Abstract
Limited reaction of glutaraldehyde with the Ca2+-ATPase (Mr approximately 110,000) of sarcoplasmic reticulum results in intramolecular cross-linking at the active site, which can be detected by an anomalous increase in apparent molecular weight (Mr approximately 125,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ross D.C., and McIntosh D.B. (1987) J. Biol. Chem. 262, 2042-2049). ATP, ADP, AMPPCP, trinitrophenyladenosine triphosphate, and decavanadate inhibited the cross-link in a manner suggestive of a homogeneous class of inhibitory sites, with K0.5 values for inhibition in agreement with Kd values for binding to the active site. Cross-link formation was inhibited in proportion to phosphoenzyme levels formed from Pi (E2-P) whereas stoichiometric phosphorylation from CaATP (E1-P) had no effect. Inhibition was observed at millimolar concentrations of CaATP, indicative of nucleotide binding to E1-P. MgATP, in the presence of Ca2+, inhibited cross-linkage in the micromolar and millimolar concentration ranges, the former attributable to E1 X ATP and E2-P formation and the latter to ATP binding mainly to E1-P. The inability to cross-link the active site only of the E2-P intermediate suggests a unique active site conformation, possibly a closed active site cleft, which we suggest is linked to low affinity, inwardly orientated Ca2+-binding sites.
Highlights
From the Medical ResearchCouncil, Biomembrane Research Unit and Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa
ATPase (Mr= 110,000) of sarcoplasmic reticulum re- presence of Ca2+ triggeras conformational changewhich may, sults in intramolecular cross-linking at the active site, as a result of an interdomain movement,appose the aspartyl which can be detected by an anomalous increase in apparent molecular weight (Mr= 125,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis
The lower limit of the range of concentration of ATPase polypeptides is derived fromthe maximum level of phosphoenzyme obtained from Pi in the presence of dimethyl sulfoxide (5 nmol/mg of protein, see below), and theupper limit is based on the assumption that all ATPases are active and that they constitute 90% of the total protein (8.2 nmol/mg of protein)
Summary
Materinls-Glutaraldehyde (Grade 11, 25% aqueous solution), hydazine, AMPPCP and Triton X-100 were purchased from Sigma. SR vesicles were prepared from rabbit back and hind limb white muscle [28]. They were stored as a suspension (10-20 mg of protein/ ml) in 10 mM imidazole,pH 7.4, and 0.3 M sucrose at -60 "C. Cross-linking-SR vesicles (0.4mg of protein/ml) were reacted with 5 D M glutaraldehyde for 4 min, unless otherwise specified, at 25 "C in a medium indicated in the legendsto the figures.The reaction was terminated by a 5-10-fold molar excess of hydrazine. Gel EZectrophoresk-This was camed out according to Laemmli [29] as described before [30]. Coomassie Blue stained and dried gels were scanned with a Vitatron TLD. Phosphoenzyme-Phosphorylation from [y-"PIATP and from ["PIPi was measured by filtration on a glass fiber filter after acid quenching [33]
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