Monoclonal antibodies to dog heart sarcoplasmic reticulum

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.

Section: Discussionsupporting

confidence: 56%

“…The production of inhibitory mAbs against the (Ca2+ Mg2+)-ATPase has been less successful. Although a number of mAbs against (Ca2+ + Mg2+)-ATPase have been produced (Zubrzycka-Gaarn et al, 1984;Levitsky et al, 1987;Dulhunty et al, 1987) only three were inhibitory.…”

Section: Introductionmentioning

confidence: 99%

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

Colyer

Mata

Lee

et al. 1989

“…This finding suggested that there exist important immunological differences between the Ca2+-transport enzymes of the SR/ER of fast-twitch skeletal muscle and smooth muscle. However, there was recently a report that some monoclonal antibodies against the SR ATPase of fast-twitch skeletal muscle [9] or cardiac muscle [10] could also recognize epitopes on the smooth-muscle enzyme.…”

Section: Introductionmentioning

confidence: 99%

Smooth muscle expresses a cardiac/slow muscle isoform of the Ca2+-transport ATPase in its endoplasmic reticulum

Wuytack

Kanmura

Eggermont

et al. 1989

Smooth muscle expresses in its endoplasmic reticulum an isoform of the Ca2+-transport ATPase that is very similar to or identical with that of the cardiac-muscle/slow-twitch skeletal-muscle form. However, this enzyme differs from that found in fast-twitch skeletal muscle. This conclusion is based on two independent sets of observations, namely immunological observations and phosphorylation experiments. Immunoblot experiments show that two different antibody preparations against the Ca2+-transport ATPase of cardiac-muscle sarcoplasmic reticulum also recognize the endoplasmic-reticulum/sarcoplasmic-reticulum enzyme of the smooth muscle and the slow-twitch skeletal muscle whereas they bind very weakly or not at all to the sarcoplasmic-reticulum Ca2+-transport ATPase of the fast-twitch skeletal muscle. Conversely antibodies directed against the fast-twitch skeletal-muscle isoform of the sarcoplasmic-reticulum Ca2+-transport ATPase do not bind to the cardiac-muscle, smooth-muscle or slow-twitch skeletal-muscle enzymes. The phosphorylated tryptic fragments A and A1 of the sarcoplasmic-reticulum Ca2+-transport ATPases have the same apparent Mr values in cardiac muscle, slow-twitch skeletal muscle and smooth muscle, whereas the corresponding fragments in fast-twitch skeletal muscle have lower apparent Mr values. This analytical procedure is a new and easy technique for discrimination between the isoforms of endoplasmic-reticulum/sarcoplasmic-reticulum Ca2+-transport ATPases.

“…An approach that can be used to study the function of the Ca2" pump is to raise monoclonal antibodies and to study their effect on the activity of the system. This& approach has given valuable information with other cation-transporting ATPases [12,13]. Previously this laboratory has reported inhibition of the plasma membrane Ca2" pump by a polyclonal antibody [14].…”

Section: Introductionmentioning

confidence: 99%

Inhibition of the purified human red-cell Ca2+ pump by a monoclonal antibody

Caride

Enyedi

Penniston

1989

1. A monoclonal antibody (1G4) was raised against the red-cell Ca2+ pump, and it reacted with the pump, as verified by Western blot analysis and by the e.l.i.s.a. method. 2. At 1 mM-ATP and 10 microM-Ca2+, 1G4 inhibited the activity of the purified Ca2+ pump by 40%. 3. Ca2+ pump inhibition by the antibody was non-competitive with regard to Ca2+, calmodulin and the high-affinity portion of the ATP curve. Thus its mechanism was quite different from that of the antibody previously reported [Verbist, Wuytack, Raemaekers, VanLeuven, Cassiman & Casteels (1986) Biochem. J. 240, 633-640], which partially caused inhibition by competition at the ATP site. 4. Antibody 1G4 reduced the steady-state level of phosphorylated intermediate and increased by 50% the calmodulin-activated p-nitrophenyl phosphatase activity of the pump. 5. The experimental results are consistent with the hypothesis that 1G4 inhibits the Ca2+ pump by decreasing the rate of the transition from the E2 form to the E1 form, causing a higher concentration of E2. 6. Analysis by Western blot of the pattern of cross-reaction of 1G4 after tryptic digestion of the pump showed that this antibody reacts with bands of Mr 90,000, 85,000, 50,000 and 33,000. After chymotryptic digestion, the antibody reacts almost exclusively with a fragment of Mr 105,000 that is fully active but is not responsive to calmodulin. Altogether, the results indicate that 1G4 binds to an epitope involved in the functional properties of the enzyme but which is not related to the calmodulin-binding domain.