We have previously isolated from human pancreatic juice a secretory glycoprotein of 19 KD (P19), devoid ofknown enzymatic activity. P19 gave by proteolysis a protein of 141W (P14), at first named protein X and also called pancreatic thread protein or pancreatic stone protein. Specific rabbit immunosera prepared against P19 and P14 were applied to localize these proteins in human small intestine. By comparison, antibodies directed against some human pancreatic enzymes (amylase, lipase, chymotrypsin, trypsinogen 1, trypsinogen 2, and trypsin 1) were also tested.

Lactoferrin was examined for its effect on the growth of a human colon adenocarcinoma cell line (HT 29) in culture and its action was compared to that produced by transferrin and two different iron solutions (ferrous sulfate and ferric chloride). When transferrin was replaced by either iron solutions the cell grew in proportion to the quantity added and the maximal effect obtained was identical to that produced by transferrin alone. When transferrin was replaced by lactoferrin the cells were unable to proliferate for a long time. However, in the presence of low-concentration iron solutions, lactoferrin stimulated the cell growth, and the effect was more pronounced with the ferric chloride solution.

Proteins with trypsin-like immunoreactivity (first detected by a specific immunoenzymatic assay) were isolated from CAPAN-1 and CFPAC-1 cell culture-conditioned media by chromatography on an immunoadsorbent prepared with a polyclonal antibody directed against trypsin 1. The adsorbed proteins were devoid of free trypsin activity but trypsin activity was present after enterokinase activation demonstrating that the immunoreactive trypsin present in cell supernatants corresponds to trypsinogens. When characterised by Western blotting using a monoclonal antibody directed against human trypsin 1 two protein bands corresponding to trypsinogen 1 (23 kDa) and trypsinogen 2 (25 kDa) gave a positive reaction. These results demonstrate the presence of trypsinogens 1 and 2 in CAPAN-1 and CFPAC-1 cells and in their culture-conditioned media.

The binding of '251-lactoferrin to HT29-D4 cells, a clone of HT29 cells, was studied and compared to the binding of '"1-transferrin to the same cells. The binding of the two iron-transport proteins is saturable and reversiblc suggesting the presence of specific reccptors for each protein. Scatchard analysis suggests the existence of binding sites for lactoferrin with the relatively high equilibrium dissociation constant, K d l of 408 nM. Additionally, the cell is capable of binding large amounts of lactoferrin with very low affinity, probably in a non-receptor intermediale fashion. The dissociation constant of transferrill and its receptor was calculated 9.29 nM which corresponds well to values found in the literature. In contrast to lactoferrin, the cell was capable of binding only low amounts of transferrin in a non-receptor intermediate fashion. Aftcr chemical crosslinking of lactoferrin to the cell surface, the radiolabeled lactoferrin was found in a complex of molecular inass 300 kDa. Crosslinking of transferrin resulted in a complex of much higher molecular mass.These data clearly show a binding site for lactoferrin different from the transferrin receptor. Only if competition cxperiments were performed with a high molar excess of both ligand proteins did a small percentage of either of [he two ligands crossreact with the receptor for glycoproteins.Iron plays a critical role in many biological processes. Free iron under physiological conditions is in the hydroxide[Fe(OH)3] form and is insoluble. Iron is transported after chelating by a carrier system of iron-binding proteins. Lactoferrin, also known as lactotransferrin [l], is a member of the family or iron-binding proteins, that also includes transferrin, for which a critical role in iron metabolisin in higher animals has been shown. Transferrin is essential for the growth of many cell lines in serum-free medium and cellsurface receptors for transferrin are expressed on proliferating cclls in vitro and irz vivo [2, 31.The biological functions of lactoferrin, an important component excreted by granulocytes and several other cell types, are still poorly understood. Only its bacteriostatic role, attributed to its high affinity for iron (K, = 10' ' M-I) depriving bacteria of iron essential for growth, is well described 151.the other, possibly due to a structural similarity of the two These data suggest that lactoferrin may play the role of iron-carrier protein in this model. In this publication we present evidence for the presence of specific lactoferrin receptors on HT29 cells by comparing lactoferrin and transferrin interactions with the cells.

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