Time‐dependent fluorescence depolarization and lifetime studies of myosin subfragment‐one in the presence of nucleotide and actin

Mendelson, Robert A.; Putnam, Susan; Morales, Manuel F.

doi:10.1002/jss.400030209

Cited by 55 publications

(37 citation statements)

References 17 publications

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“…Inhibition of K + (EDTA)-ATPase and activation of Ca 2+ -ATPase indicated specific labeling of the SHi thiols. The Ca 2+ -ATPase of fully labeled cardiac myosin (2 dyes/myosin) was activated between 6-and 7-fold, in agreement with previous findings for skeletal myosin (Mendelson et al, 1975) which indicated (as in the case of the spin label) that a fairly specific labeling of SH, had occurred.…”

Section: Fluorescence-labeled Cardiac Myosinsupporting

confidence: 90%

“…The effect of actin addition on saturation of the spin-labeled myosin EPR spectrum (Seidel, 1973) was calculated from R3 (actomyosin)/R-j (myosin) where R3 is the ratio of the amplitude of peak 3 (the midpeak) at 180 mW (high microwave power) to the amplitude of the same peak recorded at 3.2 raW (low power). Fluorescence depolarization measurements were made at 5°C using the nanosecond-decay fluorimeter previously described (Mendelson et al, 1975).…”

Section: Methodsmentioning

confidence: 99%

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EPR and fluorescence depolarization studies on bovine cardiac myosin.

Stone¹,

Mendelson²,

Botts³

et al. 1981

Circulation Research

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SUMMARY To test for possible differences in local conformation and SI flexibility, bovine cardiac and rabbit skeletal myosins were labeled with a fluorophore (1,5-IAEDANS*) and a spin label having iodoacetamide reactivity. The marked activation of the Ca 1+ -ATPase (G-to 8-fold) and inhibition of the K + (EDTA)-ATPase (80-90%) by both labels indicated specific labeling of the fast-reacting thiols (SH,) of both myosins. Fluorescence depolarization studies of 1,5-IAEDANS-labeled cardiac myosin indicated that, like skeletal myosin, the SI moieties of cardiac myosin exhibit considerable segmental flexibility with respect to the rod portion of the molecule. This indicates that segmental flexibility may be a property of all myosins. Cardiac and skeletal myosins immobilized spin labels to approximately the same extent, indicating a similarity in eteric restraints around the SHj thiol of the two myosins. The magnitude of the changes in spin label mobility accompanying binding of MgADP and hydrolysis of MgATP was reduced in cardiac myosin relative to skeletal myosin. This suggests that the lower catalytic center activity of cardiac myosin is associated with more restricted conformational changes accompanying formation of M*«ADP and M**"ADP»P,. From measurements of spin label mobility, the affinity of cardiac and skeletal myosin for ADP were similar: Kj (ADP) -7 UM, n -1.8. The EPR spectrum of spin labels attached to cardiac and skeletal myosin showed similar saturation effects upon actin binding indicating immobilization of myosin heads occurs with both proteins. Circ Res 49: 677-6S4, 1981

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Section: Fluorescence-labeled Cardiac Myosinsupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

EPR and fluorescence depolarization studies on bovine cardiac myosin.

Stone¹,

Mendelson²,

Botts³

et al. 1981

Circulation Research

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“…This suggested these SH groups could move from 14 A to as close as 7 A upon binding of MgADP. We have recently provided further support for the proximal distance between SH-1 and SH-2 of [3][4][5] A based on evidence of simultaneous chelation of these two SH groups by a single-exchange-inert Co(III) phenanthroline complex (13,14).…”

mentioning

confidence: 81%

Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1.

Wells¹,

Yount²

1979

Proc. Natl. Acad. Sci. U.S.A.

146

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Studies with reagents that crosslink two thiol groups have shown that it is possible to trap nucleotides at the active site of myosin chymotryptic subfragment 1. Subfragment 1 incorporates nearly stoichiometric quantities of I'4CIATP or 114CIADP in a manner that depends linearly on the extent of inactivation by either NN'-p-phenylenedimaleimide or Co(II)phenanthroline/ICo(IIIXphenanthroline)2CO31+ complexes. The incorporated radioactive nucleotide is retained after gel filtration, even when the enzyme derivatives are stored in the presence of EDTA or nonradioactive nucleotides (t 1/2 -5 days). The nucleotide incorporated is not covalently bound because HC104 denaturation allows immediate release of bound nucleotide. The nucleotide retained is ADP because the 'y-phosphate of ly-32PJATP is lost after trapping. Subfragment 1 inactivated as above does not bind the competitive inhibitor adenosine 5'-#,B,y-imidoltriphosphate, indicating that the active site is blocked. It is proposed that a jawlike nucleotide cleft closes on MgADP or MgATP, which can be locked shut by crosslinking two thiol groups by reaction with NN'-p-phenylenedimaleimide or cobalt phenanthroline complexes. There is a large body of information available indicating that the myosin molecule and its active proteolytic subfragments, double-headed heavy meromyosin and single-headed subfragment 1 (SF-1), undergo spectroscopically sensitive conformational changes during ATP binding, hydrolysis, and product release (refs. 1-5; see ref. 6 for review). Binding of MgADP results in a well-documented (7-10) increase in the reactivity of one of two critical SH groups, so-called SH-2, suggesting this thiol moves from a buried to an exposed position.Reisler and coworkers (11) have reported that these critical thiols, SH-1 and SH-2, can be crosslinked by a 12-to 14-A thiol crosslinking reagent, N,N'-p-phenylenedimaleimide (pPDM). Furthermore, Burke and Reisler (12) reported that crosslinking with shorter bifunctional thiol reagents occurred optimally when the crosslinking was performed in the presence of MgADP. This suggested these SH groups could move from 14 A to as close as 7 A upon binding of MgADP. We have recently provided further support for the proximal distance between SH-1 and SH-2 of 3-5 A based on evidence of simultaneous chelation of these two SH groups by a single-exchange-inert Co(III) phenanthroline complex (13,14).Inactivations with pPDM (11) or cobalt phenanthroline complexes (13) are greatly stimulated by the addition of MgADP. Surprisingly, as shown here, adenosine 5-[t,-yimidoltriphosphate (AdoPP[NHIP) does not bind to SF-1 inactivated in the presence of MgADP with either reagent. These apparently conflicting observations are explained by the finding that nucleotide is trapped at the active site during nucleotidestimulated inactivation. We provide evidence that the nucleotide is specifically trapped at the hydrolytic site and is only slowly released over a period of days. The trapped nucleotide is rapidly released when the enzyme deriva...

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“…The fluorometer used in this work is substantially that previously used to study the segmental flexibility of myosin (5), and recently modified to study the species in an actin-(S-1) ATPase system (6). In each experiment 2.5 X 107 photons were collected in 25 min.…”

mentioning

confidence: 99%

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

Highsmith¹,

Mendelson²,

Morales³

1976

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Time‐dependent fluorescence depolarization and lifetime studies of myosin subfragment‐one in the presence of nucleotide and actin

Cited by 55 publications

References 17 publications

EPR and fluorescence depolarization studies on bovine cardiac myosin.

EPR and fluorescence depolarization studies on bovine cardiac myosin.

Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1.

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

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