Specific dicyclohexylcarbodiimide inhibition of the E-P + H/sub 2/O equilibrium E + P /sub i/ reaction and ATP equilibrium P /sub i/ exchange in sarcoplasmic reticulum adenosinetriphosphatase

Journal Article · Tue Feb 12 04:00:00 EST 1985 · Biochemistry; (United States)

OSTI ID:5023422

Scofano, H M; Barrabin, H; Lewis, D; Inesi, G

Treatment of sarcoplasmic reticulum adenosinetriphosphatase (ATPase) with N,N'-dicyclohexylcarbodiimide is known to produce total inhibition of calcium binding and enzyme activity. However, we now find that treatment with lower reagent:protein ratios produces selective inhibition of hydrolytic P /sub i/ cleavage, enzyme phosphorylation with P /sub i/, and ATP in equilibrium P /sub i/ exchange, while calcium binding and enzyme phosphorylation with ATP remain largely unaffected. This specific inhibition is attributed to derivatization of residues which are normally involved in acid-base-assisted catalysis of the hydrolytic reaction and its reversal, but are not involved in calcium binding or in the mechanism of phosphoryl transfer from ATP to the enzyme. This specific inhibition is prevented by the presence of micromolar calcium during the incubation with the inhibitor, evidently through an allosteric effect of calcium binding on the catalytic site. We also find that the initial adducts formed between ATPase residues and N,N'-dicyclo(/sup 14/C)carbodiimide undergo further degradation with release of radioactive product into the medium, while the protein residues remain inactivated probably by linkage with neighboring residues. Therefore, the stoichiometry of radioactive labeling underestimates the actual number of inactivated residues.

• OSTI ID: 5023422
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 4; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ALKALINE EARTH METALS
ANIMALS
ATP-ASE
BIOCHEMICAL REACTION KINETICS
CALCIUM
CARBON 14 COMPOUNDS
CELL CONSTITUENTS
CHEMICAL REACTIONS
ELEMENTS
ENZYME INHIBITORS
ENZYMES
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MAMMALS
METALS
MUSCLES
ORGANOIDS
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHOHYDROLASES
PHOSPHORUS COMPOUNDS
PHOSPHORYLATION
RABBITS
REACTION KINETICS
SARCOPLASMIC RETICULUM
TRACER TECHNIQUES
VERTEBRATES