Evidence for two distinct affinities in the binding of divalent metal ions to myosin.

Watterson, J. G.; Kohler, Laure; Schaub, Marcus C.

doi:10.1016/s0021-9258(18)50391-4

Cited by 39 publications

(7 citation statements)

References 53 publications

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“…In skeletal muscle the regulation of contraction is attributed to changes in the affinity of the troponin complex for Ca 2+, whereas in molluscs the Ca 2+ on/off switch is regulated by binding of Ca 2+ to the light chains of myosin (Kendrick-Jones et al, 1970). However, myosin light chains from skeletal muscle also bind Ca 2+ with two distinct affinities in the absence of Mg 2+ in the same range as found here for the ABRM, i.e., with binding constants of l0 s M -1 and l0 s M -x (Watterson et al, 1979). Furthermore, it is interesting…”

Section: Discussionsupporting

confidence: 76%

The regulation of tension in a chemically skinned molluscan smooth muscle: effect of Mg2+ on the Ca2+-activated tension generation.

Cornelius¹

1980

The Journal of General Physiology

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Chemically skinned anterior byssus retractor muscle (ABRM) preparations were prepared by treatment with the nonionic detergents saponin and Triton X-100. Both maximum peak tension and rate of contraction were found to be greater in saponin-treated ABRM than in ABRM treated with Triton X-100. Active tension was initiated at a concentration of free Ca 2+ above 0.1 #M, and maximum tension development was found at a [Ca 2+] ~32 #M. During exposure of the muscle preparation to optimal Ca 2+ concentration, a high and almost constant tension level was sustained. The force recovery was high after a quick release during this period indicating the presence of an "active" state rather than a "catch" state. Actually, a state equivalent to the catch state in the living ABRM could not be induced, if the Ca 2+ concentration was above 0.1/zM. Variations in the ionic strength in the range of 0.07-0.28 M had no influence on active state and only slightly affected the maximum tension developed. The influence of Mg a+ on the Ca2+-activated tension was examined by studying the tension-pCa relation at two concentrations of free Mg 2+ (0.43 and 4.0 mM). The tension-pCa relation was found to be S-shaped with tension increasing steeply over -l pCa unit, indicating the existence of cooperativity between Ca 2+ sites. Increasing the free concentration of Mg 2+ shifted the tensionpCa relation to lower pCa as in striated muscles, demonstrating a decreasing Ca 2+ sensitivity with increasing Mg 2+. At [Mg 3+] --4.0 mM the half-maximum tension was found at [Ca 2+] = 0.43 #M, decreasing to 0.20 pM at [Mg 2+] ffi 0.43 raM. At both Mg 3+ concentrations studied, plots of log P~t/(1 -Pr,l) vs. log [Ca 2+] were nonlinear with a shape indicating a rather complicated model for cooperativity, probably involving four sites for Ca a+. These Caa+-Mg 2+ interactions are most probably taking place at the myosin head itself because troponin is absent in this myosin-regulated muscle.

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Section: Discussionsupporting

confidence: 76%

The regulation of tension in a chemically skinned molluscan smooth muscle: effect of Mg2+ on the Ca2+-activated tension generation.

Cornelius¹

1980

The Journal of General Physiology

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“…Ca2+ or Mg2+ was able to prevent RLC' formation in myosin by 50% at free ion concentrations of 1.1 X 10~8 and 4.0 X 10~7 M, respectively. From these data, the apparent affinity constants were calculated for Ca2+ (K = 9.1 X 107 M_1) and for Mg2+ (K = 2.5 X 106 M-1)-The higher affinity for Ca2+ and the lower for Mg2+ fall well into the ranges reported for direct binding studies on myosin (Watterson et al, 1979;Holroyde et al, 1979). High-affinity binding of divalent metal ions to the RLC in myosin then causes a change in the reactivity of their two thiols with DTNB when compared with the metal-free state.…”

