Young Age-Related Glycoproteins Revealed in Normal Human Urine by Isoelectric Focusing

Koichiro KISHI, Shoei ISEKI
1982 Proceedings of the Japan Academy. Series B, Physical and biological sciences  
Normal During the course of genetic studies on glycoproteins in normal human urine by isoelectric focusing electrophoresis, ' we were able to find young age-related glycoproteins in normal urine. Materials and methods. Urinary samples for this study were collected from healthy individuals (98 males and 25 females including 15 families) living in Tokyo and ranging from 2 months to 68 years of age. The urine samples were immediately filtered at 4°C, and concentrated by ultrafiltration with
more » ... lter (Toyo Co., Ltd.) . The concentrated urine samples were dialyzed overnight against 0.1 % glycine and centrifuged. Supernatants were freeze-dried and dissolved in one thousandth of their original volumes in a 2 M urea. Gels (120 x 90 x 0.5 mm) were prepared as follows. Two solutions, A and B were prepared. Solution A contained 2.3 g polyacrylamide (Nakarai), 0.066 g of bis-acrylamide (Bio-Rad), and 10 g urea (Bio-Rad), which was brought to a total volume of 30 ml with destilled water. Solution B contained 0.02 g of ammonium persulfate (Nakarai) and 4 ml of 0.004% riboflavin (Wako) solution. Four ml of solution A was mixed with 0.2 ml of Pharmalyte of pH range 2.5-5 (Pharmacia). To polymerize the gel, 0.44 ml of solution B was added to the solution A-Pharmalyte mixture. The combined solution was poured into a glass plate mold and photopolymerized for 2 hrs under a fluorescent lamp. Isoelectric focusing was carried out on the FBE-3000 equipment (Pharmacia) at 5°C. The anode electrode strip contained 0.1 M H2SO4 and the cathode 0.5 M NaOH. After one hour prefocusing with 200 V-900 V, 10 pl of concentrated urine samples were applied on 4 x 10 mm filter papers (Whatman 3MM) at a distance of 1 cm from the anode. After another one hour focusing at 1,200 V with the filter papers, the papers were removed. The gel was then fixed in 20% trichloroacetic acid for 24 hrs, and stained with 0.1% Coomassie Brilliant Blue R 250 (Merck) for protein or with the Schiff's reagent for carbohydrate. Neuraminidase treatment was carried out as follows. Twenty five pl of concentrated urine sample was mixed with 100 pl of 0.1 M sodium acetate buffer, pH 5.5, containing 0.15 M NaC1 and 0.007 M CaCI2. Two pl of neuraminidase (0.5 U/pl, BDH)
doi:10.2183/pjab.58.229 fatcat:hmnjts2mnjdsreayud436546vm