Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca²⁺‐dependent adenosine triphosphatase of sarcoplasmic reticulum

Abstract

Incorporation of 4.5 nmol fluorescein isothiocyanate/mg rabbit sarcoplasmic reticulum, or of 7.4 nmol/ mg purified ATPase, was sufficient to inhibit the activity completely. These results are not consistent with the suggestion (Pick, U. and Karlish, S.J.D. (1980) Biochim. Biophys. Acta 626, 255-261) that 2 mol ATPase were inhibited by each mole of reagent incorporated. A single labelled peptide was purified from the inhibited ATPase and it was shown that Lys 3/190, 10 residues from the N-terminus of tryptic fragment B, was the reactive lysine residue. This site is close to a potential nucleotide-binding fold in the ATPase sequence. A similar peptide showing only 2 conservative replacements was isolated from the sarcoplasmic reticulum of the lobster. (Ca 2 + + Mg 2 + )A TPase inhibition Reactive lysine residue Fluorescein isothiocyanateIntegral membrane protein Nucleotide-binding fold (Sarcoplasmic reticulum)

Tryptic cleavage inhibits but does not uncouple Ca2+ATPase of sarcoplasmic reticulum

“…Each of the resulting large tryptic fragments (A2, A1 and B) possesses a different functional site: the A2 fragment has Ca2+-binding properties [2,31, the A1 fragment bears the phosphorylation site [4] and the B fragment, labelled with fluorescein 5'-isothiocyanate [5], carries the nucleotide-binding site. Comparison of the sequences of different ion pumps shows that the T1 site is located in a region of variable sequence that may mark the boundary between the nucleotide binding and the phosphorylation domain, whereas the T2 site resides in another variable segment within the transduction domain [6,7].…”

Labeling the Ca2+-ATPase of Skeletal Muscle Sarcoplasmic Reticulum with Maleimidylsalicylic Acid

“…It has been proposed that the nucleotide domain is located in the surroundings of Lys 515 , where FITC, a well known inhibitor of ATP binding, reacts (13,40), and which is far within the primary structure of the MSA reaction site. Very recently, the crystal structure of the ATPase at 2.6 Å resolution has been reported (41), where the phosphorylation domain appears to be linked to the nucleotide domain but more than 25 Å away from the bound nucleotide.…”

“…The ATP-binding site has been probed with different reagents, one of the best characterized being FITC, which reacts with Lys 515 to inhibit ATP binding with high specificity (12,13). However, FITC binding does not alter the catalysis of smaller substrates such as acetylphosphate (14).…”

Low Temperature Molecular Adaptation of the Skeletal Muscle Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA 1) in the Wood Frog (Rana sylvatica)

“…Therefore, based on these considerations and the overall sequence identity, we suggest that the nomenclature used for R. sylvatica and R. clamitans Ca 2ϩ -ATPases that are encoded by the cloned cDNAs be SERCA1a as it was proposed earlier for R. esculenta (7). The hydrophobic putative transmembrane domains (M1-M10), the phosphorylation site (residues 349 -355), as well as sites known to bind fluorescein isothiocyanate or the ATP analogues FSBA and CIRATP in SERCA1 (27)(28)(29) are conserved in R. sylvatica SERCA1a. There are five nonconservative amino acid substitutions (Asn, Gln, Ala, Ser, and Leu) characteristic for R. sylvatica (Fig.…”