Studies on Endogenous Lectins from 3T3 Fibroblasts
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Three distinct, lactose-specific carbohydrate binding proteins, CBP 35 (Mr = 35,000), CBP 16 (Mr = 16,000) and CBP 13.5 (M r = 13,500), have been isolated from 3T3 fibroblasts on the basis of their binding to asialofetuin (ASF)-Sepharose columns. In this study, several variations of the original isolation procedure were explored. The results showed that the CBPs can be isolated independent of the presence of calcium ions, β-mercaptoethanol, and the detergent Triton X-100. Therefore, the original isolation procedure was appropriate for the isolation of optimal amounts of the mixture containing all three CBPs. CBP 35, as well as fractions containing a mixture of CBP 16 and CBP 13.5, both showed agglutination activity when assayed with rabbit erythrocytes; these agglutinations were inhibitable by lactose. None of the CBPs showed β-galactosidase activity using 4-methylumbelliferyl-β-D-galactopyranoside as a substrate at pH 4.6 or 7.0. All the β-galactosidase activity found in the cell extract was recovered in fractions that did not bind to the ASF-Sepharose column. No sialyl transferase activity was indicated for any of the CBPs in our assay. Immunoprecipitation of partially purified CBPs with monospecific antisera against CBP 35 precipitated only CBP 35 while monospeciftc antisera against chicken lactose lectin I precipitated only CBP 16. These results suggest that CBP 16 may be the murine analog of chicken lactose lectin I and that CBP 16 and CBP 35 are not structurally related. Western transfer and immunoblotting of subcellular fractions of 3T3 cells indicated the presence of CBP 35 in all of the membrane containing fractions and a cross reactive higher molecular weight polypeptide (Mr =~ 130,000) in the plasma membrane fraction. Tissue distribution of CBP 35 in A/J mice was also determined by Western transfer and immunoblotting. CBP 35 was present in the lung, spleen and thymus. An immunologically cross reactive protein (Mr~= 68,000) was seen in the brain and kidney.