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S Highsmith

Publications and source records attributed to S Highsmith.

52 records · Page 3Linked to original sources
PubMed1982

Detergent-solubilized sarcoplasmic reticulum ATPase. Hydrodynamic and catalytic properties.

Solubilization of the ATPase of sarcoplasmic reticulum vesicles with the nonionic detergent dodecyl non-aoxyethylene alcohol (C12E9) resulted in a large (about 5-fold) increase in its Ca2+ ATPase activity. Measurements using a calcium ionophore suggest this activation was the result of rendering the vesicles permeable to calcium. Complete activity is preserved at a detergent concentration range in which the detergent is complexed with the monomeric form of the ATPase, as measured by Sepharose 6B chromatography. Using a calibrated column, we found the C12E9 complex to have a Stokes radius of 55 A. As measured by time-resolved fluorescence anisotropy decay experiments, it had a rotational correlation coefficient of 214 ns, which is equivalent to a Stokes radius of 59 A. The axial ratio of the corresponding ellipsoid of revolution is calculated to be 5 to 6, indicating the complex is quite asymmetric. Like the vesicular form of the ATPase, the detergent-solubilized monomeric form bound with high affinity about 9 nmol of Ca2+/mg of protein. Also, like the vesicular enzyme, the solubilized form displayed a Ca2+ dependence of the activation of ATP hydrolysis which was cooperative (Hill coefficient 1.8). These results suggest that the calcium sites interact intramolecularly.

Animals↗
PubMed1982

Bending motions and internal motions in myosin rod.

Depolarized light scattering and high-resolution 1H NMR measurements were made on solutions of light meromyosin (LMM) and myosin rod in 0.6 M KCl-0.010 M pyrophosphate, pH 9.5, at 20 degrees C. The light scattering data indicated LMM is a rigid molecule. Myosin rod is best described as a once-broken rod with domains that can freely diffuse in conical volumes. The maximum angle that the cone surface makes with the myosin rod axis is 128 degrees, indicating the bending motion of the domains is largely unrestrained. NMR data indicated the fraction of the structure that could be in a random-coil configuration was equal and less than 0.04 for both hydrodynamically rigid LMM and bending myosin rod. Thus, the flexible bending of myosin rod appears to not be due to a random-coil structure.

Animals↗
PubMed1981

Internal motions in myosin. 2.

The high-resolution 1H NMR detected internal motions in myosin and myosin subfragment 1 (S1) [Highsmith, S., Akasaka, K., Konrad, M., Goody, R., Holmes, K., Wade-Jardetzky, N., & Jardetzky, O. (1979) Biochemistry 18, 4238--4244] were unperturbed by induced changes in the rate of protein tumbling, and the mobile regions proved inaccessible to added surface-directed paramagnetic probes. The rate of tumbling was changed by changing the solvent viscosity for S1 or by aggregation to thick filaments for myosin. Neither manipulation caused a measurable broadening of the narrow lines in the spectrum. Sulfhydryl-directed covalently attached nitroxide spin-labels, soluble nitroxide spin-labels, and MnCl2 were used to probe the surface. Unique labeling at the fastest reacting thiol of S1 had no effect on the NMR spectrum. Multiple labeling of thiols caused a small but detectable broadening of the narrow peaks. Soluble spin-labels and MnCl2 had a very small effect on the narrow bands even in great excess. The results substantiate the notion that myosin has internal motions that are independent of the overall rate of rotation and suggest that the mobile structure is mainly in the interior of the S1 moiety. This supports a model in which actin quenches the internal motions of myosin by changing the structure of myosin upon binding.

Animals↗
PubMed1979

Internal motions in myosin.

High-resolution proton nuclear magnetic resonance (1H NMR) measurements were made on myosin, heavy meromyosin (HMM), myosin subfragment 1 (S1), light meromyosin (LMM), and actin. A strong signal from amino acid side chains undergoing motions too fast to be accounted for by simple rotations of groups on a rigid backbone was obtained from myosin. Comparison of myosin, HMM, S1, and LMM showed that the mobile region is located almost entirely in S1 and accounts for approximately 22% of its structure. Adenosine triphosphate (ATP) and ATP analogues had no measurable effect on the S1 spectrum. Actin, on the other hand, quenched the internal motions of S1. When S1 was titrated with actin, an association was obtained which was in agreement with other measured values. The actin effect was reversed by adding magnesium pyrophosphate (MgPPi) or adenyl-5'-yl imidophosphate (MgAMPPNP). Quantitative treatment of the broad signals from myosin and its subfragments substantiated the existence of two flexible regions in myosin. The highly mobile portion of myosin may be located in the "swivel" between S1 and the rest of myosin or in the actin binding site or in both. These possibilites are discussed, and a new possible mechanism for muscle cross bridge elasticity is proposed.

Actins↗
PubMed1978

The effects of divalent cations on the rotational mobility of myosin, heavy meromyosin and myosin subfragment-1 and on the binding of heavy meromyosin to actin.

The effects of the divalent cations Mg2+, Mn2+ and Ca2+ on the Brownian rotational motion of fluorescently labeled myosin, heavy meromyosin and myosin subfragment-1 were measured by the method of time-resolved fluorescence depolarization. When Mg2+ was added to solutions of myosin or heavy meromyosin and EDTA, their rotational mobility increased. Ca2+ had no effect. Mn2+ increased the mobility of heavy meromyosin but decreased that of myosin. None of these divalent cations effected the mobility of subfragment-1. The binding of heavy meromyosin to actin was affected very little by Mg2+ or EDTA over a wide range of conditions. Divalent cations appear to change the swivel about which the heads of myosin rotate, presumably by binding to light chain 2 (also called DTNB light chain). However, the heads are still able to bind actin in nearly the same way whether Mg2+ is present or not. The concentration of free Mg2+ for the mid-point of the change in heavy meromyosin mobility is in good agreement with that for EDTA activation of ATPase activity. This suggests that EDTA activation is due to removal of Mg2+ bound to myosin itself.

