Consequences of reacting the thiols of myosin subfragment 1

Botts, Jean; Ue, Kathleen; Hozumi, Tetsu; Samet, Jeffrey H.

doi:10.1021/bi00590a020

Cited by 33 publications

(26 citation statements)

References 31 publications

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“…Conversely, two reagents were examined which modify sulfhydryl residues in proteins (Smith & Kenyon, 1974;Smith, Maggio & Kenyan, 1975;Botts, Ue, Hozumi & Samet, 1979;Strauss, 1984). The reagents chosen were MMTS and NEM.…”

Section: Resultsmentioning

confidence: 99%

“…The reagents chosen were MMTS and NEM. These reagents differ significantly in their structure and mode of modification (Smith & Kenyon, 1974;Smith et al, 1975;Botts et al, 1979;Strauss, 1984). As shown in Table 2, the effect of modification on Na+-Ca 2+ exchange differed strikingly between the two reagents.…”

Section: Resultsmentioning

confidence: 99%

“…The anomalous results with MMTS may be explained on the basis of reagent size. All of the sulfhydryl modiffers with the exception of MMTS incorporate relatively bulky groups at these structurally sensitive locations (Smith & Kenyon, 1974;Smith et al, 1975;Botts et al, 1979). It is possible that the modification of sulfhydryl residues with the relatively small reagent MMTS would have a different steric effect on Na+-Ca 2+ exchange, which would result in the observed stimulation.…”

Section: Discussionmentioning

confidence: 99%

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Role for sulfur-containing groups in the Na+−Ca2+ exchange of cardiac sarcolemmal vesicles

1986

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Different amino acid residues in cardiac sarcolemmal vesicles were modified by incubation with various chemical reagents. The effects of these modifications on sarcolemmal Na+-Ca2+ exchange were examined. Dithiothreitol, an agent that maintains sulfur-containing residues in a reduced state, caused a time- and concentration-dependent decrease in Na+-Ca2+ exchange. The treatment with dithiothreitol resulted in a decrease in Vmax values but did not alter the Km for Ca2+ for the Na2+-Ca2+ exchange reaction. If Na+ replaced K+ as the ion present during the modification of sarcolemmal membranes with dithiothreitol, there was substantially less of an inhibitor effect on Na+-Ca2+ exchange. Similar results were obtained with reduced glutathione, a reagent that also maintains sulfur-containing residues in a reduced state. Two sulfhydryl modifying reagents, methylmethanethiosulfonate and N'-ethylmaleimide, were capable of altering Na+-Ca2+ exchange, and the type of ion present during modification significantly affected the extent of this alteration. Almost all of the chemical reagents investigated that modified other amino acid resides (carboxyl, lysyl, histidyl, tyrosyl, tryptophanyl, arginyl and hydroxyl) had the capacity to alter Na+-Ca2+ exchange after preincubation with the sarcolemmal membrane vesicles. However, the sulfur residue-modifying reagents were the only compounds to exhibit significant differences in their action on Na+-Ca2+ exchange, depending on whether Na+ or K+ was present in the preincubation modification medium. The tryptophan modifier, N-bromosuccinimide, was the sole reagent that elicited a substantial increase in membrane permeability. The evidence is consistent with the hypothesis that sulfur-containing residues interact with a Na+-binding site for Na+-Ca2+ exchange in cardiac sarcolemmal vesicles.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Role for sulfur-containing groups in the Na+−Ca2+ exchange of cardiac sarcolemmal vesicles

1986

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“…An immediate stimulus for this study was the S-1 modification reported most recently, the generalized lysine methylation (1) used to prepare myosin crystals for structural studies (2). White and Rayment (3) have reported that such methylation markedly increases the Mg2+-ATPase activity of S-1, in a manner kinetically similar to that produced by attaching a bulky group to Cys-707 (4,5). Do the seemingly disparate modulations such as those induced by generalized lysine methylation, modification of Cys-707, transition from pH 7.5 to 9.2, and many other agencies (6, 7) share a common feature with one another and perhaps with F-actin binding?…”

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confidence: 99%

Effect of lysine methylation and other ATPase modulators on the active site of myosin subfragment 1.

