Binding of fatty acids and tryptophan to alpha-fetoprotein from fetal pigs.

Using gel electrophoresis and autoradiography, we have shown that plamitic, stearic, oleic and arachidonic acids as well as tryptophan bind to alpha-fetoprotein derived from fetal swine serum. It is also shown that these ligands bind to albumin from both fetal and adult swine serum. The results suggest that alpha-fetoprotein in the fetus has transport functions similar to albumin in the adult.

Synthesis of alpha-fetoprotein, albumin and total serum protein in neonatal pigs.

Studies on the incorporation of 3H-leucine established that synthesis of alpha-fetoprotein (AFP) does occur in the neonatal pig. This synthesis probably accounts for the increase in AFP concentration from 0.46 mg/ml on the day after birth to the maximum value of 1.02 mg/ml found 5 days after birth. After the 5th day, the concentration decreases successively and AFP cannot be detected by electroimmunoassay in the sera of 8-week-old pigs. During the first 3 weeks of extrauterine life, the albumin concentration in serum increases from 3 mg/ml to approximately 30 mg/ml due mainly to increased synthesis of albumin. The concentration of AFP in the fetal pig serum decreases from 3 mg/ml at 6.5 cm crown-to-rump (CR) length to 0.8 mg/ml at 28 cm CR length, whereas the albumin concentration increases from 0.2 mg/ml at 6.5 cm CR length to 1.0 mg/ml at 28 cm CR length. The total serum protein content in the fetus is approximately 20 mg/ml irrespective of gestational age. During the first day after birth there is a marked increase in both the synthesis and the concentration of total serum proteins. The concentration rises to 70 mg/ml during the first day and then slowly declines to 65 mg/ml during the 3 weeks following birth.

High affinity of alpha-foetoprotein for arachidonate and other fatty acids.

Fatty acid analysis of purified bovine alpha-foetoprotein showed it to contain 2.7 mol of fatty acid/mol of alpha-foetoprotein. Purified alpha-foetoprotein focused at isoelectric point 4.8. Removal of bound ligands from alpha-foetoprotein by charcoal treatment changed its isoelectric point to 5.2. This change could be reversed by addition of exogenous fatty acids to the defatted alpha-foetoprotein. Albumin isolated from the same foetal calf serum source as alpha-foetoprotein contained 1.4 mol of fatty acid/mol of protein. alpha-Foetoprotein and albumin contained comparable amounts of fatty acids with 14 to 18 carbon atoms, but alpha-foetoprotein contained 16 times as much of the long-chain polyunsaturated fatty acids as albumin. alpha-Foetoprotein was found to have slightly higher affinity for palmitate and linoleate and severalfold higher affinity for arachidonate than albumin. These findings suggest that alpha-foetoprotein may play a role in the foetal metabolism of the long-chain polyunsaturated fatty acids.

Nonhelical, fibronectin-binding basement-membrane collagen from endodermal cell culture.

A novel method of affinity chromatography on insolubilized collagen-binding fragments of fibronectin was utilized to isolate a random-coil collagenous protein from culture media of mouse teratocarcinoma-derived endodermal cells. These cells also produced another collagenous protein, which did not bind to fibronectin but could be isolated by differential salt precipitation. The affinity-purified collagen differs from its conventionally isolated counterpart in that it is not triple-helical in structure, its polypeptides are not disulfide-crosslinked and it has affinity for fibronectin in its native state. Both collagens resemble previously characterized type IV basement-membrane collagens with respect to their amino acid composition, cyanogen bromide peptides, chain size, immunological reactivity and tissue localization. The random-coil collagen is directly active in promoting the attachment of some lines of cells, but for attachment of the endodermal cells addition of fibronectin is required. This suggests that the presence of nonhelical, fibronectin-binding collagen may have biological significance in the interaction of cells with the extracellular matrix.