Isolation and characterization of tryptic fragments of the adenosine triphosphatase of sarcoplasmic reticulum

Journal Article · Thu Jan 01 04:00:00 EST 1976 · J. Biol. Chem.; (United States)

OSTI ID:7330259

Stewart, P S ^[1]; MacLennan, D H; Shamoo, A E

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1. Univ. of Toronto

Exposure of sarcoplasmic reticulum to trypsin in the presence of 1M sucrose results in degradation of the M/sub r/ = 102,000 ATPase enzyme to two fragments of M/sub r/ = 55,000 and 45,000 with subsequent appearance of fragments of M/sub r/ = 30,000 and 20,000. These fragments were purified by column chromatography in sodium dodecyl sulfate. Antibodies were raised against the ATPase and the M/sub r/ = 55,000, 45,000, and 20,000 fragments. There was no antigenic cross-reactivity between the M/sub r/ = 55,000 and 45,000 fragments. There was antigenic cross-reactivity between the M/sub r/ = 20,000 and 55,000 fragments, indicating an origin of the M/sub r/ = 20,000 fragment in the M/sub r/ = 55,000 fragment. None of the antibodies inhibited (Ca/sup 2 +/ + Mg/sup 2 +/)-dependent ATPase or Ca/sup 2 +/ transport. The M/sub r/ = 20,000 fragment and the M/sub r/ = 55,000 fragment were active in Ca/sup 2 +/ ionophore assays. The active site of ATP hydrolysis was labeled with (..gamma..-/sup 32/P)ATP and the site of ATP binding was labeled with tritiated N-ethylmaleimide. In both cases radioactivity was found in the intact ATPase and in the M/sub r/ = 55,000 and 30,000 fragments, indicating that the M/sub r/ = 30,000 fragment was also derived from the M/sub r/ = 55,000 fragment. Amino acid composition data showed that the M/sub r/ = 45,000 fragment containedabout 60% nonpolar and 40% polar amino acids, while the M/sub r/ = 55,000 fragment and the M/sub r/ = 20,000 fragment contained about equal amounts of polar and nonpolar amino acids. Studies of the reaction of various antibodies at the external surface of sarcoplasmic reticulum vesicles showed that the ATPase was exposed, whereas calsequestrin and the high affinity Ca/sup 2 +/-binding protein were not. The use of antibodies against the various fragments indicated that the M/sub r/ = 55,000 fragment was in large part exposed, whereas the M/sub r/ = 20,000 and the 45,000 fragments were only poorly exposed.

• OSTI ID: 7330259
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 251:3; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANTIBODIES
ATP
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DECOMPOSITION
DRUGS
ENZYMES
ESTERASES
HYDROGEN ISOTOPES
HYDROLASES
HYDROLYSIS
IMIDES
IMMUNE REACTIONS
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LYSIS
NEM
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDE HYDROLASES
PHOSPHATASES
RADIOISOTOPES
RADIOSENSITIZERS
REACTION KINETICS
RESPONSE MODIFYING FACTORS
SOLVOLYSIS
TRACER TECHNIQUES
TRITIUM
TRYPSIN
YEARS LIVING RADIOISOTOPES