Isolation of cyanogen bromide and tryptic peptides containing the essential thiol groups from isolated myosin heads

Kunz, Peter A.; Walser, J.T.; Watterson, J. G.; Schaub, Marcus C.

doi:10.1016/0014-5793(77)80658-3

Cited by 21 publications

(14 citation statements)

References 25 publications

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“…The effects of IAAm and its derivative spectroscopic compounds (IAEDANS and IASL) were studied in particular since IAAm has been shown to selectively modify SH-1 (Kunz et al, 1977) and these derivatives have been used in a number of spectroscopic studies. The modification conditions used in these experiments resembled those used to label muscle fibres, i.e.…”

Section: Resultsmentioning

confidence: 99%

“…We modified rabbit myosin SH-1 in conditions similar to those used to modify myofibrils and glycerinated muscle fibres with spectroscopic probes (low ionic strength, 40 mM NaCI, at 4 ~ C). IAAm and its derivative compounds were extensively studied as they are known to selectively modify SH-1 (Kunz et al, 1977). The specificity of the modification was determined on the basis of the typical alterations in the K +-and Ca2+-ATPase activities, the K+-ATPase decreased linearly with increasing amount of label bound or added and the Ca2+-ATPase was significantly elevated (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…0142-4319/89 $03.00 + .12 9 1989 Chapman and Hall Ltd. be specifically modified in vitro (Sekine et al, 1962;Kunz et al, 1977). The extent of reaction of SH-1 with a given reagent can be quantitatively monitored by characteristic alterations in the non-physiological myosin ATPase activities (Sekine & Kielley, 1964).…”

Section: Introductionmentioning

confidence: 99%

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SH-1 modification of rabbit myosin interferes with calcium regulation

Titus¹,

Ashiba²,

Szent‐Györgyi³

1989

J Muscle Res Cell Motil

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The reactive thiol of the myosin head, SH-1, can be selectively labelled in glycerinated rabbit muscle fibres. This residue has been used as an attachment site for either fluorescent or spectroscopic probes which report on head movements and orientations in various functional states of muscle. We have specifically modified SH-1 in vitro, using purified rabbit myosin and conditions similar to those employed in the labelling of muscle fibres (low ionic strength [40 mM NaCl] at 4 degrees C), with stoichiometric amounts of either [14C]-iodoacetamide, 5-(2[iodoacetyl)amino)ethyl) aminonaphthalene-1- sulphonic acid (IAEDANS), or 4-(2-iodoacetamido-2,2,6,6-tetramethyl piperidinooxyl (IASL). The specificity of modification was determined by measuring the well-defined alterations in the high salt ATPase activities of myosin and by localizing both IAAm and IAEDANS to the 20-kDa C-terminal subfragment 1 (S1) which contains SH-1. The low ionic strength actin-activated Mg2+-ATPase of SH-1-modified rabbit myosin was measured in the presence of the thin filament regulatory, complex, troponin-tropomyosin. A significant increase in this activity in the absence of calcium, concomitant with a decrease in activity in the presence of calcium, was observed as the extent of SH-1 modification was incrementally increased from zero to one mole of label bound per mole of SH-1. The elevated myosin Mg2+-ATPase, which results from SH-1 modification, does not account for the increased actin-activated Mg2+-ATPase in resting conditions (i.e. in the absence of calcium).(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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SH-1 modification of rabbit myosin interferes with calcium regulation

Titus¹,

Ashiba²,

Szent‐Györgyi³

1989

J Muscle Res Cell Motil

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“…In a first step, the reactive SHi group of chymotryptic S-1 (containing LC1 and LC3) was protected by three reagents that react quantitatively and specifically with this thiol. These were the fluorescent dye 1,5-IAEDANS (20), iodoacetamide (22), and N2ph-F (21); N2ph-F allows a reversible blocking of the SH1 thiol (25). The isolated S-1 derivatives exhibited about 5% the K+-ATPase activity of unmodified S-1, whereas their Ca2+-ATPase was enhanced to 350-450% the control value.…”

