Effect of Human Serum B12 Binders on Uptake of Vitamin B12 by Isolated Perfused Rat Liver

Abstract

It has been reported that three binders of "7Co BIZ are separated by DEAE-cellulose column chromatography following addition of labeled vitamin to normal serum (1, 2). These binders are known as transcobalamin I1 (TCII), transcobalamin I (TCI) and main protein peak binder (MPPB) . TCII and MPPB have an electrophoretic mobility of ,%globulin, while TCI has the mobility of a-globulin ( 2 ) . The molecular size of MPPB is approximately 120,000, similar to that of TCI, and larger than TCII (36,OOO) ( 2 ) .Further observations reveal that intravenously administered TCII 57C0 BIZ and MPPB 57C0 B12 are cleared rapidly from plasma while T C I 57C0 B12 shows a much slower pattern (2-4). Finkler and Hall (5) reported that HeLa cells take up TCII but not TCI bound TO BIZ. Retief et al. (6) observed that TCII delivers TO BIZ to erythrocytes at a faster rate than TCI. Haught et al. ( 7 ) recently reported that rat T C I I has no function in B12 uptake by perfused rat liver.In this study uptake by isolated perfused rat liver of VO B12 bound to the three binders was investigated. Naterials and Methods. Preparation of L 7 C~ B1! binders. The binder used in each perfusion experiment was prepared from 30 ml of serum, as follows. Serum from normal subjects or pernicious anemia (PA) patients was kept a t -20" before use. 300 pg of TO BIZ (sp act around 200 mCi/mg; purchased from Philips-Duphar, Holland) were adtded/ ml to 30 rnl of thawed serum. The solution was allowed to stand at 37" for at least 15 min, then was dialyzed in the cold room against 0.0175 M sodium phosphate buffer pH 6.3, for 24 hr.DEAE-cellulose, Schleicher and Schuell, No. 70, of ion exchange capacity between 0.90 to 0.95 mEq/g, was packed after proper preparation, into 3 X 6-cm columns ( 2 ) .The following buffers were used for elution a t 4 O and flow rate of 30 ml/hr: 0.0175 AL? sodium phosphate buffer, pH 6.3 (600 ml) ; 0.04 M sodium phosphate buffer, p H 5.9 (1000 m l ) ; 0.1 M sodium phosphate buffer, pH 5.8 (500 ml); and 0.4 M sodium phosphate buffer, p H 5.2 (700 ml). Buffer sohtions were made in distilled water containing 0.09% methylparaben and 0.01 % propylparaben (Tenneco Chemicals). TCII was eluted with the 0.04 M buffer, MPPB with the 0.1 M buffer and TCI with the 0.4 M buffer (2). Eluates containing binders were dialyzed and freeze dried, No further attempt a t purification was made.Liver perfusion. Rat liver perfusions were carried out as 2 hr experiments according to the procedure described by Miller et al.

Transport function of transcobalamin II

“…Since in none of ot promote B-12 the earlier work were the B-12 binding components of simple presence IF or serum separated, it cannot be determined which Possibly some were the active principles. Lcted from perfu-the "R" binder in human gastric juice (16), the third binder in human serum (15), and human TC II (15).…”

Separation of Transcobalamins on Small DEAE-cellulose Columns

“…MPPB has a molecular size of about 120,000 and aclts like TCII when injected intravenously into human subjects or when it is perfused through the isolated rat liver ( 3 , 4). TCI has a molecular size of about 121,000, but differs from TCII and TCI in that clearance from plasma is slow following intravenous injeotion into normal human subjects, and is poorly bound to isolated rat liver when perf used through the organ (3,4).…”

“…lar size of approximately 3 6,000, disappears rapidly from the circulation following intravenous injection into normal human subjects, and is bound to rat liver when perfused through the isolated organ (3,4 ) . MPPB has a molecular size of about 120,000 and aclts like TCII when injected intravenously into human subjects or when it is perfused through the isolated rat liver ( 3 , 4).…”