Location of an essential carboxyl group along the heavy chain of cardiac and skeletal myosin subfragments 1

Körner, Marie; Thiem, N. V.; Cardinaud, R.; Lacombe, Guy

doi:10.1021/bi00294a024

Cited by 17 publications

(11 citation statements)

References 42 publications

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“…Recently the substructure of cardiac SF1 has been investigated by examining the course and extent of tryptic cleavage. These studies showed that the cardiac SF1 is attacked at essentially the same two restricted regions of its associated heavy chain as was found previously for skeletal SF1 (Flink & Morkin, 1982;Korner et al, 1983) and results in a tryptic SF1 species comprised of the light chain and the three protease-resistant fragments of about 27K, 50K, and 21K arranged in this order in the linear sequence. These findings suggest that the conformation of the cardiac protein bears a high degree of similarity to that of skeletal SF1.…”

supporting

confidence: 75%

Stability and substructure of cardiac myosin subfragment 1 and isolation and properties of its heavy-chain subunit

Mathern¹,

Burke²

1986

Biochemistry

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The substructure and the thermal stability of the subunit interactions of bovine cardiac myosin subfragment 1 (SF1) have been examined. The results are in agreement with previous reports that the cardiac protein is cleaved in a very similar manner [Flink, I. L., & Morkin, E. (1982) Biophys. J. 37, 34; Korner, M., Thiem, N. V., Cardinaud, R., & Lacombe, G. (1983) Biochemistry 22, 5843-5847] but at a much faster rate [Applegate, D., Azarcon, A., & Reisler, E. (1984) Biochemistry 23, 6626-6630] than the skeletal protein. Additionally, it is found that the long-lived, steady-state intermediates formed by these proteins with MgATP at high ionic strength differ in their susceptibilities to tryptic attack especially at the 27K/50K junction of the associated heavy chains, suggesting a different conformation for these intermediates of the cardiac and skeletal SF1's. The thermal stability of the subunit interactions under conditions approaching the physiological state was examined by thermal ion-exchange chromatography of cardiac SF1 at 39.5 degrees C in the presence of MgATP. This results in the separation of part of the protein as the isolated heavy chain which is found to exhibit high levels of ATPase activity in the absence and presence of actin. Tryptic digestion of cardiac SF1 prior to thermal ion-exchange chromatography produces greater dissociation, with the heavy chain in this case being isolated as a complex of 27K, 50K, and 18-20K fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

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supporting

confidence: 75%

Stability and substructure of cardiac myosin subfragment 1 and isolation and properties of its heavy-chain subunit

Mathern¹,

Burke²

1986

Biochemistry

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“…The observation that the rate of thermal inactivation of the ATPase of SI appears to parallel the decay of the 50-kDa fragment (Figure 4) suggests that the integrity of the 50-kDa segment is crucial for this function as suggested by Setton and . A number of recent studies based on chemical modification (Korner et al, 1983;Hiratsuka, 1986b), proteolysis (Chaussepied et al, 1986a,b), and photoaffinity labeling (Mahmood & Yount, 1984) have pointed to the importance of this region to the ATPase function. Moreover, it is known that this segment also provides a contact with actin in both the absence and presence of nucleotide (Yamamoto & Sekine, 1979b;Mornet et al, 198 la,b;Chen et al, 1986).…”

Section: Discussionmentioning

confidence: 99%

Substructure of skeletal myosin subfragment 1 revealed by thermal denaturation

Burke¹,

Zaager²,

Bliss³

1987

Biochemistry

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The stability of myosin subfragment 1 (S1) to thermal denaturation has been followed by limited tryptic proteolysis. Digestions done during the thermal denaturation show that at temperatures at and above 37 degrees C there is a marked increase in the susceptibility of S1 to tryptic degradation, as evidenced by the loss of all bands corresponding to the normally trypsin-resistant fragments of 50, 27, and 21 kDa of the heavy chain and to the light chain. The enhanced digestion of S1 appears to be due to a general unfolding of all segments of S1, although the 50-kDa segment appears to unfold at a lower temperature than the remainder of the S1 structure. Digestions done after 30-min exposure to higher temperatures or after subsequent cooling to 25 degrees C show marked differences in the susceptibility of the S1 to trypsin. This suggests that, on cooling, a substantial portion of the S1, but not the 50-kDa segment, is capable of refolding to a state corresponding closely to that in the native S1. These data indicate that in terms of thermal denaturation the S1 behaves as though it is comprised of two domains--an unstable 50-kDa domain and a more stable domain comprised of the 27- and 21-kDa segments of the heavy chain interacting with the light chain, as proposed recently by Setton and Muhlrad [Setton, A., & Muhlrad, A. (1984) Arch. Biochem. Biophys. 235, 411-417]. The rates of thermal inactivation of the ATPase of S1 are found to correspond closely to the decay rates for the 50-kDa fragment, suggesting that this segment in S1 is closely associated with the ATPase function of the protein.

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“…A specific modification of serine 324 was revealed by a nucleotide analog containing a reactive group instead of its ribose ring (Nakamaye et al, 1985;Yount, et al, 1987). A carboxyl group was specifically modified and identified as residing in this fragment; its modification led to the loss of ATPase activities (Korner et al, 1983). The fluorescent reagent 4-flu0ro-7-nitrobenz-2-oxal,3-diazole was also incorporated and reacted selectively with two lysyl residues in this fragment (Hiratsuka, 1986a).…”

Section: The 50k Sequencementioning

confidence: 99%