A monoclonal antibody to the Ca²⁺ ‐ATPase of cardiac sarcoplasmic reticulum cross‐reacts with slow type I but not with fast type II canine skeletal muscle fibers: An immunocytochemical and immunochemical study

Abstract

Ca2+-ATPase of the sarcoplasmic reticulum was localized in cryostat sections from three different adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with monoclonal antibodies to the Ca2+-ATPase. Type I (slow) myofibers were strongly labeled for the Ca2+-ATPase with a monoclonal antibody (I1 DS) to the Ca2+-ATPase of canine cardiac sarcoplasmic reticulum; the type I1 (fast) myofibers were labeled at the level of the background with monoclonal antibody I1 DS. By contrast, type I1 (fast) myofibers were strongly labeled for Ca2+-ATPase of rabbit skeletal sarcoplasmic reticulum. The subcellular distribution of the immunolabeling in type I (slow) myofibers with monoclonal antibody I1 DS corresponded to that of the sarcoplasmic reticulum as previously determined by electron microscopy. The structuraI similarity between the canine cardiac Ca2+-ATPase present in the sarcoplasmic reticulum of the canine slow skeletal muscle fibers was demonstrated by immunoblotting . Monoclonal antibody (I1 DS) to the cardiac Ca2+-ATPase binds to only one protein band present in the extract from either cardiac or type I (slow) skeletal muscle tissue. By contrast, monoclonal antibody (I1 H11) to the skeletal type I1 (fast) Ca2+-ATPase binds only one protein band in the extract from type I1 (fast) skeletal muscle tissue. These immunopositive proteins coelectrophoresed with the Ca2+-ATPase of the canine cardiac sarcoplasmic reticulum and showed an apparent M, of 115,000. It is concluded that the Ca2+-ATPase of cardiac and type I (slow) skeletal sarcoplasmic reticulum have at least one epitope in common, which is not present on the Ca2+-ATPase of sarcoplasmic reticulum in type I1 (fast) skeletal myofibers. It is possible that this site is related to the assumed necessity of the Ca2+-ATPase of the sarcoplasmic reticulum in cardiac and type I (slow) skeletal myofibers to interact with phosphorylated phospholamban and thereby enhance the accumulation of Ca2+ in the lumen of the sarcoplasmic reticulum following 6 -adrenergic stimulation.

Thapsigargin discriminates strongly between Ca2+‐ATPase phosphorylated intermediates with different subcellular distributions in bovine adrenal chromaffin cells

“…Since the 56-35 kDa tryptic digestion fragments have been shown to originate from the SERCA 2-type Ca2+-ATPases (see section 2), we probed the immunoblots of gradient fractions using the IID8 monoclonal antibody specific for this class of pumps [24]. Specific staining was detected corresponding to molecular mass 100 kDa in fractions 3-8 across the gradient, and is illustrated for fractions 4 and 7 in Fig.…”

“…This conclusion is supported by the SERCA 2-like immunostaining with the IID8 monoclonal antibody in fractions 4 and 7. This antibody, raised against the dog cardiac sarcoplasmic reticulum Ca2+-ATPase (SERCA 2a) [24], has been shown to react with an epitope common to SERCA 2a and SERCA 2b isoenzymes in several species, including pig [29], rabbit [31] and rat [32]. Therefore, both the tryptic digestion and the immunological data suggest that in the present work, the differential sensitivity to thapsigargin of the 2 Ca2+-ATPase E-P forms in bovine adrenal medulla microsomes reveals a new level of heterogeneity within the SERCA 2 class of isoenzymes.…”

Separation of active and inactive (nonphosphorylating) Ca2+‐ATPase in sacroplasmic reticulum subfractions from low‐frequency‐stimulated rabbit muscle

“…This could result from an inactivation of selected regions of the SR in all muscle fibers or from an inactivation of the SR of a specific fiber population. Because TA and EDL muscles are composed of approximately 95% fast-twitch (type IIA and type liB) fibers which are identical in their fast caa+-ATPase isoform composition [14,15], it appears unlikely that the inactivation affects, selectively, fast or slow isoforms. Also, a fast-to-slow Ca2*-ATPas¢ isozyme transition does not occur after 4-day low-frequency stimulation [15].…”

Isoform-specific up-regulation of plasma membrane Ca2+ATPase expression during colon and gastric cancer cell differentiation

“…1:2000 dilution of the pan-anti-PMCA (5F10) and the anti-SERCA2 (IID8), 1:1000 dilution of the anti-PMCA4 (JA9) and 1:1500 dilution of the anti-PMCA4b (JA3) monoclonal antibodies (ascites) were used. The IID8 antibody [37] was from Affinity BioReagents, Neshanic Station, NJ, USA. The 5F10, JA9 and JA3 antibodies were obtained as described earlier [38,39].…”