Stoichiometry and mapping of the nucleotide sites in sarcoplasmic reticulum ATPase with the use of UTP.
Purified sarcoplasmic reticulum ATPase was phosphorylated by either ATP or UTP under otherwise identical conditions. Calcium, pH, and nucleotide concentrations were adjusted to permit maximal steady-state accumulation of phosphoenzyme (EP). Either 4 or 8.5 nmol of EP/mg of protein were obtained with ATP or UTP, respectively. Tryptic digestion of phosphorylated ATPase followed by acid gel electrophoresis showed that EP from UTP was on fragment A1, similar to the report in the literature for EP from ATP. Phosphorylation with Pi in the absence of calcium gave EP levels similar to those obtained from UTP. Thus, comparison of EP levels from different substrates measured in parallel in the same preparation reveal that with ATP half of the sites are phosphorylated. Illumination of the ATPase with UV light in the presence of [3H]UTP caused photolabeling of the ATPase at a maximal level of 1 nmol of [3H]UTP incorporated/mg of ATPase. The UTP concentration dependence for photolabeling was the same as that for promoting catalysis. ATP when present in the illumination protected with a competitive pattern against photolabeling with UTP. Tryptic digestion and autoradiography of photolabeled ATPase revealed that UTP was covalently attached to tryptic fragment A2. The data indicate that a peptide sequence of fragment A2 is involved in the binding of the nucleoside moiety of UTP and possibly belongs to the nucleotide domain of the ATPase in addition to the sequence of fragment A1 which contains the phosphorylation residue.
Ferreira
S T
ST
Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Brazil.
Verjovski-Almeida
S
S
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000345:Affinity Labels; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010777:Photochemistry; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013696:Temperature; D013861:Thiocyanates / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism; D014500:Uracil Nucleotides / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000378:metabolism*
D000345:Affinity Labels; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010750:Phosphoproteins; D013861:Thiocyanates; D014500:Uracil Nucleotides; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium; D014544:Uridine Triphosphate