Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T

Biochemistry ◽  
1988 ◽  
Vol 27 (18) ◽  
pp. 6983-6987 ◽  
Author(s):  
John Leszyk ◽  
John H. Collins ◽  
Paul C. Leavis ◽  
Terence Tao
Keyword(s):  
Skeletal Muscle ◽  
Ternary Complex ◽  
Troponin T ◽  
Troponin C ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Binary Complex

scholarly journals The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

Disparate contributions of Tyr10and Tyr109to fluorescence intensity of rabbit skeletal muscle troponin C identified using a genetically engineered mutant

FEBS Letters ◽  
1994 ◽  
Vol 354 (2) ◽  
pp. 135-139 ◽  
Author(s):  
David Keleti ◽  
Venu G. Rao ◽  
Hong Su ◽  
Arvind B. Akella ◽  
Xiao-Ling Ding ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Fluorescence Intensity ◽  
Troponin C ◽  
Genetically Engineered ◽  
Rabbit Skeletal Muscle

scholarly journals Involvement of caldesmon at the actin-myosin interface

1992 ◽  
Vol 287 (2) ◽  
pp. 633-637 ◽  
Author(s):  
M C Harricane ◽  
E Fabbrizio ◽  
C Arpin ◽  
D Mornet
Keyword(s):  
Ternary Complex ◽  
Actin Binding ◽  
Cross Linking ◽  
Binary Complex ◽  
Binding Region ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Spacer Arm ◽  
Protein Components ◽  
Chemical Cross Linking

Addition of myosin subfragment 1 (S-1) to the actin-caldesmon binary complex, which forms bundles of actin filaments resulted in the formation of actin/caldesmon-decorated filaments [Harricane, Bonet-Kerrache, Cavadore & Mornet (1991) Eur. J. Biochem. 196, 219-224]. The present data provide further evidence that caldesmon and S-1 compete for a common actin-binding region and demonstrate that a change occurs in the actin-myosin interface induced by caldesmon. S-1 digested by trypsin, which has an actin affinity 100-fold weaker than that of native S-1, was efficiently removed from actin by caldesmon, but not completely dissociated. This particular ternary complex was stabilized by chemical cross-linking with carbodi-imide, which does not have any spacer arm, and revealed contact interfaces between the different protein components. Cross-linking experiments showed that the presence of caldesmon had no effect on stabilization of actin-(20 kDa domain), whereas the actin-(50 kDa domain) covalent association was significantly decreased, to the point of being virtually abolished.

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