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Hydrolysis of the emulsified mixture of short-chain triacylglycerols by porcine pancreatic lipase in the presence of procolipase and micellar sodium taurodeoxycholate has been studied. Increase in the content of tributyrin and trioctanoin in the mixture with triacetin had highly cooperative effects on the formation of the interfacial lipase procolipase complex. Abrupt enhancement of the complex stability was observed in the presence of 0.4-0.6 mol mol-1 of tributyrin or 0.58 mol mol-1 of trioctanoin in the substrate phase. The affinity of lipase towards interfacially bound procolipase for the trioctanoin containing 0.07-0.42 mol mol-1 of triacetin was approximately three times higher than that for pure trioctanoin. The cooperative processes involved in complex formation did not contribute to the affinity of the interfacial lipase/(pro)colipase complex towards substrate molecules and its catalytic activity.

X-ray crystal structures of bovine pancreas prophospholipase A2 (proPLA2) inhibited by two amide-type inhibitors, [(R)-2-dodecanoyl-amino-1-hexanolphosphocholine (DAHPc) and (R)-2-dodecanoylamino-1-hexanolphosphoglycol (DAHPg)], were determined to R = 0.208 and 0.215 using reflections with up to 2.1 A resolution, respectively. Both complex crystals lacked defined electron densities for the prosequence of the N-terminal and for a loop region consisting of residues 65-70, retaining the disordered feature observed in free proPLA2 despite stabilization due to complex formation. The polar and nonpolar moieties of the amide-type inhibitors were located in the calcium-binding pocket and in the N-terminal alpha-helical hydrophobic region of the enzyme, respectively. As for the amide group of the inhibitor, which is lacking in the true substrate, a strong hydrogen bond was formed between the NH of the inhibitor and the unprotonated N(delta1) atom of His-48, resulting in the tight binding of the inhibitor to proPLA2, as well as to PLA2. The 20-30 times more potent inhibitory activity of DAHPg than DAHPc toward PLA2 could be explained by hydrogen bond formation between the glycol OH of DAHPg and the carbonyl O of Asp-49. The seven residues of the N-terminal prosequence of proPLA2, though disordered, block the access of a water molecule to Ala-1 of PLA2 or change the hydrogen-bonding property of Ala-1 alpha-amino group, resulting in breakage of the water-mediated hydrogen-bond network which is commonly formed in PLA2. The results of molecular dynamics (MD) calculation in an aqueous solution at 300 K indicate that this, rather than the close contact between the prosequence and the residues 65-70 loop region, is the main reason why the latter region becomes flexible in proPLA2, compared with in PLA2.

Procolipase was identified in the stomach by in situ hybridisation. A strong autoradiographic labelling of chief cells was seen in the fundus region, declining more distally and being almost absent in antrum. There was no labelling seen in the intestine. Colipase activity was estimated in rat gastric juice following pentagastrin stimulation and was found to average 2 microM. Furthermore, enterostatin, the N-terminal pentapeptide of procolipase, has been identified in the rat gut and pancreas. Extracts from gastric mucosa, intestinal mucosa and pancreas were purified by gel filtration (Sephadex G25), ion-exchange chromatography (CM-Sepharose) and HPLC (C18 reverse phase). Using an ELISA assay with antibodies directed against enterostatin, two forms of the peptide were identified both in the gut and in the pancreas, with the amino-acid sequences APGPR and VPGPR, respectively. APGPR was found to be the predominant form of enterostatin, whereas only a small amount had the structure VPGPR. Enterostatin in the form of APGPR, when injected intracerebroventricularly in female Sprague-Dawley rats, significantly reduced high-fat food intake in a two-choice situation of low-fat (14% fat by energy) and high-fat (38% fat) food. It is concluded that procolipase is produced in the stomach and secreted into the gastric juice. This is also a novel source of enterostatin.

