either the vesicular or the detergent-solubilized CaATPase. The midpoints of the transition from vesicular to monomeric CaATPase as a function of increasing detergent concentration were determined from fluorescence polarization and light-scattering measurements. The dependence of these midpoints on the CaATPase concentration indicated a stoichiometry of 262 +/- 35 molecules of C12E9 per CaATPase in the detergent-protein complex. Both measurements gave the same result. The decrease of fluorescence polarization with increasing saturation of the FITC binding sites for vesicular and detergent-solubilized CaATPase was analyzed in terms of energy-transfer depolarization to determine the spatial arrangements of CaATPase molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The CaATPase activity of rat-incisor odontoblast vesicles.

An homogenate of rat incisor odontoblasts had Ca2+ and Mg2+ ATPase activity and suitable storage conditions kept it stable for several days. Over 90 per cent of the activity was retained in a vesicle-rich microsomal fraction that removed about 85 per cent of the total material from the homogenate. This fraction was further characterized: the resolved Ca2+-activated ATPase activity, above the basal MgATPase activity, was 0.30 mumol Pi/min-mg total protein, and 50 per cent activated at free [Ca2+] equal 0.8 microM. This calcium dependency is consistent with an intracellular Ca2+-regulated enzymatic activity. The calcium ionophore, A23187, had no measurable effect on the CaATPase activity, which suggests that the odontoblast vesicles do not concentrate Ca2+ in a lipid bilayer compartment. Direct measurement of the uptake of 45Ca2+ by the filtration method and parallel measurements of CaATPase activity on the same preparations under identical conditions indicated that the odontoblast-derived vesicles have a coupling ratio of 0.024 Ca2+/ATP. This low coupling ratio and the lack of detectable compartmentalization of calcium indicate that the CaATPase activity of the odontoblast microsomes is not associated with a calcium pump. The [Ca2+] dependence of the activity suggests the CaATPase is under intracellular Ca2+ control, but its function is unknown.

Animals↗
PubMed1986

Sarcoplasmic reticulum interacts with the Ca(2+) indicator precursor fura-2-am.

Fura-2-am, the pentaester precursor of the fluorescent Ca(2+) indicator fura-2, is modified when it is exposed to isolated skeletal muscle sarcoplasmic reticulum vesicles. The modified fura-2-am has enhanced fluorescence, is not sensitive to Ca(2+), and is partially bound to the SR membrane. The isolated product is further converted into fura-2 by esterase. It is suggested that the SR-induced modification is a selective enzymatic hydrolysis of only some of the five ester moieties on fura-2-am. A structure is proposed to account for the results. The potential for this effect of SR on fura-2-am to cause complications in measurements of in vivo intracellular free [Ca(2+)] is noted.

Animals↗
PubMed1986

Myosin subfragment 1 has tertiary structural domains.

Transient electrical birefringence measurements were made on skeletal muscle myosin subfragment 1 (S1) at 3.7 degrees C in 10 mM tris(hydroxymethyl)aminomethane-acetate and 0.10 mM MgCl2, pH 7.0. The specific birefringence for 4.5 microM S1 was determined from steady-state measurements to be (8.1 +/- 0.3) X 10(-7) (cm/statvolt)2. For electric fields in the range of 2.47-24.7 statvolts/cm, the alignment was due to a large permanent dipole moment for S1, estimated to be 8500 +/- 2000 D. The duration and the strength of the transient electric field was varied, and the temporal response of the decay of the birefringence signal was analyzed. The rate of rotational motion after the field was removed increased with increasing field strength for short (0.35-microseconds) pulses and decreased with increasing pulse lengths for all field strengths. The rate of decay from a steady-state birefringence signal was independent of field strength. A model of S1 structure is proposed, which is consistent with these data and most other data on S1 structure. In this model, S1 is composed of two tertiary structural domains that are connected by a flexible linkage with a substantial restoring force. The electric dipole moments on the two domains are arranged head to tail. The segmental movement of the domains is restricted to certain directions. The average conformation of the molecule is elongated, but it can be made more compact by the torque exerted by an electric field. The structural changes depend on the strength and duration of the pulse.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Solvent accessibility of the adenosine 5'-triphosphate catalytic site of sarcoplasmic reticulum CaATPase.