Bivin

Khoroshev

et al. 1994

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

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Many and diverse modifications of the myosin subfragment 1 (S-1) increase (modulate) its ATPase activity, including interaction of this particle with actin; a recent addition to these modifications is the extensive lysine modification of S-1 that seems prerequisite to crystallizing it for structure analysis. In this study we first established kinetically the ATPase modulations induced by various treatments of the myosin S-1 enzyme, and we also measured two properties of the S-1 active site-the affinity with which the site binds (a fluorescent analog of) the enzymatic nucleotide product and the access that a fluorescence quencher has to the bound ADP product-in an effort to get at the mechanism of modulation. Modulations achieved by substituting Ca2+ for the normal Mg2+ cocatalyst or by substituting Cl-for the normal carboxylate anion seem due to the product being held more loosely by the modulated enzyme. In other illustrative modulations (lysine methylation, or alkylation of or transition from neutral pH to pH 9.2) nucleotide product affinity and access to quencher do change, but not in a pattern explained simply by a lifting of product inhibition. Lysine methylation results in weaker binding of nucleotide product.Various modifications of myosin subfragment 1 (S-1) (including complexation with actin) modulate its ATPase rate, but the underlying mechanisms of modulation are unknown. This study looks for attendant active-site changes that may suggest a general mechanism. An immediate stimulus for this study was the S-1 modification reported most recently, the generalized lysine methylation (1) used to prepare myosin crystals for structural studies (2). White and Rayment (3) have reported that such methylation markedly increases the Mg2+-ATPase activity of S-1, in a manner kinetically similar to that produced by attaching a bulky group to 5). Do the seemingly disparate modulations such as those induced by generalized lysine methylation, modification of Cys-707, transition from pH 7.5 to 9.2, and many other agencies (6, 7) share a common feature with one another and perhaps with F-actin binding? Here we have studied three intentionally disparate modulations-methylation, alkylation of Cys-707, and the pH transition. Our efforts centered on two characterizations of the ATPase site, both deriving from an earlier observation (8) that, in the presence of an inoffensive concentration of the collisional fluorescence quencher acrylamide, a fluorescent nucleotide increased its emission upon binding to (being engulfed by) the ATPase site. By using this effect one can obtain the binding constant of the nucleotide to the site.Further, as Rosenfeld and Taylor (9) later showed, it is also possible to extract "Stern-Volmer quenching constants" of the nucleotide interacting with the site, thus measuring the access of the quencher to the bound nucleotide. These two measures-binding constant and Stern-Volmer constantexpress the physical state of the ATPase site and can be measured before ("control") and after modulating t...

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“…Another feature of interest is the relation of SH1 to the N site because modification of the former has a profound effect on catalysis at the latter. We, on the basis of modification experiments (48), and Tao and Lamkin, on the §The reasoning is instructive because it points up an ambiguity in all "distance mapping": To specify lattices of 1, 2, 3, and 4 points one requires the additional measurement of, respectively, 0, 1, 2, and 3 distances-i.e., a total of six distances. In placing the 4th point there is an unresolvable ambiguity (is it "above" or "below" the triangle formed by the previous 3?).…”

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confidence: 99%

On the mechanism of energy transduction in myosin subfragment 1.

Botts¹,

Takashi²,

Torgerson³

et al. 1984

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

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It is proposed that the myosin subfragment 1 moiety of the muscle contractile apparatus is a self-contained "engine" whose operational plan is based on the interactive nature of ATP (or degradation intermediate) binding and actin binding, made possible by an intersite communication system. It is suggested that the spatial information required for examining this engine can, at least provisionally, be derived from fluorescence resonance energy transfer measurements interpreted by the Forster equation and that the existence of an intersite communication system can be deduced from piecewise detection of interacting pairs of points. A Theory of How the Myosin Subfragment 1 (S-i) Engine WorksIt has been thought that myosin S-1 moieties in active muscle fibers, pivoting about their attachments to subfragment 2 (S-2) moieties, deliver mechanical impulses to adjacent actin filaments, thus producing the relative interfilament translation ("slide") that characterizes isotonic muscle contraction (see ref. 1 for a review). Since there is no evidence that the gross shape (as distinct from local conformation) of S-1 changes during interaction with nucleotides (2) or actin (3), it is plausible to think that in its "purposeful" motion, as in its thermal motion, the cross-bridge rotates about S-1/S-2 as a quasirigid body. Because S-1 is elongate, a natural positional parameter is the angle that its "long" axis makes with the filament axis (4). There are now investigations showing that during isometric activity (i) this angle fluctuates (5) (7). Furthermore, the motional frequencies observed in i and iii are what might be expected from independent biochemical (8) and mechanical (9) observations and are magnitudes slower than thermally activated frequencies. Thus points i-iii support the idea that during activity cross-bridges move. They also suggest that the motion is describable by angles that position S-1 relative to the fiber axis (10), but in what follows it is necessary only that positional parameters relating S-1 to actin change with time repetitively.It is known that each S-1 bears an ATPase (N) site separate from the site (A) at which it binds actin (11) and that binding at these sites is interactive (12, 13). So it is tempting to think that the observed motions of S-1 are not those of an appendage passively moved by another agency, but rather that S-1 is a self-contained "unitary engine" strategically placed in the muscle apparatus. Previously we have elaborated on this hypothesis (14). We have noted that to examine this hypothesis we must look at common knowledge about S-1 in an uncommon way. For example, instead of considering S-1 motion in a fiber-based coordinate system we should position ligands (actin, nucleotides) in an S-1-based system, and we should think of enzymatic intermediates (15) as sequentially bound N-site ligands. These simple reinterpretations suggest a plan for the S-1 "engine": Because of the intersite communication system, binding of a particular intermediate at the N site determines a parti...

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Consequences of reacting the thiols of myosin subfragment 1

Cited by 33 publications

References 31 publications

Role for sulfur-containing groups in the Na+−Ca2+ exchange of cardiac sarcolemmal vesicles

Role for sulfur-containing groups in the Na+−Ca2+ exchange of cardiac sarcolemmal vesicles

Effect of lysine methylation and other ATPase modulators on the active site of myosin subfragment 1.

On the mechanism of energy transduction in myosin subfragment 1.

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