The absorption of cobalamin (Cbl) from the diet, its subsequent transport and cellular uptake, requires the interaction of Cbl with two soluble proteins and specific membrane receptors for each of these proteins. The objectives of this project have been to purify and characterize these Cbl binding proteins and their membrane receptors and to delineate their role in Cbl metabolism. This project will now focus on the following specific objectives: 1) Study the structure, snythesis and secretion of transcobalamin II (TCII), the transport protein required for the cellular uptake of Cbl and examine the process by which TCII facilitates the uptake of Cbl. Monoclonal antibodies will be developed to specific epitopes on the TCII molecule and peptide fragments of the protein that cross react with specific antibodies to the Cbl binding site and to the receptor binding site will be identified and sequenced to determine the amino acids which comprise the functional domains of TCII and this will be correlated with the complete amino acid sequence deduced from the complementary DNA (cDNA) (vide infra). The biosynthesis and secretion of TCII will be studied in human umbilical vein endothelial cells using [35S] methionine labeling of nascent TCII. The role of TCII in the intestinal absorption of Cbl will be examined by studying the expression of mRNA for TCII during Cbl absorption and by immunochemical techniques; 2) Purify the membrane receptor for TCII from human placenta, characterize its structure and generate an antiserum to this purified protein; 3) Isolate the TCII cDNA from a human endothelial cell library, determine the nucleotide sequence of the cDNA from which the complete amino acid sequence of TCII can be deduced. Determine the functional properties of the recombinant TCII. Study the restriction length polymorphism and determine the chromosomal location of the TCII gene. These studies will provide fundamental information about the proteins required for the cellular uptake of Cbl and the molecular genetics of TCII, all of which are important in the understanding of acquired and congenital disorders of Cbl metabolism.