The preparation of tropomyosin and troponin from natural actomyosin

Hartshorne, David J.; Mueller, Helmut

doi:10.1016/0005-2795(69)90008-7

Cited by 141 publications

(34 citation statements)

References 31 publications

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“…All buffers used contained DTT to maintain the reduced state of TnC thought to be essential for its functioning (8). The protein concentration was determined by measuring the absorbance, E 1%,280nm ϭ 3.8 cm Ϫ1 (9).…”

Section: Methodsmentioning

confidence: 99%

Binding of Tropomyosin–Troponin to Actin Increases Filament Bending Stiffness

Goldmann

2000

Biochemical and Biophysical Research Communications

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Section: Methodsmentioning

confidence: 99%

Binding of Tropomyosin–Troponin to Actin Increases Filament Bending Stiffness

Goldmann

2000

Biochemical and Biophysical Research Communications

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“…Troponin was prepared according to Greaser and Gergely (39) and was the generous gift of Dr. Terence Tao. Troponin concentration was determined from the absorbance at 280 nm using the extinction coefficient A 1% 280nm ) 4.5 cm -1 (40). Skeletal muscle tropomyosin was prepared from rabbit skeletal muscle according to Greaser and Gergely (41) and its concentration determined from its absorption at 277 nm using an extinction coefficient A 1% 277nm ) 2.4 cm -1 (42).…”

Section: Protein Preparation and Concentration Determinationmentioning

confidence: 99%

Movement of Smooth Muscle Tropomyosin by Myosin Heads

Graceffa

1999

Biochemistry

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It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.

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“…The dialysis was continued for 6h with two changes of buffer to effectively eliminate the remaining ATP in the actin sample. Tropomyosin was purified from the residue remaining after extracting the actin from the acetone-dried powder according to the procedure reported by Bailey 1 1) and modified by Hartshorne and Mueller,12) and then stored at -20°C after its lyophilization. Myosin was extracted with a Guba-Straub solution and then purified according to the procedure of Briskey and Fukazawa.…”

Section: Methodsmentioning

confidence: 99%