Section: Resultsmentioning

confidence: 79%

“…In isolated RLC, they have no effect on the DTNB-induced RLC' formation at all. Yet, the isolated RLC is still able to bind Ca2+ or Mg2+ ions, albeit with affinities that are almost 3 orders of magnitude lower (Alexis & Gratzer, 1978) than in intact myosin (Watterson et al, 1979;Holroyde et al, 1979). The isolated RLC is therefore assumed to adopt a conformation in solution where its two thiols are close together.…”

Section: Discussionmentioning

confidence: 99%

Disulfide formation within the regulatory light chain of skeletal muscle myosin

Huber¹,

Brünner²,

Schaub³

1989

Biochemistry

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Thiol-disulfide exchange reactions between myosin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) lead to the formation of 5-thio-2-nitrobenzoic acid (TNB)-mixed disulfides as well as to protein disulfide bonds. After incubation with DTNB, myosin was treated with an excess of N-ethylmaleimide (NEM) before electrophoretic analysis of the protein subunits in sodium dodecyl sulfate (SDS) without prior reduction by dithiothreitol (DTT). Without NEM treatment, thiol-disulfide rearrangement reactions occurred in the presence of SDS between the residual free thiols and DTNB. In the absence of divalent metal ions at 25 degrees C, DTNB was shown to induce an intrachain disulfide bond between Cys-127 and Cys-156 of the RLC. This intrachain cross-link restricts partially the unfolding of the RLC in SDS and can be followed as a faster migrating species, RLC'. Densitometric evaluation of the electrophoretic gel patterns indicated that the stoichiometric relation of the light chains (including RLC and RLC') remained unchanged. The two cysteine residues of the fast migrating RLC' were no more available for reaction with [14C]NEM, but upon reduction with DTT, the electrophoretic mobility of the RLC' reverted to that of unmodified RLC and of the RLC modified with two TNB groups. Ca2+ or Mg2+ was able to prevent this disulfide formation in the RLC of myosin by 50% at a free ion concentration of 1.1 X 10(-8) and 4.0 X 10(-7) M, respectively, at 25 degrees C and pH 7.6. Intrachain disulfide formation of RLC never occurred in myosin at 0 degree C.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Our proposal is supported by different findings relevant to the LC2 light-chain function. Mg2+ binding to LC2 light chain of skeletal myosin profoundly increases the chemical reactivity of SHj thiol group (Watterson et al, 1979); conversly, the blocking of SHj and SH2 in this myosin abolishes Ca2+ binding to the light chain (Srivastava & Wikman-Coffelt, 1980); chemical modification of SH[ in gizzard myosin mimics the physiological phosphorylation of the regulatory light chain and induces the activation of the Mg2+-ATPase by actin (Seidel 1979); proteolytic removal of LC2 light chain from cardiac myosin enhances actin-myosin interaction but also causes changes in the ATPase activities which are similar to those due to SH2 modification but which are reversed upon recombination of the light chain (Malhotra et al, 1979). In this set of observations the SHt and SH2 thiols present in the 20K fragment behave as sensitive probes of the proposed interaction of the 20K domain with the LC2 light chain.…”

Section: Discussionmentioning

confidence: 99%

Proteolytic approach to structure and function of actin recognition site in myosin heads

Mornet¹,

Bertrand²,

Pantel³

et al. 1981

Biochemistry

172

132

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Evidence for two distinct affinities in the binding of divalent metal ions to myosin.

Cited by 39 publications

References 53 publications

The regulation of tension in a chemically skinned molluscan smooth muscle: effect of Mg2+ on the Ca2+-activated tension generation.

The regulation of tension in a chemically skinned molluscan smooth muscle: effect of Mg2+ on the Ca2+-activated tension generation.

Disulfide formation within the regulatory light chain of skeletal muscle myosin

Proteolytic approach to structure and function of actin recognition site in myosin heads

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