Porcine colipase, the protein cofactor of pancreatic lipase, was isolated from pancreas freshly collected on animals and from a side fraction from the production of insulin (Novo Nordisk A/S). Samples of purified colipase were analyzed for homogeneity by polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography (RPLC), quantitative N-terminal sequence determination and mass spectrometry. The activating properties of colipase preparations were assayed against tributyrin, triolein or the commercial Intralipid emulsion, in presence of bile salt. Two fractions of colipase with the same specific activity were purified from fresh pancreas. The major fraction (85%) contained one single protein corresponding to fragment 1-93 of the 95-residue form of colipase (procolipase) previously characterized in porcine pancreatic juice. The other fraction (15%) corresponded to fragment 1-91 of procolipase. Also, two fractions of colipase were purified from the side fraction supplied by Novo. These fractions consisted of the 95-residue proform of colipase and of fragment 1-93, respectively, both specifically cleaved at the Ile79-Thr80 peptide bond with partial removal of isoleucine at position 79 and serine at position 78. Procolipase split at the 79-80 bond retained full activity on tributyrin and triolein and on the Intralipid emulsion but the kinetics of hydrolysis of triacylglycerol substrates showed much longer lag periods than those observed with native procolipase. Also, all forms of procolipase split at the 79-80 bond showed one peak in RPLC but their retention time was markedly decreased as compared to that of native procolipase which indicated a weaker hydrophobic binding capacity. The value of the retention time was of the same order of magnitude as that of inactive reduced procolipase. Treatment of native procolipase by pancreatic endopeptidases showed that elastase is likely responsible for specific cleavage at the 79-80 bond of procolipase purified from the Novo extract. Limited proteolysis by trypsin of the proforms of colipase split at the 79-80 bond reduced the lag period. Results presented in this communication provide the first direct evidence showing that the finger-shaped peptide segment between half-cystine residues at positions 69 and 87 is involved in colipase-lipid interaction as previously hypothesized from the three-dimensional structure of the protein.

Intestinal fat digestion is carried out by the concerted action of pancreatic lipase and its protein cofactor colipase. Colipase is secreted from pancreas as a procolipase and is transformed into colipase by the trypsin cleavage of the Arg5-Gly6 bond during liberation of an N-terminal pentapeptide. The kinetic parameters for the lipase-colipase system compared to the lipase-procolipase system has been compared using trioctanoin and Intralipid as substrates. It was found that at pH 7.0 the Kmapp using Intralipid as substrate was the same for procolipase and colipase, 0.06 mM and 0.05 mM, respectively. At pH 8.0, however, the Kmapp were different-0.23 mM for procolipase and 0.08 mM for colipase. In a similar way the binding between colipase and lipase had a dissociation constant of 2.4 x 10(-6) M at pH 7.0, while for procolipase--lipase binding the dissociation constant was 4.1 x 10(-6) M with no significant difference. At pH 8.0 the binding between colipase and lipase was stronger, Kd being 2.0 x 10(-7) M, while weaker for procolipase and lipase, Kd being 1.0 x 10(-5) M. It is concluded that at the physiological pH value as is found in the intestine, the activation of procolipase to colipase has no influence on the hydrolysis of trioctanoin or Intralipid in the presence of bile salt.

Bovine pancreatic phospholipase A2 and its zymogen were studied by laser photo-CIDNP 1H-NMR. Resonances of Trp3 and Tyr69 protons of the two proteins were assigned. By varying the delay between a short light pulse and the observation pulse, time dependencies of the CIDNP signals were obtained from which effective T1 values could be derived. The photo-CIDNP chemical shifts, intensities and relaxation data pointed to environmental differences for the Tyr69 residues in the two proteins, while only small differences were noted for the Trp3 residues. The more buried position of Tyr69 in the enzyme relative to the zymogen was related to the ability of the enzyme to bind to micellar aggregates, to which the zymogen is unable to bind.

Antibodies against porcine procolipase B were produced in rabbits. The antiserum was used to immunoinactivate various forms of native and trypsin-treated porcine colipase. Our results indicate that all forms of the porcine cofactor bind to anti-porcine procolipase B antibodies. Human colipase showed lower affinity for the antibodies than porcine colipase. No cross-reactivity was observed between pig and horse, cow, dog or chicken colipases. Immunological studies on porcine colipase, carried out in the presence of lipid, provided evidence that antibodies bind to colipase at or near the lipase binding site. The binding of antibodies to colipase is not affected by the adsorption of the cofactor at a lipid interface. Using a predictive method for identification of the antigenic determinants, it was found that, in pig colipase, regions at positions 42-48 and 70-74 might represent antigenic sites. In the horse protein, the peptide segment 42-48 was also recognized as a possible antigenic site. An immunoadsorbent gel column was prepared for a one-step isolation of porcine colipase. In contrast to purification methods described so far, immunoaffinity chromatography yielded only one form of the porcine cofactor when starting from a pancreatic extract. This protein preparation has structural, biochemical and immunochemical properties similar to that of porcine procolipase A previously isolated from pancreas in the presence of detergent.

