What question did this study set out to answer?

To structurally and functionally map sarcoplasmic reticulum ATPase domains using enzymatic proteolysis and chemical derivatization with dicyclohexylcarbodiimide.

March 8, 1988Biochemistry

Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosine triphosphatase

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Authors

JAJ. García de AncosBritish Medical Association GIGiuseppe InesiElectrophysiology

Discussion

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Implication

In vitro biochemical study uncovers domain-specific labeling in sarcoplasmic reticulum ATPase, indicating that A1 and A2 fragments form the functional calcium-binding site.

Key Points

To structurally and functionally map sarcoplasmic reticulum ATPase domains using enzymatic proteolysis and chemical derivatization with dicyclohexylcarbodiimide.
Digested sarcoplasmic reticulum ATPase using trypsin, chymotrypsin, subtilisin, or thermolysin and mapped peptide fragments using active-site 32P and fluorescein isothiocyanate markers.
Derivatized ATPase using [14C]dicyclohexylcarbodiimide (DCCD) under mild versus drastic reaction conditions in the presence or absence of calcium and exogenous nucleophiles.
Mild DCCD treatment selectively inhibited ATPase hydrolytic catalysis without substantial incorporation of the 14C radioactive label.
Drastic DCCD treatment impaired calcium binding, induced extensive protein cross-linking, and selectively labeled tryptic fragments A1 and A2.
Presence of calcium during derivatization prevented functional inactivation, blocked 14C labeling of fragment A2, and inhibited internal cross-linking of fragment A1.

Cite This Study

Ancos et al. (1988) studied this question.

synapsesocial.com/papers/6a1b58e3db618aee6aabc75e https://doi.org/10.1021/bi00405a061

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March 8, 1988Biochemistry

Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosine triphosphatase

View Full Paper

Ask AI

Bookmark

Authors

JAJ. García de AncosBritish Medical Association GIGiuseppe InesiElectrophysiology

Discussion

Member takes

Implication

In vitro biochemical study uncovers domain-specific labeling in sarcoplasmic reticulum ATPase, indicating that A1 and A2 fragments form the functional calcium-binding site.

Key Points

To structurally and functionally map sarcoplasmic reticulum ATPase domains using enzymatic proteolysis and chemical derivatization with dicyclohexylcarbodiimide.
Digested sarcoplasmic reticulum ATPase using trypsin, chymotrypsin, subtilisin, or thermolysin and mapped peptide fragments using active-site 32P and fluorescein isothiocyanate markers.
Derivatized ATPase using [14C]dicyclohexylcarbodiimide (DCCD) under mild versus drastic reaction conditions in the presence or absence of calcium and exogenous nucleophiles.
Mild DCCD treatment selectively inhibited ATPase hydrolytic catalysis without substantial incorporation of the 14C radioactive label.
Drastic DCCD treatment impaired calcium binding, induced extensive protein cross-linking, and selectively labeled tryptic fragments A1 and A2.
Presence of calcium during derivatization prevented functional inactivation, blocked 14C labeling of fragment A2, and inhibited internal cross-linking of fragment A1.

Cite This Study

Ancos et al. (1988) studied this question.

synapsesocial.com/papers/6a1b58e3db618aee6aabc75e https://doi.org/10.1021/bi00405a061

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