Characterization of a cysteine‐containing peptide after affinity labelling of Ca2+‐ATPase of sarcoplasmic reticulum with the disulfide of 3′(2′)‐O‐biotinyl‐thioinosine triphosphate

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Abstract
3''(2'')-O-Biotinyl-thioinosine triphosphate is a substrate of the Ca2+ pump of sarcoplasmic reticulum. Its disulfide inactivates the Ca2+-ATPase with two different velocities. The rapidly inactivated sulfhydryl group cannot be protected by ATP and is therefore considered to be outside the ATP binding site. The slowly reacting sulfhydryl group interacts with the disulfide of 3''(2'')-O-biotinyl-thioinosine triphosphate with a dissociation constant of Kd = 137 .mu.M and an inactivation velocity constant of 1.7 .times. 10-3 s-1. It is protected by ATP with two different dissociation constants of the enzyme-ATP complex of Kd = 221 .mu.M and 1130 .mu.M. The slowly reacting sulfhydryl group is therefore considered to be part of the ATP binding site. Since it was impossible to isolate a tryptic peptide by affinity purification on matrix-bound avidin after affinity labelling with the disulfide of 3''(2'')-O-biotinyl-thioinosine triphosphate, differential labelling with ido[2-14C]acetic acid after affinity labelling with the disulfide of 3''(2'')-O-biotinyl-thioinosine triphosphate was carried out. Tryptic digestion and FPLC purification led to the isolation of a radioactive carboxymethyl derivative of the cysteine-containing peptide ANACNSVIR. This peptide is equivalent to the cDNA-derived sequence 468-476 of Ca2+-ATPase [Brandl et al. (1986) Cell 44, 597-607] and is located between the phosphorylation site, Asp351, and Lys515, a part of the putative purine binding subsite of ATP. Although the carboxymethylation of Cys471 is hindered by (biotinyl-s6ITP)2, the strong dilution of the specific radioactivity of iodo[2-14C]acetic acid in the isolated peptide 468-476 argues against its direct interaction with the ATP analogue. It is therefore proposed that Cys471 undergoes ATP-dependent conformational changes.