Delivery of 57 Co B 12 to Lymphoblasts Derived from Mice with Transplanted 1210 Ascites Tumor Cells by Transcobalamins I,II,and III 1

Abstract

It has been shown in this and other laboratories that uptake of radioactive vitamin B₁₂ from serum in various cell systems is mediated through a protein identified as trans-cobalamin II (TC II). Two other binders in serum, TC I and TC III, do not provide this function (1-8). With the availability of BDF1 mice with transplanted L1210 ascites tumor cells in this laboratory, an attempt was made to determine whether lymphoblasts from the abdominal cavity of these animals would accept radioactive B₁₂ from any of the labeled transcobalamins derived from human serum. A recent report with these same fractions utilizing PHA-stimulated human lymphocytes indicated that only TC II ⁵⁷Co B₁₂ was capable of accomplishing this function (7).

Materials and Methods. Preparation of vitamin B₁₂ binders. This has been reported in detail elsewhere (9). Thirty milliliters of normal serum were used in each column chromatographic separation. Serum used in each experiment was drawn from an individual donor considered to be hematologically normal. Five hundred pico-grams. of ⁵⁷Co B₁₂ (specific activity 120 mCi/ mg; purchased from the Radiochemical Centre, Amersham, England) were added per milliliter to 30 ml of serum. The solution was allowed to stand at 37° for 20 min, dialysed at 4° against four liters of 0.0175 M sodium phosphate buffer, pH6.3, for 48 hr. The buffer was changed twice during this period.

DEAE-cellulose (Schleicher and Schuell, No. 70, of ion exchange capacity between 0.90 and 0.95 mequiv/g) was packed after preparation into 3 × 60-cm columns (ea). The following buffers were used for elution at 4° with flow rate of 30 ml/hr: 0.0175 M sodium phosphate buffer, pH 6.3 (600 ml); 0.04 M sodium phosphate buffer, pH 5.9 (1,000) ml; 0.1 M sodium phosphate buffer, pH 5.8 (500 ml); and 0.4 M sodium phosphate buffer, pH 5.2 (700 ml).