Thrombic Digestion of Myosin Alkali Light Chains

Winstanley, Monica A.; Trayer, Ian P.

doi:10.1042/bst0070703

Cited by 10 publications

(3 citation statements)

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“…These data are consistent with the NMR studies (Prince et al, 1981) and the affinity chromatography studies (Winstanley & Trayer, 1979;, which suggest that the Al light chain, but not the A2 light chain, interacts with actin. Also, it has been recently demonstrated that the Al light chain can be chemically cross-linked to the C-terminal region of actin (Sutoh, 1982).…”

Section: Discussionsupporting

confidence: 88%

Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide

Chalovich¹,

Stein²,

Greene³

et al. 1984

Biochemistry

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Myosin subfragment 1 (S-1) can be fractionated into two isozymes, (A1)S-1 containing alkali light chain 1 and (A2)S-1 containing alkali light chain 2. The predominant difference in the behavior of the two isozymes of S-1 is that, at low ionic strength, the actin concentration required for half-maximal ATPase activity is considerably lower for (A1)S-1 than for (A2)S-1; that is, the apparent binding constant KATPase for (A1)S-1 is greater than KATPase for (A2)S-1 [Weeds, A.G., & Taylor, R.S. (1975) Nature (London) 257, 54-56]. This difference disappears at high ionic strength [Wagner, P. D., Slater, C. S., Pope, B., & Weeds, A.G. (1979) Eur. J. Biochem. 99, 385-394]. In the present study we investigated whether the difference in the KATPase values of (A1)S-1 and (A2)S-1 is due to a difference in the actual affinity of these S-1 isozymes for actin. Binding was measured in the presence of ATP and AMP-PNP and in the absence of nucleotide at varied ionic strengths. We found that at low ionic strength where KATPase is several times stronger for (A1)S-1 than for (A2)S-1, the binding of (A1)S-1 to actin is correspondingly stronger than that of (A2)S-1 irrespective of the nucleotide present. Furthermore, as the ionic strength is increased, just as the difference between the KATPase values for (A1)S-1 and (A2)S-1 disappears so too does the difference in the affinity of the two isozymes for actin.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionsupporting

confidence: 88%

Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide

Chalovich¹,

Stein²,

Greene³

et al. 1984

Biochemistry

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“…Thus, it is likely that the N-terminal segment of AL1 binds to actin at and around its acidic residues at positions 360, 362, and 363. Consistent with the above result, affinity chromatography of the thrombic digest of AL1 demonstrated a direct interaction between a fragment of AL1 (residues 1-79) and actin (Winstanley & Trayer, 1979;Henry et al, 1980). NMR studies have also demonstrated that the N-terminal segment of AL1 (residues 1-41) interacts with actin in the acto-Sl rigor complex (Prince et al, 1981).…”

Section: Discussionmentioning

confidence: 55%

Identification of myosin-binding sites on the actin sequence

Sutoh¹

1982

Biochemistry

343

313

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The rigor complex of actin and trypsin-treated myosin subfragment 1 (S1) whose heavy chain was cleaved into three fragments (20K, 25K, and 50K) was cross-linked with a zero-length cross-linker, 1-ethyl-3-[3-(dimethyl-amino) propyl]carbodiimide. The cross-linking reaction generated three types of cross-linked products with apparent molecular weights of 65K, 68K, and 95K. The 65K, 68K, and 95K products were covalently linked complexes of actin-20K fragment of the S1 heavy chain, actin-alkaline light chain 1, and actin-50K fragment of the S1 heavy chain, respectively. Cross-linking sites of S1 heavy and light chains on the actin sequence have been determined by digesting the cross-linked products with cyanogen bromide or with hydroxylamine and then mapping resulting peptides on sodium dodecyl sulfate gels. The result indicates that some of the N-terminal acidic residues of actin at positions 1, 2, 3, 4, and 11 are cross-linking sites of the 20K and 50K fragments of the S1 heavy chain while some of its C-terminal acidic residues at positions 360, 362, and 363 are cross-linking sites of the alkaline light chain 1.

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“…This sensitive site is different from another single site (Arg76-Ala77), cleaved by thrombin in isolated LCl [48]. The ionic concentration effect we have described above requires the filament structure, since varying ionic strength had no effect on LC1' cleavage in S1 and HMM- part from the expected behaviour since their LC1' cleavage exhibits a significant ionic concentration effect.…”

Section: Cleavages In Lc1mentioning

confidence: 97%

The proteolytic susceptibility of specific sites in myosin light chains is modulated by the filament conformation

Roulet

Burgat

Cardinaud

1993

European Journal of Biochemistry

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The proteolytic susceptibilities of specific sites in the LC1 and LC2 N-termini were modulated by ionic strength in myosin (a species able to form filaments) but not in S1. (a) In the presence of Ca'+ or Mg", the proteolytic susceptibility (apparent initial reaction rate) showed a sharp discontinuity at a critical ionic concentration similar for LCl', LC2 ' and LC2" cleavages. (b) The susceptibility of LC1' and LC2" was higher at low ionic concentration in the more compact structure of the filament than in the dissociated form at high ionic concentration. (c) The ionic concentration effect was no longer observed with species unable to form filaments. (d) This effect occurred at a critical ionic concentration markedly different from the critical concentration at which the monomer-filament equilibrium was found.These observations lead to the following conclusions. (a) The ionic concentration effect is an attribute of the filament structure. (b) In the filament the faster cleavage at sites (LC1' and LC2") near the LC1 and LC2 N-termini are due to an extended configuration of the N-terminal segment binding to a site in the filament structure. (c) The slower rate of formation of LC2' in the filament indicates that the N-terminal segment of LC2 binds more tightly to the structure than that of LC1. (d) The critical ionic concentration is not that of the filament -monomer equilibrium but corresponds to the order-disorder transition of the heads in the filament. These results suggest that the N-termini of the light chains (here in striated muscles) play a role in a secondary regulatory mechanism. The analysis of these regions may contribute to our understanding of the altered activity and regulation seen in such diseases as idiopathic dilated cardiomyopathy [Margossian, S. S., White, H. D., Caulfield, J. B., Norton, P., Taylor, s. & Slayter, H. s. (1992) Circulation 85, 2720-17331.The central event in muscle contraction is the interaction of actin and myosin in the presence of Mg-ATP. Since the part of myosin which interacts with actin is the head, the complex of actin and S1 is currently considered as a model system of the interaction between actin and myosin. There have been several attempts to solve the structure of this complex [ l , 21. The choice of this model was further supported by the observation that, on S1-coated surfaces, S1 appears to be a sufficient structure to sustain the movement of actin filaments [3], suggesting that the stroke results essentially from a transconformation of the myosin head (review in [4]).However Toyoshima et al. [3] have reported that the sliding velocity was highest with heavy meromyosin (HMM; 6-8 p d s , 30°C) and is only 33-25% of this value with S1. Thus the higher integrated structure found in HMM may be suspected of imparting a better efficiency to the system where a contribution of the S2 hinge segment could be suggested. In myosin thick filament, a more highly integrated system, physiological effectors were observed to govern the position of the heads close to or out fro...

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Thrombic Digestion of Myosin Alkali Light Chains

Cited by 10 publications

References 0 publications

Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide

Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide

Identification of myosin-binding sites on the actin sequence

The proteolytic susceptibility of specific sites in myosin light chains is modulated by the filament conformation

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