The CaATPase of rabbit skeletal sarcoplasmic reticulum was labeled at or near the ATP catalytic site with fluoresceinyl isothiocyanate (FITC), and the accessibility of the attached probe to the bulk solvent was determined by I- quenching of its fluorescence. The quenching of free FITC was also measured. In both cases, the quenching was of the Stern-Volmer type and collisional quenching rate constants were obtained over the pH range 5-8 in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and with added Ca2+, vanadate, or phosphate. The fluorescence intensity and susceptibility to quenching by I- of free FITC were insensitive to the added ligands. In all cases, the intensity decreased with pH, as predicted from the known properties of FITC mono- and dianions. The collisional quenching rate constants increased at lower pH, as expected for I- quenching of a molecule with decreasing negative charge due to protonation. When FITC was attached to the CaATPase, the FITC fluorescence intensity and I- collisional quenching rate constants were sensitive to ligand binding as well as pH. The changes in fluorescence intensity with acidity, when compared to the results for free FITC, indicated the pKa of the FITC was reduced 0.6 unit when it was attached to the CaATPase. Excited-state lifetime measurements indicated that ligand effects at constant pH were not due to protonation-induced changes in FITC quantum yield but to conformational changes of the CaATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Transient electrical birefringence characterization of heavy meromyosin.

Heavy meromyosin (HMM) and myosin subfragment 1 (S1) were prepared from myosin by using low concentrations of alpha-chymotrypsin. The light chain distribution in HMM was identical with that of myosin, within experimental error, when analyzed on 12% polyacrylamide gels after electrophoresis. Specific birefringences and birefringence decay times were measured by transient electrical birefringence in 5 mM KCl, 5 mM tris(hydroxymethyl)aminomethane (pH 7), and 1 mM MgCl2 at 4 degrees C under gentle conditions that reduced the CaATPase activity by less than 10%. For solutions of HMM, by use of electric field pulses shorter than 0.5 microseconds, the birefringence decay signal from the S1 portions of HMM could be resolved and the rotational motions of the S1 moieties observed directly. The rotation relaxation time, adjusted to 20 degrees C, was 0.34 microseconds; this is in quantitative agreement with previous hydrodynamic results obtained by using covalently attached probes. The assignment of the fast decay time obtained with HMM to the S1 portions was confirmed by birefringence decay measurements on free S1, for which the relaxation time was 0.13 microseconds, corrected to 20 degrees C. The specific birefringences for S1 and HMM, respectively, were 0.37 X 10(-6) and 12.8 X 10(-6) (cm/statvolt)2. Thus, for much longer electric field pulses, the signal from HMM is due almost entirely to its subfragment 2 (S2) portion, and its rotational dynamics can also be monitored directly by using electrical birefringence. The decay of the signal from the S2 portion could be adequately fit without evoking bending of the S2 portion of HMM other than at its junction with S1.

Birefringence↗
PubMed1985

High-affinity and low-affinity vanadate binding to sarcoplasmic reticulum Ca2+-ATPase labeled with fluorescein isothiocyanate.

