Translocation of bFGF to the nucleus is G1 phase cell cycle specific in bovine aortic endothelial cells.

Primary cultures of adult bovine aortic endothelial (ABAE) cells require bFGF to grow. G1-arrested cells, obtained after 48 h without serum and bFGF, were found to enter S phase and grow synchronously for at least two generations on addition of bFGF. In growing cells bFGF was detected both in the cytoplasm (90%) and in the nucleus (10%) where it accumulates in the nucleolus. It was not detected in the nucleus of confluent cells. bFGF uptake was continuous in the cytoplasm throughout the cell cycle with a maximum in G2, while nuclear uptake occurred only in late G1. Cytoplasmic bFGF (18.4 kd) is cleaved into a 16.5 kd peptide in G1 (t1/2 = 30 min). In the nucleus the 18.4 kd form was the only one detected 2 h following bFGF addition and was then cleaved into the 16.5 kd in early S phase. These results are consistent with the possibility that in addition to the classical pathway of signal transduction, bFGF is directly translocated to the nucleus in late G1, and could play a role in replication and/or in transcription of rDNA.

Inhibitory properties and antigenic specificity of monoclonal antibodies to pancreatic colipase.

To understand the mechanism by which colipase acts as a protein cofactor for anchoring pancreatic lipase at triacylglycerol/water interface, we have used an immunochemical approach. Ten monoclonal antibodies (Mabs) against porcine pancreatic procolipase were produced. Purified immunoglobulins and Fab fragments were studied for their capacity to inhibit colipase-dependent lipase activity. These studies were carried out by using procolipase, the secretory form of the cofactor, and its trypsin-treated form obtained by removal of the amino terminal pentapeptide by trypsin. Reactivities of Mabs with both forms of the cofactor were also studied by immunoenzymatic methods. Mabs 6.1, 49.20. 75.8, 270.13 and 419.1 were found to inhibit lipolysis by preventing the binding of procolipase or trypsin-treated colipase to the lipid substrate. Mab 72.11 inhibited procolipase binding but had no effect on trypsin-treated colipase. Mab 72.11 reacted with procolipase in ELISA but showed no reactivity with trypsin-treated colipase. Finally, preincubation of Mab 72.11 with porcine procolipase prevented specific cleavage at the Arg5-Gly6 bond by trypsin. It could be concluded, that the five first residues of procolipase are structural elements of the antigenic determinant recognized by Mab 72.11. Results of ELISA additivity tests (cotitrations) further indicated that epitopes for Mabs 6.1, 72.11, 270.13 and 419.1 and for Mabs 49.20 and 75.8 are located in two distinct antigenic regions of the procolipase molecule. It appears then that the lipid binding domain of the pancreatic lipase protein cofactor comprises two regions. The first region corresponds to the amino terminal fragment of the protein. The second region is likely identical with the peptide segment at position 51-59 as previously hypothesized from NMR and spectrophotometric studies. Studies carried out on procolipase chemically modified at tyrosine residues provided evidence that epitopes for Mabs 49.20 and 75.8 are in or close to the region which contains tyrosines at positions 55 and 59, and that the two peptide regions essential for interfacial binding are spatially adjacent in the procolipase and the trypsin-treated form of the cofactor. General conclusions are in accordance with the location of antigenic regions of procolipase determined by predictive methods.

Production and characterization of four monoclonal antibodies against porcine pancreatic colipase.

Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase.

Inhibition of pancreatic colipase by antibodies and Fab fragments. Selective effects of two fractions of antibodies on the functional sites of the cofactor.

Rabbit antiserum was raised against porcine pancreatic colipase and Fab fragments were prepared by papain digestion of purified antibodies followed by purification on protein A-Sepharose. Fab fragments showed inactivation toward porcine colipase activity similar to that of antiserum and purified antibodies. From inactivation studies carried out by incubating porcine colipase and lipase with Fab fragments in the absence of lipid or in the presence of triolein and sodium deoxycholate, it could be concluded that polyclonal antiporcine colipase antibodies contain fractions that bind specifically to epitopes at or near the functional regions of the porcine cofactor. Studies with an enzyme-linked immunosorbent assay showed that cross-reactivity of horse or chicken colipase with antiporcine colipase antiserum was lower than that of the human or porcine protein. Results of immunoactivation kinetic studies performed with the same proteins, fully confirmed these observations. Partial cross-reactivity between porcine and chicken colipases allowed us to fractionate antibodies by immunoaffinity chromatography on immobilized chicken colipase. Fraction I contains antibodies absorbed on porcine colipase not accessible when the cofactor is bound to lipid. Antibodies of fraction II, nonadsorbed on chicken colipase, inactivate porcine colipase preincubated with triolein/deoxycholate. Lipase had a protective effect against inactivation. Antibodies of fraction II bind likely to epitopes close to the specific region of colipase interacting with lipase. Our conclusions are in good agreement with analysis of the sequence of porcine, equine and human colipases by calculating local hydrophilicity indices.

