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First published online 18 March 2003
doi: 10.1242/jcs.00392

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Transient association of the sarcoplasmic reticulum Ca²⁺ ATPase with the Na⁺/K⁺-ATPase and H⁺/K⁺-ATPase ß-subunits during its biogenesis in Xenopus oocytes

¹ Department of Biochemical Engineering and Science, Kyushu Institute of Technology Iizuka, Fukuoka 820-8502, Japan
² Department of Molecular Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan

View larger version (60K): [in a new window]   Fig. 1. Immunoprecipitation and western blot of the SR Ca2+ ATPase. (Left) Oocytes injected with both cRNAs for the SR Ca2+ ATPase and the ß-subunit of the Na+/K+ ATPase (each 10 ng oocyte–1) were incubated in modified Barth's medium at 19°C for 10 hours. The oocytes were subsequently transferred to Barth's medium containing [35S]methionine and [35S]cysteine, and labeled for 60 minutes. The extracts of the labeled oocytes were immunoprecipitated and the precipitates were separated by SDS-PAGE followed by autoradiography. (Right) The separated proteins in lanes 1-4 of the left panel were transferred to PVDF membrane. After washing the blotted PVDF membrane three times with 2% SDS containing 0.7% 2-mercaptoethanol at 70°C for 30 minutes, the proteins were stained with antiserum specific for the SR Ca2+ ATPase raised in goat. The primary antibody was detected with anti-goat secondary antibody (20,000x dilution) that was labeled with peroxidase.

View larger version (82K): [in a new window]   Fig. 2. Short-term labeling of the SR Ca2+ ATPase. The oocytes that had been directly injected with radiolabeled amino acids as described in Materials and Methods, were incubated in modified Barth's medium for 3-30 minutes. The microsomes from the labeled oocytes were immunoprecipitated with antisera specific for the SR Ca2+ ATPase (lanes 1-4) and for the ß-subunit of the Na+/K+ ATPase (lanes 5-8).

View larger version (54K): [in a new window]   Fig. 3. Pulse-chase of the SR Ca2+ ATPase. The oocytes that had been labeled for 1 hour were chased for 1-48 hours as described in Materials and Methods. Lanes 1-8 contain immunoprecipitates with antiserum to the SR Ca2+ ATPase; lanes 9-16 contain immunoprecipitates with antiserum to the ß-subunit of the Na+/K+ ATPase.

View larger version (40K): [in a new window]   Fig. 4. Association of the H+/K+ ATPase ß-subunit and the SR Ca2+ ATPase. The oocytes were injected with cRNAs and radiolabeled as described in Fig. 1, except that antisera to the ß-subunit of the H+/K+ ATPase was used instead of that of the Na+/K+ ATPase.

View larger version (68K): [in a new window]   Fig. 5. Prevention of the synthesis of the SR Ca2+ATPase by the anti-ß-subunit antibody. Oocytes were injected with cRNA for the SR Ca2+ATPase (10 ng oocyte–1) and incubated in modified Barth's medium at 19°C. After incubation for 10 hours, the oocytes were injected with varying volumes of antibody, adjusted to a total volume of injection at 23 nl with water and allowed to stand for 60 minutes. The volumes of antibody injected were 0 nl, 0.08 nl, 0.23 nl, 0.77 nl, 2.3 nl, 7.7 nl and 23 nl (from left to right). The oocytes were then labeled with [35S]methionine plus [35S]cysteine for 30 minutes, followed by immunoprecipitation with anti-Ca2+-ATPase antiserum.

View larger version (77K): [in a new window]   Fig. 6. Tryptic digestion of the SR Ca2+ATPase. The oocytes were injected with cRNAs and radiolabeled as in Fig. 1. The microsomes from the oocytes were digested with trypsin (Worthington: L-1-tosylamide-2-phenylethy-chloromethyl-ketone-treated) at ratios of trypsin to protein of 0.01 and 0.1 on ice for 60 minutes. The digests were immunoprecipitated with antiserum to the SR Ca2+ ATPase (lanes 1-6) or to the ß-subunit of the Na+/K+ ATPase (lanes 7-9).