Use of anti-peptide antibodies to study the transmembranous topography and structure-activity relationships of (Ca2+-Mg2+)-ATPase

Abstract

Polyclonal antibodies were raised against peptides corresponding to segments of (Ca2+-Mg2+)-ATPase from fasttwitch adult rabbit skeletal muscle by the following methods. Peptides corresponding to residues 1 1 1, 504-5 1 1 and 985-994 were synthesized by the method of Merrifield (1986) or Sheppard (Atherton et af., 1981). For peptide 1-11, a cysteine residue was added to the C-terminus to facilitate coupling to the carrier protein and the same modification was made to the N-termini of peptides 504-51 1 and 985-994. These last two peptides correspond to the T, trypsin cleavage site and the C-terminus of the (Ca2'Mg2')-ATPase, respectively (Brandl et af., 1986). All three peptides were coupled to keyhole limpet haemocyanin with m-maleimidobenzoyl-N-hydroxysuccinamide ester by the method of Green et af. (1982). Peptides 567-582 and 38 1-400 (corresponding to segments of the protein on the nucleotide-binding and phosphorylation domains, respectively) were used without coupling to a carrier. New Zealand White rabbits were immunized by intradermal injection with 500 pg of peptide or peptide linked to carrier in Freund's complete adjuvant. Animals were boosted with a repeat intramuscular injection 6-8 weeks later and the serum was collected 5-10 days later. Except for peptide 504-5 1 1, specific antibodies were obtained, which in e.1.i.s.a. (Hudson & Hay, 1983) were capable of recognizing the peptides against which they were raised with minimal cross-reactivity with the other peptides. Anti-peptide antibodies against peptide 504-5 1 1 not only recognized the peptide against which they were raised but also peptide 381-400. This was observed on two occasions with serum from separate rabbits. No reciprocal cross-reactivity was observed with antibodies against peptide 38 1-400. An examination of the sequence of these peptides reveals no obvious similarities. All anti-peptide antibodies, except those against peptide 567-582, strongly recognized both the ATPase and sarcoplasmic reticulum (SR) in e s.a., suggesting that not only are these segments of the ATPase on the cytoplasmic face of the SR vesicles, which with the exception of the C-terminus segment, has already been substantiated by other techniques (Klip et af., 1980; Tong, 1980; Allen et al., 1980; Brandl et al., 1986), but they are also exposed on the surface of the cytoplasmic domains. The reason for the poor recognition of the ATPase by anti-peptide 567-582 antibodies is unclear. This anti-peptide antibody was also unable to recognize denatured ATPase, making it unlikely that the lack of recognition was due to the epitope being inaccessible. To further investigate the transmembranous location of the C-terminus, binding of the anti-peptide antibodies to