Studies on chicken pancreatic lipase and colipase.

Lipase and colipase have been purified to homogeneity from chicken pancreatic tissue. The enzyme has a molecular weight (48 000) and catalytic properties similar to those of pancreatic lipase from higher mammals. Hydrolysis of triolein by chicken lipase is strongly inhibited by various bile salts, including sodium taurochenodeoxycholate, which is present in large proportion in chicken bile. Inhibition is reversed by colipase. With triolein as enzyme substrate, in the presence of sodium deoxycholate, no difference was observed in the ability of pure colipase from chicken, horse or pig to fully activate bile-salt-inhibited lipase from the same species. However, kinetic studies of the hydrolysis of a lecithin-stabilized emulsion of triacylglycerol (Intralipid) by chicken lipase show that the lag period is much longer in the presence of porcine colipase than with the chicken cofactor. This might reflect the higher ability of the avian enzyme to associate with colipase from the same species than with mammalian cofactors when the triacylglycerol substrate surface is covered with amphiphilic lecithin. From our study, the chicken pancreatic lipase/colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. It is then likely that they are of comparable physiological significance in fat digestion in avian and mammalian species.

Evidence for the existence of procolipase in chicken pancreas and pancreatic juice.

Purified antibodies raised against chicken colipase were coupled to Sepharose 4B and colipase was isolated in a single step by immunoaffinity chromatography from an extract of chicken pancreas prepared under conditions where trypsin activation is avoided. The purified protein has a single amino terminal residue of alanine and its biochemical properties are similar to those of the precursor form of colipase (procolipase) previously isolated from porcine and equine pancreas or pancreatic juice. Further evidence for the existence of procolipase was obtained from kinetic studies of the hydrolysis of the Intralipid emulsion by untreated and trypsin-treated chicken pancreatic juice.

Isolation and characterization of colipase from porcine and human pancreatic juice by immunoaffinity chromatography.

Pure colipase was prepared by immunoaffinity chromatography from porcine and human pancreatic juice. A single form of the porcine colipase was obtained, having the structural and biological properties of previously characterized porcine procolipase A. Two forms of activated colipase (N-terminal Gly) were isolated from human pancreatic juice by the same procedure. The existence of two forms of activated colipase might arise from rapid activation of a precursor form of human colipase during collection of the pancreatic juice.

Limited trypsinolysis of porcine and equine colipases. Spectroscopic and kinetic studies.

Porcine and equine colipases have been submitted to mild tryptic digestion. Proteolysis occurs at the Arg5-Gly6 bond with the loss of the N-terminal pentapeptide. Studies of native and trypsin-treated colipases by circular dichroism and laser chemically induced dynamic nuclear polarization indicate that proteolysis induces conformational changes in the region of the tyrosine cluster. Experiments in the presence of phospholipid provide further evidence showing that these residues are in or close to the region of the protein interacting with aggregated lipids. Kinetic studies of the reaction of bile salt-inhibited lipase with emulsified triolein in the absence and in the presence of lecithin show that tryptic hydrolysis of the protein cofactor increases its affinity for the enzyme in the presence of lipid substrate. In both cases, it was found that the apparent dissociation constant of the lipase-colipase complex is decreased by one order of magnitude. Our results confirm that the biological activity of the lipase cofactor is enhanced by specific tryptic cleavage in the amino terminal region of the polypeptide and support the suggestion by Borgström et al. (Borgström, B., Wieloch, T., Erlanson-Albertsson (1981) FEBS. Lett. 108, 407-410) that the secreted form of colipase is a precursor.

Horse pancreatic lipase. Interaction with colipase from various species.

Horse pancreatic lipase has been purified from tissue homogenates. Molecular and catalytic properties of horse lipase are comparable to those of the pancreatic lipases previously isolated. Kinetic studies of the inhibition of horse lipase activity by bile salts and of reactivation by pure colipase from three species (horse, ox and pig) allowed to calculate the apparent dissociation constant (Kd) of the lipase-colipase complex in the presence of the substrate (triolein). Identical values of Kd were found in all three cases (Kd = 1.1 10(-9) M). These values are lower by several orders of magnitude than that published for the binding between lipase and colipase in the absence of substrate. Qualitative experiments show that the activation of horse lipase can be accomplished by rat, dog and chicken colipase as well. The interaction between lipase and colipase is enhanced when the complex is adsorbed at the lipid-water interface. This specific protein-protein interaction is preserved in heterologous mixtures using colipases from other animal species.

Isolation and partial structural characterization of chicken pancreatic colipase.

Colipase has been isolated from acidic extracts of chicken pancreatic tissue homogenized with Triton X-100. The cofactor fully activates bile salt inhibited mammalian lipases. The amino terminal sequence of the avian protein has been determined up to position 39 and compared to the homologous region of the mammalian colipases (pig, horse, man) previously studied. From this comparison, it appears that a high degree of homology exists between the proteins.