Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosinetriphosphatase
Two series of experiments were carried out to characterize (a) peptide fragments of sarcoplasmic reticulum (SR) ATPase, based on proteolysis with different enzymes and distribution of known labels, and (b) specific labeling and functional inactivation patterns, following ATPase derivatization with dicyclohexylcarbodiimide (DCCD) under various conditions. Digestion with trypsin or chymotrypsin results in the initial cleavage of the SR ATPase in two fragments of similar size and then into smaller fragments, while subtilisin and thermolysin immediately yield smaller fragments. Peptide fragments were assigned to segments of the protein primary structure and to functionally relevant domains, such as those containing the /sup 32/P at the active site and the fluorescein isothiocyanate at the nucleotide site. ATPase derivatization with (/sup 14/C)DCCD under mild conditions produced selective inhibition of ATPase hydrolytic catalysis without significant incorporation of the /sup 14/C radioactive label. This effect is attributed to blockage of catalytically active residues by reaction of the initial DCCD adduct with endogenous or exogenous nucleophiles. ATPase derivatization with (/sup 14/C)DCCD under more drastic conditions produced inhibition of calcium binding, /sup 14/C radioactive labeling of tryptic fragments A/sub 1/ and A/sub 2/ (but not of B), and extensive cross-linking. The presence of calcium during derivatization prevented functional inactivation, radioactive labeling of fragment A/sub 2/, and internal cross-linking of fragment A/sub 1/. It is proposed that both A/sub 1/ and A/sub 2/ fragments participate in formation of the calcium binding domain and that the labeled residues of fragment A/sub 2/ are directly involved in calcium complexation. A diagram is constructed, representing the relative positions of labels and functional domains within the ATPase protein.
- Research Organization:
- Univ. of Maryland, Baltimore (USA)
- OSTI ID:
- 5408877
- Journal Information:
- Biochemistry; (United States), Vol. 27:5
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
IMIDES
AUTORADIOGRAPHY
BIOCHEMISTRY
DERIVATIZATION
ATP-ASE
CARBON 14 COMPOUNDS
ELECTROPHORESIS
ENZYMATIC HYDROLYSIS
PHOSPHORUS 32
SARCOPLASMIC RETICULUM
ACID ANHYDRASES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
ENZYMES
HYDROLASES
HYDROLYSIS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LYSIS
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
PHOSPHOHYDROLASES
PHOSPHORUS ISOTOPES
RADIOISOTOPES
SOLVOLYSIS
550601* - Medicine- Unsealed Radionuclides in Diagnostics
550201 - Biochemistry- Tracer Techniques