Plasma membrane calcium pump and 28-kDa calcium binding protein in cells of rat kidney distal tubules

Abstract

In an effort to extend our studies on Ca2+ pumps to animal models, we developed a new monoclonal antibody (5F10) prepared against the human erythrocyte Ca2+-Mg2+-adenosinetriphosphatase (ATPase) that recognizes a protein of approximately 140 kDa in rat kidney homogenates. Enzyme-linked immunosorbent assays show that monoclonal antibody 5F10 binds purified Ca2+-Mg2+-ATPase and rat kidney membrane extracts in a concentration-dependent manner. In paraffin-embedded tissue sections, antibody 5F10 binds to an epitope in the basolateral membranes of rat kidney distal convoluted tubule principal cells. The antibody does not bind to intercalated cells. The latter cells were characterized by the presence of large amounts of carbonic anhydrase C. Polyclonal antibodies directed against chick intestinal 28-kDa vitamin D-dependent calcium binding protein (28-kDa CaBP) also bind epitopes in distal convoluted tubule cells, connecting tubules, and portions of collecting duct but not intercalated cells. Western blot and 45Ca blot analysis of renal cytosolic proteins showed that the polyclonal 28-kDa CaBP-directed antibody detects a protein which also binds calcium. Western blot analysis with monoclonal antibody 5F10 shows binding to both the authentic purified erythrocyte Ca2+ pump (approximately 138 kDa) and to tryptic fragments of this pump. Antibody JA3, previously used for staining of human kidney tubules, reacts with a different set of tryptic fragments, showing that the two antibodies are directed against different regions or conformational determinants on the pump molecule. We show that Ca2+-Mg2+-ATPase and 28-kDa CaBP are present in the principal cells of the distal convoluted tubule of the rat and are absent in intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetes in rat pregnancy alters renal calcium and magnesium reabsorption and bone formation in adult offspring

“…Expression of PMCA The monoclonal anti-PMCA antibody detected a broad protein band at around 140 kDa (∼133-147 kDa) and a smaller band at 92 kDa, consistent with previous observations [19]; these bands were absent from negative control blots (data not shown). The relative density of the major immunoreactive species at ∼140 kDa, representing PMCA [19], was significantly greater in the OD rats than in the OC rats, in both neonatal (Fig.…”

“…The relative density of the major immunoreactive species at ∼140 kDa, representing PMCA [19], was significantly greater in the OD rats than in the OC rats, in both neonatal (Fig. 2a) and adult ( Fig.…”

“…This may, in part, be related to the greatly increased renal calcium and magnesium loss suffered by the diabetic mother, as compared with her non-diabetic pregnant counterparts [16]; an effect that could influence fetal renal function. Active transcellular calcium transport in the kidney occurs mainly in the distal portion of the nephron [17] and involves three key proteins: epithelial calcium channel 1 (ECaC1), cal-bindin-D 28K and plasma membrane calcium ATPase (PMCA) [18][19][20]. Altered PMCA [20] and calbindin-D 28K [21] expression has been observed in diabetic rats.…”

Ca2+Dynamics along Identified Synaptic Terminals inDrosophilaLarvae

“…We observed similar motor terminal staining when using a monoclonal antibody prepared against the human erythrocyte PMCA (Borke et al, 1989). This staining pattern indicates that the PMCA protein is concentrated in the presynaptic terminals; however, we cannot rule out that PMCA concentrated in the postsynaptic membrane contributes to the observed staining pattern.…”

Plasma Membrane Ca2+-ATPase Associates with the Cytoskeleton in Activated Platelets through a PDZ-binding Domain

“…Kim et al (10) demonstrated that both PMCA1b and PMCA4b bind to PDZ domains, although PMCA4b was observed to have significantly higher affinity. To determine the relative distribution of the two isoforms in the cytoskeleton, monoclonal antibodies 5F10 and JA9 were employed (22,23); 5F10 recognizes both isoforms, whereas JA9 only binds PMCA4. As shown in Fig.…”