Abstract
We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca2+ transport pathway and an unsaturable Ca2+ flux component (Lockwich et al., 1994. J. Membrane Biol. 141:289–296). In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E. coli bulk phospholipids, by using a detergent dilution method. PrL exhibited 3–5-fold higher 45Ca2+ influx than control liposomes (without protein). Ca2+ uptake into PrL was dependent on the [protein] in PrL and steady state [Ca2+] in PrL was in equilibrium with external [Ca2+]. These data demonstrate that a passive, protein-mediated Ca2+ transport has been reconstituted from BLMV into PrL. 45Ca2+ influx into liposomes did not saturate with increasing [Ca2+] in the assay medium. In contrast, PrL displayed saturable 45Ca2+ influx and exhibited a single Ca2+ flux component with an apparent K ca=242 ± 50.9 μm and V max=13.5 ± 1.14 nmoles Ca2+/mg protein/ minute. The K ca of Ca2+-transport in PrL was similar to that of the high affinity Ca2+ influx component in BLMV while the V max was about 4-fold higher. The unsaturable Ca2+ flux component was not detected in PrL. 45Ca2+ influx in PrL was inhibited by divalent cations in the order of efficacy, Zn2+>Mn2+>Co2+=Ni2+, and appeared to be more sensitive to lower concentrations of Zn2+ than in BLMV. Consistent with our observations with BLMV, the carboxyl group reagent N,N′-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted Ca2+ transport in PrL. Importantly, in both BLMV and PrL, DCCD induced a 40–50% decrease in V max of Ca2+ transport without an alteration in K ca. These data strongly suggest that the high affinity, passive Ca2+ transport pathway present in BLMV has been functionally reconstituted into PrL. We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca2+ influx in the rat parotid gland basolateral plasma membrane.
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We thank Dr. Bruce Baum for his constant support and encouragement. We also thank Ms. Grace Park and all our colleagues for their assistance during the course of this work.
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Lockwich, T., Chauthaiwale, J., Ambudkar, S.V. et al. Reconstitution of a passive Ca2+-transport pathway from the basolateral plasma membrane of rat parotid gland acinar cells. J. Membarin Biol. 148, 277–285 (1995). https://doi.org/10.1007/BF00235045
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DOI: https://doi.org/10.1007/BF00235045