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J C Seidel

Publications and source records attributed to J C Seidel.

At least 37 records · Page 2Linked to original sources
PubMed1979

Enzymatic activities and ATP-induced fluorescence enhancement of myosin from fast and slow skeletal and cardiac muscles.

The maximal ATP-induced enhancement of fluorescence and the dependence of this enhancement on ATP concentration were determined for myosins from fast and slow skeletal and cardiac muscle of the rabbit. With myosins from slow and cardiac muscle modifications in the preparative procedure and chromatography on DEAE-Sephadex were required to obtain preprations which were free of actin, which exhibited the maximal fluorescence enhancement and which bound two moles of ATP per mole of myosin. Since the fluorescence enhancement of cardiac and slow muscle myosins is labile at slightly alkaline pH, it was also necessary to minimize incubation at pH greater than 7 in order to attain the maximal enhancement. With fast muscle myosin the changes in preparative procedure, together with chromatography, led to a 50 to 100% increase in the steady-state rate of ATP hydrolysis and fluorescence enhancement, without changing the maximal binding of ATP. From a comparison of the rate of steady-state hydrolysis of ATP with the rate of decay of the enhanced fluorescence, it appears that for all three myosins, both ATP binding sites have the same enzymatic activity, the steady-state rate per site being slower for cardiac and slow muscle myosins than for fast muscle myosin.

Adenosine Triphosphatases↗
PubMed1976

Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity.

The Ca2+ binding component (TnC) of troponin has been selectively labeled with either a spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, or with a fluorescent probe, S-mercuric-N-dansyl cysteine, presumably at its single cysteine residue (Cys-98) in order to probe the interactions of TnC with divalent metals and with other subunits of troponin. The modified protein has the same Ca2+ binding properties as native TnC (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628), viz. two Ca2+ binding sites at which Mg2+ appears to compete (Ca2+-Mg2+ sites, KCa = 2 X 10(7) M-1) and two sites at which Mg2+ does not compete (Ca2+-specific sites, KCa = 2 X 10(5) M-1). Either Ca2+ or Mg2+ alters the ESR spectrum of spin-labeled TnC in a manner that indicates a decrease in the mobility of the label, Ca2+ having a slightly greater effect. In systems containing both Ca2+ and Mg2+ the mobility of the spin label is identical with that in systems containing Ca2+ alone. The binding constants for Ca2+ and Mg2+ deduced from ESR spectral changes are 10(7) and 10(3) M-1, respectively, and the apparent affinity for Ca2+ decreases by about an order of magnitude on adding 2 mM Mg2+. Thus, the ESR spectral change is associated with binding of Ca2+ to one or both of the Ca2+-Mg2+ sites. Addition of Ca2+ to the binary complexes of spin-labeled TnC with either troponin T (TnT) or troponin I (TnI) produces greater reduction in the mobility of the spin label than in the case of spin-labeled TnC alone, and in the case of the complex with TnI the affinity for Ca2+ is increased by an order of magnitude. The fluorescence of dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-labeled TnC is enhanced by Ca2+ binding to both high and low affinity sites with apparent binding constants of 2.6 X 10(7) M-1 and 2.9 X 10(5) M-1, respectively, calculated from the transition midpoints. The presence of 2 mM Mg2+, which produces no effect on dansyl fluorescence itself, in contrast to its effect on the spin label, shifts the high affinity constant to 2 X 10(6) M-1. Spectral changes produced by Ca2+ binding to the TnC-TnI complex furnish evidence that the affinity of TnC for Ca2+ is increased in the complex. The reactivity of Cys-98 to the labels and to 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is decreased by Ca2+ or Mg2+ both with native TnC and in 6 M urea. The reaction rate between Cys-98 and Nbs2 decreases to one-half the maximal value at a Ca2+ concentration that suggests binding to the Ca2+-Mg2+ sites. Formation of a binary complex between TnI and TnC reduces the rate of reaction, and there is a further reduction by Ca2+. The effect of Ca2+ takes place at concentrations that are 1 order of magnitude lower than in the case of TnC alone. These results suggest that the Ca2+ binding site adjacent to Cys-98 is one of the Ca2+-Mg2+ binding sites.