Previous experiments demonstrated the existence of at least two pools of secretory proteins in the exocrine pancreas. We have measured the specific activities of amylase released under resting conditions and of amylase in the zymogen granules. Specific activity of resting secretion was twice that found under stimulated conditions or in zymogen granules. Secretory proteins were pulse-labeled and amylase was measured after precipitation of the enzyme with glycogen. Pancreatic juice collected at 45-50 min post-pulse contained 10-25-times the amylase activity found in zymogen granules. These results confirm the existence of at least two distinct pools of secretory proteins in the exocrine pancreas and suggest the existence of an intracellular route of secretory proteins which would bypass the zymogen granule compartment.

We report the ability of focused soft X-rays to visualize at spatial resolution well beyond that of the optical microscope (less than 100 nm) the interior of a small, whole biological object without fixation, staining, dehydration or sectioning. Quantitative estimation of its protein content with unique femtogram sensitivity is also reported. The present results represent a significant step towards the goals of natural imaging and chemical mapping of biological structures with soft X-rays.

The N-terminal sixteen residues of the amino acid sequence of reduced and alkylated human pancreatic proelastase 2 have been established, and N-terminal amino acid residue has been shown to be carboxymethylcysteine. A peptide containing an amino acid sequence corresponding to the first twelve residues of proelastase 2 was isolated following activation of proelastase 2 with trypsin, performic acid oxidation, and gel filtration. This peptide was not released prior to performic acid oxidation, suggesting that it remains attached to the major peptide chain via a disulfide bond containing the N-terminal half-cysteine in a manner similar to that found for chymotrypsinogens. However, the amino acid sequence of the activation peptide is not strongly homologous to either porcine chymotrypsinogen A or porcine proelastase 1.

Limulus amebocyte lysate was fractionated by heparin-Sepharose chromatography into four components (fractions A, B, C and D). Major coagulation factors, i.e., proclotting enzyme, coagulogen, and proclotting enzyme activating factor precursor (proactivator) in the lysate were eluted, respectively, in fraction A, fraction B and fraction C. Clotting enzyme activity was detected only following recombination of fraction A and fraction C in the presence of endotoxin. The conversion of proactivator to its active form (activator) was an endotoxin-dependent reaction and was inhibited by polymyxin B. Either proactivator is an endotoxin-sensitive factor or another endotoxin-sensitive factor, which activates proactivator, is present in fraction C. Optimal pH for proclotting enzyme activation by activator was broad and ranged from pH 6.0 to 8.0, while that for the endotoxin-mediated activation of proactivator was pH 7.0. No initial latent period was observed during activation of the proactivator or proenzyme. The activator was inhibited by benzamidine, leupeptin, soybean trypsin inhibitor and diisopropyl fluorophosphate, suggesting that the activator is a trypsin-type serine protease. Trypsin, but not thrombin, urokinase, plasmin, papain or alpha-chymotrypsin activated the proclotting enzyme. Therefore, limited proteolysis, i.e., of an arginyl- or lysyl-X bond(s), of the proenzyme molecule is probably involved in its activation.

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.

A true collagenase (EC 3.4.24.3) which had been discovered previously in bone culture fluids and extracts of whole bone has now been localized to the cellular component of bone. The cellular enzyme bears the same characteristics as that of bone collagenases described earlier. Moreover, it is directly extractable in relatively large quantities. Attempts to demonstrate the presence of a bone cell procollagease were unsuccessful. It was also observed that the cells secrete significant amounts of collagenase in vitro. With increasing incubation time the extracellular collagenase levels rise and the intracellular collagenase levels drop.