Conditions were found that allowed both the fluorescence detection of vanadate binding to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum and the vanadate-induced formation of two-dimensional arrays of the enzyme. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca2+-ATPase increased with high-affinity vanadate binding (Ka = 10(6) M-1) as reported by Pick and Karlish (Pick, U. and Karlish, S.D. (1982) J. Biol. Chem. 257, 6120-6126). The Ca2+ and Mg2+ dependencies for high-affinity vanadate binding were similar but not identical to those for orthophosphate. In addition, it was found that there is low-affinity (Ka = 380 M-1) vanadate binding, which causes a 25% decrease in fluorescence. The Ca2+ and Mg2+ dependencies of the low-affinity vanadate binding were different from those of orthophosphate or high-affinity vanadate binding. The covalent attachment of fluorescein isothiocyanate (FITC) in the ATP site of the Ca2+-ATPase did not affect the formation of two-dimensional arrays, as detected by negatively stained electron micrographs. Vanadate concentrations high enough to saturate the low-affinity binding caused two-dimensional arrays as reported by Dux and Martonosi (Dux, L. and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). In addition, freeze-fracture replicas of quick-frozen specimens showed rows of indentations in the inner leaflet of the bilayer that corresponds to the arrays seen on the outer leaflet. This appearance of indentations suggests that low-affinity vanadate binding causes a transmembrane movement of the Ca2+-ATPase. By contrast, high-affinity vanadate binding was shown to cause neither array formation nor the appearance of indentations.

Animals↗
PubMed1984

Nd3+ and Co2+ binding to sarcoplasmic reticulum CaATPase. An estimation of the distance from the ATP binding site to the high-affinity calcium binding sites.

Nd3+ binding to sarcoplasmic reticulum (SR) was detected by inhibition of ATPase activity and directly by a fluorimetric assay. Both methods indicated that Nd3+ inhibited the ATPase activity by binding in the high-affinity Ca2+ binding sites. The stoichiometry of binding was about 11 nmol of Nd3+ bound per mg of SR proteins at pNd = 6.5. At higher [Nd3+], substantial nonspecific binding occurred. The association constant for Nd3+ binding to the high-affinity Ca2+ binding sites was estimated to be near 2 X 10(9) M-1. When the CaATPase was inactivated with fluorescein isothiocyanate (FITC), 5.3 nmol were bound per mg of SR protein. This fluorescent probe is known to bind in the ATP binding site. The stoichiometry of Nd3+ binding to FITC-labeled CaATPase was the same, within experimental error, as to the unlabeled CaATPase. Fluorescence energy transfer between FITC in the ATP site and Nd3+ in the Ca2+ sites was found to be very small. This donor-acceptor pair has a critical distance of 0.93 nm and the distance between the ATP site and the closest Ca2+ was estimated to be greater than 2.1 nm. Parallel measurements with FITC-labeled SR and Co2+, an acceptor with a critical distance 1.2 nm, suggested the ATP and Ca2+ binding sites are greater than 2.6 nm apart.

Animals↗
PubMed1984

Evidence that the ATP binding site of sarcoplasmic reticulum CaATPase has a Mg(2+) ion binding sub-site.

The CaATPase of skeletal muscle sarcoplasmic reticulum was specifically labeled in the ATP binding site with fluorescein isothiocyanate under gentle conditions (pH 7 X 5). Fluorescence energy transfer from the attached fluorescein to Nd3+ indicated that a cation binding site was about 1 X 0 nm away from the fluorescein. Thus it appears that the ATP site includes a cation binding site. At 25 degrees C in 0 X 5 M KCl, the association constants for Nd3+, Ca2+ and Mg2+ were 3 X 3 X 10(5) M-1, 84 M-1 and 35 M-1, respectively, making it possible that, in vivo, the site binds Mg2+.

Adenosine Triphosphate↗
PubMed1983

Actin-induced changes in the dynamics of myosin subfragment-1 detected by nuclear magnetic resonance.

Analysis of high resolution 1H NMR spectra for myosin and myosin subfragment-1 (S-1) indicates that S-1 has an unusual structure, about 20% of which is mobile. The rest of the myosin molecule and F-actin are rigid by comparison. A wide variety of perturbations do not affect the S-1 internal mobility and suggest that the mobile structure is located in the interior of S-1. Actin binding uniquely quenches the internal motions entirely. The F and G forms have a similar effect. Nucleotide binding restores the internal motions under conditions known to cause dissociation of the acto-S-1 complex. A model of force generation by the actomyosin-nucleotide system, which incorporates this striking actin-induced change in S-1 structural dynamics, is proposed and discussed.

Actins↗