Reconstitution of Troponin Activity from Three Protein Components

Greaser, Marion L.; Gergely, J.

doi:10.1016/s0021-9258(18)62075-7

Cited by 564 publications

(96 citation statements)

References 37 publications

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“…Rabbit skeletal TnC was isolated and purified according to the method described by Greaser and Gergley (1971). STnC was then labeled with fluorophores as follows.…”

Section: Preparation Of Labeled Proteinsmentioning

confidence: 99%

Ca2+ and Cross-Bridge-Induced Changes in Troponin C in Skinned Skeletal Muscle Fibers: Effects of Force Inhibition

Martyn

Freitag

Chase

et al. 1999

Biophysical Journal

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Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhodamine (5'IATR), attached to Cys98 (sTnC-5'ATR), in sTnC-5'ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca2+ and cross-bridge binding on sTnC structure, maximum Ca2+-activated force was inhibited with 0.5 mM AlF4- or with 30 mM 2,3 butanedione-monoxime (BDM) during measurements of the Ca2+ dependence of force and dichroism. Dichroism was 0.08 +/- 0.01 (+/- SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 +/- 0.002 (+/- SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca2+ sensitivity of dichroism. Similarly, inhibition of force at pCa 4.0 with 0.5 mM AlF4- decreased force to 0.04 +/- 0.01 of maximum (+/- SEM, n = 3), and dichroism was 0.04 +/- 0.03 (+/- SEM, n = 3) of the value at pCa 9.2 and unchanged relative to the corresponding normalized value at pCa 4.0 (0.11 +/- 0.05, +/- SEM; n = 3). Inhibition of force with AlF4- also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 microm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment caused dichroism at pCa 9.2 to decrease to 0.56 +/- 0.03 (+/- SEM, n = 5) of the value at pCa 9. 2, and force was 0.51 +/- 0.04 (+/- SEM, n = 6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to 0.19 +/- 0.03 (+/- SEM, n = 6), slightly above the pCa 4.0 control level; force was 0.66 +/- 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca2+].

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“…Rabbit skeletal TnC was isolated and purified according to the method described by Greaser and Gergley (1971). STnC was then labeled with fluorophores as follows.…”

Section: Preparation Of Labeled Proteinsmentioning

confidence: 99%

Ca2+ and Cross-Bridge-Induced Changes in Troponin C in Skinned Skeletal Muscle Fibers: Effects of Force Inhibition

Martyn

Freitag

Chase

et al. 1999

Biophysical Journal

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“…Troponin was prepared from rabbit skeletal muscle essentially as described by Greaser & Gergely (1971). Troponin B was obtained from troponin by the method of Hartshorne & Mueller (1968).…”

Section: Methodsmentioning

confidence: 99%

“…Essentially no differences in the behaviour of the phosphatase preparations with the two substrates were seen, suggesting that minor phosphorylated proteins in troponin It were not significant substrates for the phosphatases under the assay conditions used. In view of this finding, the term 'troponin I phosphatase' is usedi in the text, even though some early experiments were performed with troponin B. Troponin I was prepared from troponin as described by Greaser & Gergely (1971) with the modification of Eisenberg & Kielly (1972). Figure lb shows-that the purity of troponin I obtained by this method was at least 95%.…”

Section: Troponin I Phosphatase In Skeletal Musclementioning

confidence: 99%

The identification and properties of phosphatases in skeletal muscle with activity towards the inhibitory subunit of troponin, and their relationship to other phosphoprotein phosphatases

Ray¹,

England²

1976

Biochemical Journal

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1. Phosphoprotein phosphatases with activity towards the inhibitory subunit of troponin (troponin I), phosphorylase a and lysine-rich histone (fraction F1) have been fractionated from rat skeletal muscle by chromatography on Sephadex G-200 and polylysine-Sepharose. Six separate fractions were identified on the basis of substrate specificity and behaviour during chromatography. 2. All fractions showed similar Km values for any given protein substrate. The Km for troponin I (5 muM) was significantly lower than that previously reported. 3. Phosphatase activities towards troponin I and hosphorylase a did not show a requirement for bivalent-metal ions. Two of the fractions with only minor activity towards histone were activated by Mn2+. 4. Discontinuous polyacrylamide-gel-electrophoresis studies indicated that several of the fractions contained more than one phosphatase activity, and additionally showed that several of the activities could exist in different aggregation states. On the basis of these studies at least two phosphatases with activity only towards troponin I were identified. In addition, phosphorylase phosphatase (which has considerable activity towards troponin I) and a general phosphatase with activity towards all three substrates were found. 5. A fraction with mol.wt. of 150000 could be activated by freezing with 2-mercaptoethanol or by heating to 55 degrees C. This activation was accompanied by a decrease in mol.wt. to 25000. 6. The total amount of phosphatase with activity towards troponin I which was extracted would be sufficient to dephosphorylate all the troponin I present in skeletal muscle in approximately 10s.

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“…Four different proteins are involved in regulating actin sites: tropomyosin and the three subunits of troponin (Greaser and Gergely, 1971). These regulatory components interact with actin and with each other in a very specific manner.…”

Section: Actin Controlmentioning

confidence: 99%

“…There is an 18,000 dalton calcium binding component, troponin-C, that binds calcium with high affinity and undergoes calcium dependent changes in conformation. A 23,000 dalton component, troponin-I, has an inhibitory function, while a 37,000 dalton component TN-T binds to actin (Hartshorne and Mueller, 1968;Schaub and Perry, 1969;Greaser and Gergely, 1971).…”

Section: Actin Controlmentioning

confidence: 99%

Calcium regulation of muscle contraction

Szent‐Györgyi¹

1975

Biophysical Journal

186

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Calcium triggers contraction by reaction with regulatory proteins that in the absence of calcium prevent interaction of actin and myosin. Two different regulatory systems are found in different muscles. In actin-linked regulation troponin and tropomyosin regulate actin by blocking sites on actin required for complex formation with myosin; in myosin-linked regulation sites on myosin are blocked in the absence of calcium. The major features of actin control are as follows: there is a requirement for tropomyosin and for a troponin complex having three different subunits with different functions; the actin displays a cooperative behavior; and a movement of tropomyosin occurs controlled by the calcium binding on troponin. Myosin regulation is controlled by a regulatory subunit that can be dissociated in scallop myosin reversibly by removing divalent cations with EDTA. Myosin control can function with pure actin in the absence of tropomyosin. Calcium binding and regulation of molluscan myosins depend on the presence of regulatory light chains. It is proposed that the light chains function by sterically blocking myosin sites in the absence of calcium, and that the "off" state of myosin requires cooperation between the two myosin heads. Both myosin control and actin control are widely distributed in different organisms. Many invertebrates have muscles with both types of regulation. Actin control is absent in the muscles of molluscs and in several minor phyla that lack troponin. Myosin control is not found in striated vertebrate muscles and in the fast muscles of crustacean decapods, although regulatory light chains are present. While in vivo myosin control may not be excluded from vertebrate striated muscles, myosin control may be absent as a result of mutations of the myosin heavy chain.

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Reconstitution of Troponin Activity from Three Protein Components

Cited by 564 publications

References 37 publications

Ca2+ and Cross-Bridge-Induced Changes in Troponin C in Skinned Skeletal Muscle Fibers: Effects of Force Inhibition

Ca2+ and Cross-Bridge-Induced Changes in Troponin C in Skinned Skeletal Muscle Fibers: Effects of Force Inhibition

The identification and properties of phosphatases in skeletal muscle with activity towards the inhibitory subunit of troponin, and their relationship to other phosphoprotein phosphatases

Calcium regulation of muscle contraction

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