IMMUNOGICAL,GENETICAL AND BIOCHEMICAL STUDIES ON ABH GLYCOPROTEIN SUBSTANCES OFF RED CELLS

1994 Fiscal Year Final Research Report Summary

• Project/Area Number: 05454236
• Research Category: Grant-in-Aid for General Scientific Research (B)
• Allocation Type: Single-year Grants
• Research Field: Legal medicine
• Research Institution: GUNMA UNIVERSITY
• Principal Investigator: FURUKAWA Ken GUNMA UNIV.SCHOOL MEDICINE PROFESSOR, 医学部, 教授 (90008227)
• Co-Investigator(Kenkyū-buntansha): NAKAJIMA Tamiko GUNMA UNIV.SHOOL MEDICINE ASSISTANT, 医学部, 助手 (40008561) ; MATSUKI Takasumi GUNMA UNIV.SCHOOL MEDICINE ASSISTANT PROF., 医学部, 助教授 (10126617)
• Project Period (FY): 1993 – 1994
• Keywords: ABO BLOOD GROUPS / ABH BLOOD GROUP SUBSTANCES / GLYCOPROTEIN / RED CELL GHOST / RED CELL MEMBRANE

Research Abstract

RED CELL GHOSTS WERE OBTAINED BY THE METHOD OF FAIRBANKS ET AL.THE WASHED RED CELLS WERE HEMOLYZE WITH SODIUM PHOSPHATE BUFFER pH 8 AND PRECIPITATED RED CELL GHOSTS WERE COLLECTED BY ASPIRATION OF SUPERNATANT AND CENTRIFUGATION.THE GHOSTS WERE WASHED THREE TIMES WITH SODIUM PHOSPHATE BUFFER.THE RED CELL GHOSTS WERE OBTAINED AS HOMOGENOUS AND WHITE PELLETS.TWO HUNDRED ml OF WET GHOSTS WERE OBTAINED FROM 10,000 ml OF GROUP 0 AND A RED CELLS.
1) ABH ACTIVE GLYCOPROTEINS FROM RED CELL MEMBRANE WERE EXTRACTED ACCORDING TO THE METHOD OF MARCHESI ET AL.THE RED CELL GHOSTS WERE SOLUBILIZED BY SUSPENDING IN LITHIUM DIIODOSALICYLATE.THE GLYCOPROTEIN WAS EXTRACTED BY ADDITION OF 50% PHENOL TO THE SUSPENSION.THE PHENOL EXTRACTS WERE TESTED BLOOD GROUP H OR A ACTIVITY.THE INHIBITION TITER OF THE EXTRACTS WERE ONLY 1 : 4.
2) ACCORDING TO EXTRACT WATER SOLUBLE BLOOD GROUP SUBSTANCES FROM GASTRIC LININGS,90% PHENOL WAS ADDED DIRECTLY TO FREEZE DRIED RED CELL GHOSTS.THE PHENOL SOLUBLE FRACTION WAS COLLEC TED AND GLYCOPROTEIN WAS PRECIPITATED BY ADDITION OF ETHANOL AT THE FINAL CONCENTRATION OF 35% AND 70%. THE PRECIPITATE WAS COLLECTED BY CENTRIFUGATION AND LYOPHILIZED.BLOOD A OR H ACTIVITY WAS EXAMINED WITH AGGLUTINATION INHIBITION TEST.THE BLOOD GROUP ACTIVITY OF SOLUTION OF DIRECT PHENOL EXTRACT WAS VERY LOW.
3) SOLUBILIZED RED CELL GHOST WERE APPLIED ON POLYACRYLAMIDE GEL AND ELECTROPHORESIS WAS CARRIED OUT.GEL WAS STAINED BY THE METHOD OF SCHIFF AND USING STAINED ELECTROPHORETIC PATTERN UNSTAINED GEL WAS CUT INTO SMALL BLOCKS.THE SALINE EXTRACTS OF EACH BLOCK WERE TESTED BLOOD GROUP ACTIVITY BY ABSORPTION TEST OF AGGLUTININS.THE FRACTIONS OF BLOCKS WERE SHOWED VERY LOW BLOOD GROUP ACTIVITIES.
THERE ARE MANY BIOCHEMICAL REPORTS OF GLYCOPROTEINS OBTAINED FROM RED CELL MEMBRANE.IT IS PROBABLE THAT THE ACTIVITY OF OBTAINED GLYCOPROTEIN FRACTION MAY CONTAINS WATER SOLUBLE GLYCOLIPIDS.IF THE GLYCOPROTEIN BLOOD GROUP SUBSTANCES WERE PRESENT ON THE RED CELL MEMBRANES IT IS MOST PROBABLE THAT THE BLOOD GROUP SUBSTANCES IN THE SERUM WERE ABSORBED ON THE RED CELL SURFACE LIKE LEWIS BLOOD GROUP SUBSTANCES.