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The amino acid sequence of an activated colipase purified from human pancreas was determined. The protein consists of a single polypeptide chain of 86 amino acids (human colipase86) and has a molecular weight of 9289. The sequence was determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of two CNBr peptides. Sequence determination of porcine procolipase II was also performed, which showed that in the original sequence determination apparently two residues were missed. These residues were determined to be a leucine at position 37 and a serine in position 50. For comparison with porcine and equine procolipases, the residues composing human colipase are numbered from 6 to 91. No human procolipase has been isolated so far. The colipases from man, pig, horse and chicken show a high degree of homology: human colipase differs from the other proteins by substitutions of 19 (porcine), 24 (equine A) and 21 (equine B) residues, respectively.

Pig and horse colipases contain three tyrosine residues. In addition, horse colipase possesses a tryptophan residue. Some of the tyrosine residues are involved in the association of colipase and a bile salt micelle. The present report demonstrates that the aromatic residues responsible for colipase fluorescence are in an aqueous environment. In the presence of bile salt micelles, changes in colipase fluorescence properties indicate that the intrinsic fluorophores are located in a more hydrophobic environment upon colipase-micelle complex formation. In addition, the fluorescence of an NBD group fixed on lysine 60, which is very close to the aromatic region in the pig colipase, is also altered in the presence of micelles. These results show that the micelle binding site is not limited to the tyrosine residues but may be broadened to adjacent residues such as lysine 60 and also tryptophan 52 in horse colipase.

Lipase and colipase have been purified to homogeneity from canine pancreatic juice. The purity of the lipase and colipase preparations was established by acrylamide gel electrophoresis. Either lipase or colipase alone did not produce fat necrosis when injected intraperitoneally into mice. Fat necrosis was seen only when both lipase and colipase were used together. Selective removal of lipase from fresh canine pancreatic juice by immunoprecipitation with an antilipase specific antiserum also eliminated its fat necrotizing activity. Together, these results identify the fat necrotizing factors to be pancreatic lipase and colipase. Their uncontrolled release during acute pancreatitis is believed to constitute the cause of fat necrosis. The absolute amount of lipolytic activity was not found to be the crucial factor in the induction of fat necrosis. It is suggested that the colipase molecule may have other functions besides enhancing the lipolytic activity of purified lipase in causing fat cell necrosis.

A radioimmunoassay for each of the human pancreatic proteins (colipase, lipase and phospholipase A2) is described. Determinations of the mean concentration of each protein in plasma and urine from healthy individuals were carried out with the radioimmunoassays. The values obtained in plasma were 0.5 nM (5.3 micrograms/l), 0.6 nM (32 micrograms/l) and 0.3 nM (4.3 micrograms/l) for colipase, lipase and phospholipase A2, respectively. In urine, the corresponding values were found to be 0.2 nM (2.4 micrograms/l), 0.09 nM (4.4 micrograms/l) and less than 0.017 nM (0.2 micrograms/l). No physical interaction between any of the three proteins and the lipid particles of plasma was demonstrated by centrifugation experiments or gel filtration. Gel filtration of plasma depleted of fat by centrifugation showed the proteins only in their monomeric form. The corresponding porcine proteins displayed a binding to antibodies against the human proteins, but with a lower affinity than the homologous interactions. The binding was weak but could differentiate between the porcine proforms and activated ones, i.e., procolipase and colipase87.

Procedures for purification of porcine colipase II (Gly6-Gly89) and for obtaining purified anti-colipase antibodies are described. The interactions between antibodies immobilized on an Ultrogel AcA 22 column and colipase were investigated and colipase radioimmunoassay carried out. The immobilized antibody-colipase binding was preserved in the presence of mixed micelles, lipase, or both when added to the elution mixture. Bound colipase maintained its capability of interacting with mixed micelles, but not with lipase in either the presence or the absence of mixed micelles. It could be inferred that the antigenic site(s) is independent of the interfacial recognition site and close to the site of lipase recognition. Results are reported suggesting that one or both colipase histidyl residue-containing sequences are involved as antigenic determinant(s). Immunoreactive colipase, bound to a macromolecular protein complex, was found in the plasma of pig. This finding could be explained by an endocrine 'leakage' of colipase from the exocrine pancreatic cell rather than by passage through the intestinal mucosa.

Pancreatic procolipase is a cofactor for lipase and necessary for optimal fat digestion in the intestine during a meal. It is activated by trypsin in the intestine during release of an activation peptide, with the sequence Val-Pro-Asp-Pro-Arg in rat. This peptide, in the following termed VPDPR, was found to decrease food intake in rats. The human procolipase activation peptide with the sequence Ala-Pro-Gly-Pro-Arg (APGPR) had no effect on food intake in rats, nor the trypsinogen activation peptide with the sequence Phe-Pro-Val-Asp-Asp-Asp-Asp-Lys (FPVDDDDK). Procolipase added to standard pellets decreased the daily food intake in rats, whereas colipase added to pellets had no effect.

A rapid assay for pancreatic lipase (E.C., glycerol-ester hydrolase 3.1.1.3) is described. The assay is based on the color change of a pH indicator as butyric acid is released from the substrate tributyrin. A mixture made with tributyrin and the water soluble components of the assay is ideally suited for use as a rapid test as, for example, when assaying chromatography fractions. Quantitative data can be obtained by measuring the disappearance of absorbance at 557 nm versus a blank reaction. The assay has been used in the rapid preparation of colipase-free lipase and colipase.

1. Colipase was purified from canine pancreatic juice and found to have certain specificity in its reaction with various pancreatic lipases. 2. This colipase will stimulate the lipolytic activities of lipases isolated from canine, bovine and porcine pancreas but not lipases from a fungus, or from human and rat pancreases. 3. Characterization of these lipases showed (a) the molecular dimension of rat lipase is very different from the other lipases; (b) the pIs of canine, porcine and bovine lipases are almost identical but different from the pIs of rat, human and Candida (a fungus) lipases; and (c) the antiserum prepared against canine lipase will also react with lipases from human, hog and cow pancreases but not with rat and Candida lipases. 4. These physical differences can explain partly the difference in reaction between the various lipases and the canine colipase.