Binding of p-Chloromercuribenzoate to Actin*

Tonomura, Yūji; Yoshimura, Junko

doi:10.1093/oxfordjournals.jbchem.a127530

Cited by 43 publications

(19 citation statements)

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“…On the other hand, it is known that NEM inhibits F-actin-activated ATPase of myosin completely by forming a covalent bond with SH-1 of myosin (23). Further, it was reported that NEM has hardly any effect on F-actin (25). Under this situation, the most plausible postulation from the results of the present study is that the essential component is myosin.…”

Section: Discussionmentioning

confidence: 41%

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Localization of Myosin in the Internodal Cell of Nitella as Suggested by Differential Treatment with N-Ethylmaleimide

Chen¹,

Kamiya

1975

Cell Struct. Funct.

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ABSTRACT. Cytoplasmic streaming in the internodal cell of Nitella ceased quickly following the application of the SH-group modifying reagent Nethylmaleimide (NEM). Centrifugation and NEM application were combined in a single cell mounted in a double-chambered vessel. This arrangement made possible the separate treatment of the streaming endoplasm and stationary cortex with NEM. Streaming occurred normally when the endoplasm was kept intact while the cortex was treated with the reagent. In the reciprocal case, in which endoplasm was treated while cortex was untreated, no streaming occurred. The results obtained suggest that myosin molecules are not intermingled with actin filaments localized on the cortex but reside in the endoplasm, since it is known that NEM inhibits actomyosin-type ATPase conspicuously while it has little effect on F-actin. It was tentatively concluded that the cytoplasmic streaming in Nitella results from the sliding of the endoplasmic myosin molecules alongside the stationary F-actin filaments lying on the cortex.

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Section: Discussionmentioning

confidence: 41%

“…This reagent is bound with SH-groups with covalent bond and inhibits irreversibly F-actin-activated myosin-ATPase (23,28), but this reagent has practically no effect on polymerization or depolymerization of actin (25).…”

mentioning

confidence: 99%

Localization of Myosin in the Internodal Cell of Nitella as Suggested by Differential Treatment with N-Ethylmaleimide

Chen¹,

Kamiya

1975

Cell Struct. Funct.

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“…It has been widely suggested that the divalent cation participates in the binding of the ATP moiety (Martonosi & Gouvea, 1961;Strohman & Samorodin, 1962;Barany et al, 1962;Tonomura & Yoshimura, 1962;Strzelecka-Golaszewska & Drabikowski, 1967). Moreover, the affinity of actin for ATP increases when the high-affinity cation binding site is occupied (Strzelecka-Golaszewska, 1973a,b).…”

mentioning

confidence: 99%

Proton nuclear magnetic resonance and electron paramagnetic resonance studies on skeletal muscle actin indicate that the metal and nucleotide binding sites are separate

Barden¹,

Cooke²,

Wright³

et al. 1980

Biochemistry

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The distance separating the high-affinity binding sites of actin for a divalent metal ion and nucleotide was evaluated by using high-resolution proton NMR and EPR spectroscopy. Replacement of the Ca2+ or Mg2+ bound to the high-affinity divalent cation site of G-actin by trivalent lanthanide ions such as La3+, EU3+, or Gd3+ results in an increase in the mobility of the bound ATP as observed in the NMR spectra of G-actin monomers. Little difference was observed between the spectra obtained in the presence of the diamagnetic La3+ control and the paramagnetic ions Eu3+ and Gd3+ which respectively shift and broaden the proton resonances of amino acids in the vicinity of the binding site. Analysis of the NMR spectra indicates that the metal and nucleotide binding sites are separated by a distance of at least 16 A. In the past, the metal and ATP have been widely assumed to bind as a complex. Further verification that the two sites on actin are physically separated was obtained by using an ATP analogue with a nitroxide spin-label bound at the 6' position of the purine ring. An estimate of the distance was made between the site containing the ATP analogue and the paramagnetic ion, Mn2+, bound to the cation binding site. These EPR experiments were not affected by the state of polymerization of the actin. The data obtained by using this technique support the conclusion stated above, namely, that the cation and nucleotide sites on either G- or F-actin are well separated.

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“…Myosin and actin were prepared by the method of Tonomura et a1 [21] and Spudich and Watt [22], respectively. S1 was obtained by digestion of myosin filaments with achymotrypsin at a 300: 1 (by mass) ratio [23].…”

Section: Proteinsmentioning

confidence: 99%

Effect of actin on the tryptic digestion of myosin subfragment 1 in the weakly attached state

Blotnick

Mühlrád

1992

European Journal of Biochemistry

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The structure of myosin subfragment 1 (SI) in the weakly attached complex with actin was studied at three specific sites, at the 50-kDa/20-kDa and 27-kDa/SO-kDa junctions, and at the N-terminal region, using tryptic digestion as a structure-exploring tool. The structure of S1 at the vicinity of the 50-kDa/20-kDa junction is pH dependent in the weakly attached state because the tryptic cleavage at this site was fully protected by actin at pH 6.2, but the protection was only partial at pH 8.0. Since the actin protection is complete in rigor at both pH values, the results indicate that the structure of S1 at the 50-kDa/20-kDa junction differs in the two states at pH 8.0, but not at pH 6.2. Actin restores the ADP-suppressed tryptic cleavage after Lys213 at the 27-kDa/50-kDa junction in the strongly attached state, but not in the weakly attached state, which indicates structural difference between the two states at this site. ATP and ADP open a new site for tryptic cleavage in the N-terminal region of the S1 heavy chain between Arg23 and Ile24. Actin was found to suppress this cleavage in both weakly and strongly attached states, which shows that, in the vicinity of this site, the structure of S1 is similar in both states. The results indicate that the binding of S1 to actin induces localized changes in the S1 structure, and the extent of these changes is different in the various actin-S1 complexes.Myosin and actin are two cardinal proteins of muscle, whose interaction with each other, coupled with the hydrolysis of ATP, constitutes the molecular basis of contraction. The head segment of myosin, called S1, contains the distinct actinbinding and nucleotide-binding sites of myosin [I] and is sufficient, together with ATP and actin, to generate movement in vitro [2]. It is, therefore, obvious that its structural and functional characterization has great significance. The heavy chain of S1 is cleaved by limited tryptic digestion into three major fragments of 27 kDa, 50 kDa, and 20 kDa (aligned from the N-terminus in this order [3]). The fragments have become a valuable framework in which the nucleotide-binding and actin-binding sites and other specific functions can be assigned.It is generally assumed [4] that the contractile force originates from the hydrolysis of ATP taking place at the ATPbinding site of S1. Each intermediate in the ATP cycle induces specific conformational changes in S1, which define the actin-S1 relationship at each step. In the various actin-S1 complexes, the affinity of S1 for actin depends on the occupancy of the ATP-binding site. On the basis of affinity, three types of S1-actin complexes can be defined [5]: (a) rigor complex, noCorrespondence to A. Muhlrad,

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Binding of p-Chloromercuribenzoate to Actin*

Cited by 43 publications

References 0 publications

Localization of Myosin in the Internodal Cell of Nitella as Suggested by Differential Treatment with N-Ethylmaleimide

Localization of Myosin in the Internodal Cell of Nitella as Suggested by Differential Treatment with N-Ethylmaleimide

Proton nuclear magnetic resonance and electron paramagnetic resonance studies on skeletal muscle actin indicate that the metal and nucleotide binding sites are separate

Effect of actin on the tryptic digestion of myosin subfragment 1 in the weakly attached state

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