PregnancySpecific β₁-Glycoprotein (SP₁) の代謝経路に関する研究

第2報 SP₁の精製と¹²⁵I標識SP₁のマウス生体内分布および排泄に関する検討

抄録

SP₁ (pregnancy specific β₁-glycoprotein) was purified from human placentae, and the metabolism of ¹²⁵I labeled SP₁ was studied in non-pregnant and pregnant mice of ICR strain.
Purification of SP₁ was carried out by the method of Bohn (1971) and Nose (1981) with modifications. The procedure was as follows : Placental tissues were homogenized with an equal volume of 0.4% NaCl and centrifuged to remove the debris. Into this supernate, rivanol was added to 0.8% concentration. Then the solution was centrifuged, and into the supernate thus obtained, solid ammonium sulfate was added to 50% saturation. The precipitate was thoroughly dialysed against 0.01M tris-HCl buffer pH 8.0 and was applied to the column of DEAE-cellulose equilibrated with the same buffer. After washing, the proteins were eluted with 0.02M tris-HCl buffer pH 8.0 containing 0.45M NaCl. These proteins, concentrated by 503o saturated ammonium sulfate, were applied to a Sephadex G-150 column and eluted with 0.05M tris-HC1 buffer pH 8.0 containing 0.02M NaCl. The SP₁ containing fraction was applied to a DEAE-Sephadex A-50 column, and the elution was carried out with a NaCl gradient (from 0.2% to 2%) in 0.01M tris-HC1 buffer pH 7.6. Next, concentrated SP₁ rich fractions were applied to a Sephacryl S-200 column and eluted with 0.05M tris-HCl buffer pH 8.0 containing 0.5M NaCl.
The SP₁ fractions were applied to an anti SP₁ bound Sepharose 4B column and were eluted with 0.2M glycine-HCl buffer pH 2.5. Each fraction was immediately neutralized by 1.0M tris to pH 7.6. After having been dialysed against distilled water, the pooled protein fraction was lyophilized.
By immunoelectrophoresis the obtained SP₁ formed one precipitated line using anti pregnant serum as well as and SP₁ serum, and did not react with any and sera except for anti SP₁ by the Ouchterlony method. Furthermore, polyacrylamide gel electrophoresis showed one main band, and the purity was estimated approximately 98% from a densitometric pattern.
A preparation of purified SP₁ was labeled with ¹²⁵I by the Chloramine-T method. Radio-iodinated SP₁ was separated from the reaction mixture by gel filtration on a Sephadex G-50 column using 0.01M sodium phosphate buffer pH 7.4 added 1% bovine serum albumin. The radioactivity of each fraction was measured in a γ-spectrometer. The free radioactive iodine of the fractions which reacted with anti SP₁ serum was less than 1%. These fractions were pooled and stored at -20°C until use.
¹²⁵I-SP₁ (0.1ml of approximately 2.5 × 10⁶ C.P.M./ml radioactivity) was injected into the subcutaneous tissue of both non-pregnant and pregnant mice. The experiment was carried out in 6 non-pregnant mice and 4 pregnant mice as one group. The animals were killed at 0.5, 1, 2, 4, 8 and 16 hours after the injection. The organs and tissues were collected, weighed and analysed for accumulated radioactivity.
Non-pregnant mice tissue radioactivity exhibited the highest count in the kidney, followed by blood, ovary & uterus, spleen, liver, muscle and fat at 30 min. after the injection; similar tendencies were observed at other times. Radioactivity of the kidney was more than twice that of blood. A blood disappearance curve showed a half life of 8 hours.The radioactive substance was excreted in urine at a very high level from 30 min. to 4 hours, and in the faeces, it peaked at 8 hours. Pregnant mice tissue exhibited the highest count in the kidney, followed by blood, spleen, ovary & uterus, placenta, fat, muscle and fetus at 30 min. A blood disappearance curve showed a half life of about 11 hours. Radio-activity of the placenta exceeded that of blood after about 1.5 hour from the injection and reached a peak at 4 hours.