PMC:3248852 / 33357-36234 JSONTXT

{"project":"2_test","denotations":[{"id":"22054214-4558563-9450464","span":{"begin":1039,"end":1040},"obj":"4558563"},{"id":"22054214-3843705-9450465","span":{"begin":1114,"end":1116},"obj":"3843705"},{"id":"22054214-4288135-9450466","span":{"begin":1216,"end":1218},"obj":"4288135"},{"id":"22054214-14220729-9450467","span":{"begin":1527,"end":1529},"obj":"14220729"}],"text":"32PO4 Incorporation into yolk phosvitin.\nIn the phosvitin labeling studies, groups of 400 follicles were labeled with 10 μCi [32PO4] in 20 ml Ringer's solution (with and without 3.2 μM progesterone) for the times indicated at 20-22°C. Follicles were also pulse labeled for 4 h, beginning 5 h after exposure to 3.2 μM progesterone. At the end of the exposure to 32PO4 containing media, the follicles were rinsed with Ringer's solution, homogenized in 5 volumes of 7% TCA at ice bath temperatures and allowed to stand at 4-5°C for 5 min. The suspension was centrifuged, the precipitate washed 2× with 3 volumes of 7% cold TCA, and extracted sequentially with 2 volumes of CHCl3:CH3OH (2:1) and CHCl3:CH3OH:HCl (2:1:0.01). Nucleic acid was extracted with 7% TCA at 90°C for 15 min, and the final precipitate treated with 0.05 N NaOH at 100°C for 10 min to recover the alkali-labile phosphate from the remaining phosphoprotein. Aliquots were counted by liquid scintillation spectrophotometry and analyzed for phosphate as described elsewhere [5]. Protein was measured by the Bicinchoninic acid method of Smith et al. [41].\nPhosvitin was prepared from Rana ovarian follicles using a method adapted from Mano and Lipmann [42]. A more recent isolation method by McCollum et al. [43] utilized ferric ion precipitation of \"specific\" phosvitins. However, Taborsky has reported [44] that ferric ions caused dephosphorylation of egg yolk proteins at alkaline pH. It should be noted that non-heme iron is recovered with purified phosvitin [23] and is present as a contaminant in laboratory grade salts used in protein isolation. To reduce possible protein dephosphorylation during purifcation, 10 mM EDTA was added to all solutions used to isolate oocyte phosvitin.\nThe highest yields of phosvitin were obtained from Rana follicles as follows: 400 follicles (~900 mg wet weight) were homogenized in 1/2 volume of a solution of 0.15 M KCl, 0.025 M NaHCO3, 0.02 M NaF and 0.01 M EDTA (disodium salt) in a Potter-Elvehjem homogenizer at ice-bath temperatures. The homogenate was diluted with 1.5 volumes of the extraction solution and centrifuged at 13,000 × g for 60 min. 1/10 volume of 1M barium acetate was added to the supernatant and the pH adjusted to about 7.0 with dilute NH4OH. After standing for 30 min at ice-bath temperatures, the precipitate was collected by centrifugation and resuspended in 0.5 volumes of 0.2 M ammonium sulfate containing 0.05 M Tris buffer, pH 7.6. The resulting barium sulfate ppt was removed by centrifugation, and the opalescent solution dialyzed against distilled water for 48 h at 5°C. In an ice-bath, 1.5 volumes of 95% ethanol were added to precipitate the protein, which was washed with CHCl3:CH3OH (2:1), then dried and ground in a mortar. The powder contained 0.75 ± 0.04% alkali-labile phosphorous (N = 3), and represented 59 ± 4% (N = 3) recovery."}