Effects on ATPase activity of monoclonal antibodies raised against (Ca2+ + Mg2+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum and their correlation with epitope location

Abstract

A total of 28 monoclonal antibodies have been raised against the (Ca2+ + Mg2+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum. Epitope mapping, using protein fragments generated by proteolysis, indicates that these antibodies include examples binding to at least four distinct epitopes on the A1 and B tryptic fragments of the ATPase. Competition data also show that the 28 antibodies are directed against at least five spatially distinct regions. Altogether, nine inhibitory antibodies were produced: six of these inhibitory antibodies mapped to the same spatial region, although they appear to bind to two distinct epitopes located within the hinge region and the nucleotide-binding domains of current structural models; one antibody bound to an epitope located within the phosphorylation domain and the stalk-transmembranous region designated M4S4 by Brandl, Green, Korczak & MacLennan [(1986) Cell 44, 597-607]. Two of the inhibitory antibodies recognized assembled epitopes exclusively and could not be mapped. Binding to four of the five identified spatial regions was without effect on activity. These data show that the inhibition of catalytic activity by monoclonal antibodies is achieved only by binding to defined regions of the ATPase and they may therefore provide useful probes of structure-function relationships.

Intracellular Ca2+ stores of T lymphocytes: Changes induced by in vitro and in vivo activation

“…Service Corp. ,Woburn, MA; PHA and Con A from Biochrom KG, Berlin, Germany. The Ab employed had been described elsewhere: anti-calreticulin (CR) in [12]; anti-ER membrane proteins in [13]; anti-protein disulfide isomerase [PDI] in ( [14]; anti-endoplasmic-sarcoplasmic reticulum Ca2+-ATPases (SERCA) in [15]. 1251-labeled protein A, the sheep anti-mouse 1251-labeled mAb, 45Ca2+ and [3H] IP3 were purchased from Amersham Int., GB; culture sera and media from GIBCO, Basel, Switzerland; glass microfiber filters from Whatman, Maidstone, GB.…”

The Type 2 Ryanodine Receptor of Neurosecretory PC12 Cells Is Activated by Cyclic ADP-ribose

“…Caffeine, sodium nitroprusside (SNP), 8-Br-cGMP, and the remaining chemicals were from Sigma (Milano, Italy). The antibodies used have been described elsewhere: anti-protein-disulfide isomerase, a rabbit polyclonal antibody (Ab), by Villa et al (22); anti-SERCA, a mouse monoclonal Ab, by Colyer et al (23); and anti-IP 3 receptor, a rabbit polyclonal Ab, by Villa et al (24). The polyclonal rabbit antisera specific for RyR types 1-3 (described in Ref.…”

The Endoplasmic Reticulum in PC12 Cells

“…Antibodies against SERCA1 (16) 20 -40 nM) and quenched after 30 s by addition of 1 ml of ice-cold 10% trichloroacetic acid, containing 50 mM NaH 2 PO 4 and 0.5 mM ATP. Samples were allowed to stand on ice for 15 min before the precipitated material was collected by centrifugation, and washed once with 1 ml of ice-cold distilled water.…”