Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosinetriphosphatase

Journal Article · Tue Mar 08 04:00:00 EST 1988 · Biochemistry; (United States)

OSTI ID:5408877

de Ancos, J G; Inesi, G

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), Journal Name: Biochemistry; (United States) Vol. 27:5; ISSN BICHA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
ACID ANHYDRASES
ATP-ASE
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
CARBON 14 COMPOUNDS
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DERIVATIZATION
ELECTROPHORESIS
ENZYMATIC HYDROLYSIS
ENZYMES
HYDROLASES
HYDROLYSIS
IMIDES
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LYSIS
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIOISOTOPES
SARCOPLASMIC RETICULUM
SOLVOLYSIS