Binding Sites↗
PubMed1975

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.

Adenosine Triphosphatases↗
PubMed1975

The quantitative measurement of rotational motion of the subfragment-1 region of myosin by saturation transfer epr spectroscopy.

According to current models of muscle contraction (Huxley, H. E., Science 164: 1356-1366 [1969]), motion of flexible myosin crossbridges is essential to the contractile cycle. Using a spin-label analog of iodoacetamide bound to the subfragment No. 1 (S1) region of myosin, we have obtained rotational correlation times (tau 2) for this region of the molecule with the ultimate goal of making quantitative measurements of the motion of the crossbridges under conditions comparable to those in living, contracting muscle. We used the newly developed technique of saturation transfer electron paramagnetic resonance spectroscopy (Hyde, J.S., and Thomas, D.D., Ann. N.Y. Acad Sci. 22:680-692 [1973]), which is uniquely sensitive to rotational motion in the range of 10(-7)-10(-3) sec. Our results indicate that the spin label is rigidly bound to S1 (tau 2 for isolated S1 is 2 X 10(-7) sec) and that the motion of the label reflects the motion of the S1 region of myosin. the value of tau 2 for the S1 segment of myosin is less than twice that for isolated S1, while the molecular weights differ by a factor of 4, indicating flexibility of myosin in agreement with the conclusions of Mendelson et al. (Biochemistry 12:2250-2255 [1973]). Adding F-actin increses tau 2 in either myosin or isolated S1 by a factor of mearly 103, indicating rigid immobilization of S1 by actin. Formation of myosin filaments (at an ionic strength of 0.15 or less) increses tau 2 by a factor of 10-30, depending on the ionic strength, indicating a decrease of the rotational mobility of S1 in these agregates. The remaining motion is at least a factor of 10 faster than would be expected for the filament itself, suggesting motion of the S1 region independent of the filament backbone but slower than in a single molecule. F-actin has a strong immobilizing effect on labeled S1 in myosin filaments (in 0.137 M KC1), but the immobilization is less complete than that observed when F-actin is added to labeled myosin monomers (in 0.5 M KC1). A spin-label analog of maleimide, attached to the SH-2 thiol groups of S1, is immobilized to a much lesser extent by F-actin than is the label on SH-1 groups. The maleimide label also was attached directly to F-actin and was sufficiently immobilized to suggest rigid binding to actin.

Actins↗
PubMed1975

Motion of subfragment-1 in myosin and its supramolecular complexes: saturation transfer electron paramagnetic resonance.

Molecular dynamics in spin-labeled muscle proteins was studied with a recently developed electron paramagnetic resonance (EPR) technique, saturation transfer spectroscopy, which is uniquely sensitive to rotational motion in the range of 10(-7)-10(-3) sec. Rotational correlation time (tau2) were determined for a spin label analog of iodoacetamide bound to the subfragment-1 (S-1) region of myosin under a variety of conditions likely to shed light on the molecular mechanism of muscle contraction. Results show that (a) the spin labels are rigidly bound to the isolated S-1 (tau2 = 2 x 10(-7) sec) and can be used to estimate values of tau2 for the S-1 region of the myosin molecule; (b) in solutions of intact myosin, S-1 has considerable mobility relative to the rest of the myosin molecule, the value of tau2 for the S-1 segment of myosin being less than twice that for isolated S-1, while the molecular weights differ by a factor of 4 to 5; (c) in myosin filaments, tau2 increases by a factor of only about 10, suggesting motion of the S-1 regions independent of the backbone of the myosin filament, but slower than that in a single molecule; (d) addition of F-actin to solutions of myosin or S-1 increases tau2 by a factor of nearly 10(3), indicating strong immobilization of S-1 upon binding to actin. Saturation transfer spectroscopy promises to provide an extremely useful tool for the study of the motions of the crossbridges and thin filaments in reconstituted systems and in glycerinated muscle fibers.

Binding Sites↗