Section: Resultsmentioning

confidence: 99%

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

Chaussepied¹,

Mornet²,

Kassab³

1986

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

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When myosin subfragment 1 derivatives in which the reactive sulfhydryl SH1 has been blocked react with N,N'-p-phenylenedimaleimide or 5,5'-dithiobis(2-nitrobenzoic acid), the reactive sulfhydryl group SH2 of the 20-kDa domain is crosslinked with a thiol of the 50-kDa domain of the heavy chain. The crosslink induces the stable trapping of a significant amount of Rabbit skeletal muscle actin was prepared as described by Eisenberg and Kielley (17), and its concentration was estimated by using A19i, = 11.0.Crosslinking Reactions. The reaction of 1.3-fold excess Ph(NMal)2 in 50 mM Hepes (pH 8.0) was done with 50 ,uM modified or native enzyme in the presence or absence of 2.5 mM Mg2+-ATP or Mg2+-ADP at 4°C. The crosslinking of S-1 with Nbs2 was performed under similar conditions, using a 5-fold excess of reagent. The reactions were quenched and the protein derivatives were purified by filtration over Sephadex G-50 (20 x 1.5 cm) in 50 mM Hepes, pH 7.0/200 mM KCl/0.5 mM NaN3 at 40C. NaDodSO4/PAGE. Tryptic fragments of modified S-1 were separated by electrophoresis in 0.1% NaDodSO4/polyacrylamide slab gels containing a 5-18% (wt/vol) gradient of acrylamide (18). The running buffer was 50 mM Tris/100 mM boric acid (pH 8.0) (19). The densitometric scanning of the gels was carried out with a computerized model CS930 Shimadzu (Kyoto, Japan) gel scanner. The following were used as molecular mass markers: S-1 heavy chain (95 kDa), S-1 light chain 1 (LC1, 25 kDa), S-1 light chain 3 (LC3, 17 kDa), the three fragments obtained by tryptic cleavage of S-1 (50 kDa, 27 kDa, and 20 kDa), and actin (42 kDa).Specific Modifications of S-1 Thiols. The specific fluorescence labeling of SH1 with 5-[2-(iodoacetyl)aminoethyl]aminonaphthalene-1-sulfonic acid (1,5-IAEDANS) was done according to the procedure of Takashi et al. (20). Under our conditions, 1.1 mol of the dye was incorporated per mol of S-1. Fluorescence gel scanning allowed us to quantify the labeling ratio as 0.2 in the light chains and 0.9 in the heavy chain.The specific chemical modifications of SH1 by 2,4-dinitrofluorobenzene (N2ph-F) and iodoacetamide were carried out as described by Reisler et al.

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“…Properties of BD-Alkylation of S-1 with IAA and IAEDANS invariably results in the modification of only SH1 (20,26,27). Although chemical properties of BD had scarcely been studied, the reagent was expected to be useful as a specific fluorescent label for SH1 of S-1.…”

Section: Resultsmentioning

confidence: 99%

ATP-induced Opposite Changes in the Local Environments around Cys697 (SH2) and Cys707 (SH1) of the Myosin Motor Domain Revealed by the Prodan Fluorescence

Hiratsuka

1999

Journal of Biological Chemistry

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To obtain a consistent view of the nucleotide-induced conformational changes around Cys 697 (SH2) and Cys 707(SH1) in skeletal myosin subfragment-1 (S-1), the two thiols were labeled with the same environmentally sensitive fluorophore, 6-acyl-2-dimethylaminonaphthalene group, using 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AD) and 6-bromoacetyl-2-dimethylaminonaphthalene (BD), respectively. The resultant fluorescent derivatives, AD-S-1 and BD-S-1, have the same fluorophore at either SH2 or SH1, which was verified by inspections of changes in the ATPases and the localization of fluorescence after tryptic digestion and CNBr cleavage for the two derivatives. Especially, AD was found to be a very useful fluorescent reagent that readily reacts with only SH2 of S-1. Measurements of the nucleotideinduced changes in fluorescence emission spectra of AD-S-1 and BD-S-1 suggested that during ATP hydrolysis the environment around the fluorophore at SH2 is very distinct from that around the fluorophore at SH1, being defined as that the former has the hydrophobic and closed characteristics, whereas the latter has the hydrophilic and open ones. The KI quenching study of the fluorescence of the two S-1 derivatives confirmed these results. The most straightforward interpretation for the present results is that during ATP hydrolysis, the helix containing SH2 is buried in hydrophobic side chains and rather reinforced, whereas the adjacent helix containing SH1 moves away from its stabilizing tertiary structural environment.The structural element of the molecular motor myosin is the subfragment-1 (S-1) 1 moiety that contains the sites responsible for the ATP hydrolysis and binding of actin (1). A striking feature of the S-1 structure is a long helix spanning 85 Å that is stabilized by interactions with the myosin light chains (2). It has been proposed that this light chain-binding domain acts as a semi rigid "lever arm" to amplify and transmit conformational changes in the ATP and actin binding sites of S-1 (3-9).Cys 707 (SH1) and Cys 697 (SH2) are located on the two different helixes in the C-terminal segment of the S-1 catalytic domain and separated from one another by 19 Å (2). The two helixes are kinked at a conserved glycine residue (Gly 699 ) found in a bend joining two helixes. Recent experiments have led to a conclusion that the fulcrum point for the swinging motion of the lever arm is in the vicinity of the SH1-SH2 region (4 -9). Furthermore, Gly 699 (close to SH2) and Gly 710 (close to SH1) have been proposed to act as pivot points or flexible hinges for such motion (8,10,11). These data show the importance of motion in the SH1-SH2 region in the force generation cycle of myosin. However, the precise conformational changes of this region that are involved in lever arm movement are still unknown.One approach for understanding the involvement of the SH1-SH2 region in lever arm movement is a precise comparison of the individual conformational changes around SH1 and SH2 during ATPase cycle of S-1. Such a study could...

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Isolation of cyanogen bromide and tryptic peptides containing the essential thiol groups from isolated myosin heads

Cited by 21 publications

References 25 publications

SH-1 modification of rabbit myosin interferes with calcium regulation

SH-1 modification of rabbit myosin interferes with calcium regulation

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

ATP-induced Opposite Changes in the Local Environments around Cys697 (SH2) and Cys707 (SH1) of the Myosin Motor Domain Revealed by the Prodan Fluorescence

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