Actins↗
PubMed1978

Heavy meromyosin binds actin with negative cooperativity.

The association of fluorescently labeled heavy meromyosin (HMM) and F-actin was measured by time-resolved fluorescence depolarization. The effects of varying the protein concentrations, temperature, KCl concentration, and pH were determined. Measurements of HMM mobility supported a model of no interaction between the two heads in the absence of actin. Measurements of actin binding, when compared with results for myosin subfragment I, indicated that the two heads of HMM do not bind independently in the rigor complex. This could result from actin-transmitted negative cooperativity or from steric inhibition due to the structure of HMM. For HMM and actin in 0.15 7 kcl at 25 degrees C: Ka = 3.9 X 10(7) M-1, deltaHco' = 36 +/- 2 J M-1, deltaSco' = 0.26 +/- 0.02 kJ M-1 K-1; the slope of ln Ka vs. [KCl]1/2 = -3.88 and the pH of maximum association was 6.9.

Actins↗
PubMed1977

Flexibility of myosin rod, light meromyosin, and myosin subfragment-2 in solution.

Myosin rod was prepared by papain proteolysis of myosin. The components of rod, light meromyosin (LMM) and subfragment-2 (S-2), were prepared by proteolysis of myosin and rod, respectively, using trypsin treated with tosylphenylalanine chloromethyl ketone. S-2, thus prepared, was of greater molecular weight than obtained previously, so that the combined molecular weights of LMM and S-2 were equal to that of rod, and S-2 contained virtually all of the region of the rod susceptible to trypsin. Electro-optical measurements were made on the three fragments in 2 mM sodium pyrophosphate, pH 9.3 at 3 degrees, over a large range of protein concentrations. Analysis of the relaxation of birefringence, at low protein concentration where there was no aggregation, showed that LMM (relaxation time 13.1 micros) behaves as a rigid cylinder. Rod (relaxation time 41.2 micros) and S-2 (relaxation time 6.0 micros) had relaxation rates that were too fast for rigid molecules of their dimensions, and therefore are not straight rods. This implies that myosin rod is flexible in the S-2 portion, presumably in the region susceptible to proteolysis. The implications of rod flexibility for the mechanism of muscle contraction are discussed.

Birefringence↗
PubMed1976

Interactions of the actin and nucleotide binding sites on myosin subfragment 1.

The effects of selected nucleotides (N) on the binding of myosin subfragment 1 (S-1) and pure F-actin (A) were measured by time-resolved fluorescence depolarization for 0.15 M KCl, pH 7.0 at 4 degrees. The association constants K'A, KN, and K'N in the scheme (see article), were determined for the magnesium salts of ADP, adenyl-5'-yl imidodiphosphate AMP-P(NH)P, and PPi. The nucleotide binding site on S-1 was "mapped" with respect to its interaction on the actin binding site. The subsites were the beta- and gamma-phosphoryl groups of ATP bind had the largest effects. A quantitative measure of the interaction, the interaction free energy, was defined as -RT ln (KA/K'A). For ADP, K'A was 2.7 X 10(5) M-1 and the interaction free energy was -4.67 kJ M-1. For AMP-P(NH)P and PPi it was much larger. A ternary complex was shown to exist for ADP, S-1, and actin in the presence of Mg2+ and evidence from AMP-P(NH)P and PPi measurements indicated that ATP also likely forms a ternary complex. The mechanism of (S-1)-actin dissociation is discussed in light of these results.

Actins↗
PubMed1976

Affinity of myosin S-1 for F-actin, measured by time-resolved fluorescence anisotropy.

The association constant for myosin subfragment-1 (S-1) and actin was measured, using a new application of fluorescence depolarization which capitalizes on the fact that S-1 has high rotational mobility while F-actin does not. Uncoupling of the time dependences of the anisotropy decay and the association/dissociation phenomena allowed the experimentally determined anisotropy decay curve to be fitted by a sum of two terms weighted by the mole fractions of the free and bound S-1. At 4 degrees C, ionic strength 0.16 M, and pH 7.0, the association constant Ka is (1.73 +/- 0.35) X 10(6) M-1 at infinite dilution. This makes the -deltaG degrees of binding of F-actin to S-1 similar to the -deltaG degrees of binding of ATP to S-1, and the possible physiological relevance of the similarity to muscle contraction is discussed.

Actins↗
PubMed1975

Mechanism of action of bovine testicular hyaluronidase. Mapping of the active site.

The reactions of purified, homogeneous bovine testicular hyaluronidase have been studied with radioactively labeled oligomers of hyalobiuronic acid, (GlcUA-GlcNAc)n, as substrates and acceptors. Transglycosylation occurs by transfer of a glycosyl residue with retention of configuration from a leaving group to an acceptor. On the basis of detailed examination of cleavage and transglycosylation patterns for the trimer; comparison of trimer, tetramer, and polymer as substrates; comparison of acceptors; equilibrium binding; and other data, it is proposed that the enzyme's active site consists of five subsites for hyalobiuronate residues. In the terminology of Schechter, I., and Berger, A. ((1966) Biochemistry 5, 3371), these are s2-s1-s' 2-s3, where the reducing terminus is to the right, and cleavage occurs between s1 and s' 1. It is proposed that subsite s'2 has a high affinity for a substrate residue, while s1 and s'1 have low substrate affinity, and s2 and s' 3 are intermediate in affinity. This proposal is seen to have mechanistic implications. The reactions of several substrates show similar bell-shaped pH dependences, with optima in the region of pH 5 to 5.5.

Animals↗