Catherine Figarella

Purification and characterization of a carboxyl ester hydrolase from human pancreatic juice

A carboxyl ester hydrolase has been purified 20-fold from human pancreatic juice. It is a glycoprotein with a molecular weight of 100 000. It contains 9% neutral and amino carbohydrates and the amino acid composition is characterized by a high content of proline residue (12.7%). The enzyme catalyzes the hydrolysis of p-nitrophenylacetate and the activity increases in the presence of biliary salts; V is not modified but Km is decreased 10 times by addition of biliary salts. The enzyme migrates on Sephadex G-200 as a protein with a molecular weight of 300 000. This behaviour does not seem to be due to a polymerization but to a peculiar configuration of the enzyme.

Human pancreatic lipase: A glycoprotein

Human lipase has been purified from pancreatic juice. The protein has a molecular weight of 48 000 and an N-terminal residue of lysine. It has been characterized as a glycoprotein containing 4.7 mol of glucosamine, 2.8 mol of mannose, 2.9 mol of fucose, 3.0 mol. of galactose and 1.1 mol of glucose per mol of protein. Two isolipases have been separated by polyacrylamide gel electrophoresis. Their isoelectric points are 5.80 and 5.85, respectively and both are glycoproteins. Immunological cross reactions have been obtained between human lipase and other mammalian lipases (porcine, bovine, ovine, canine and rat).

On zymogens of human pancreatic juice.

Pancreatic protein composition is now known in such diverse species as cattle [ 1,2] , swine, dog [3] , rat [4] , chicken [5] and even in spiny dogfish [6], the most phylogenetically distant from man yet investigated. Comparison among these species shows that each has a full complement of enzymes and differs from the others only in enzymatic levels, chemical characteristics of proteins and number of homologous enzymes. Little information has been published on human pancreatic enzymes. Studies of extracts of acetonic powder by immunodiffusion [7] and, very recently, an identification of pancreatic enzymes in human duodenal contents [8] contributed to our knowledge of human pancreatic proteins. Since the enzymes were present in their active forms, however, it was impossible to determine the number and the chemical characteristics of the zymogens secreted by the gland. Preliminary studies of the zymogens in human pancreatic homogenates [9] have so far been limited by paucity of material. Recently, Keller et al. [lo] published a chromatographic study of proteins of human pancreatic juice, but some activation occurred during the experiment and raised some ambiguity about the number and nature of proteolytic zymogens. We succeeded in obtaining human pancreatic juice devoid of free tryptic activity. Large amounts of

Possible regulation of CFTR‐chloride channels by membrane‐bound phosphatases in pancreatic duct cells

We have studied CFTR‐Cl− channels in non‐CF CAPAN‐1 and in CFTR‐transfected CFPAC‐PLJ‐CFTR‐6 epithelial cells from human pancreas. Theophylline and IBMX induced the opening of cell‐attached CFTR‐Cl− channels. Theophylline, IBMX and the alkaline phosphatase (AP) inhibitor levamisole enhanced the activity of excised channels and reduced by 70–75% the apical membrane‐associated APs activity. Okadaic acid had no effect on APs and channel activities. A polyclonal anti‐alkaline phosphatase antibody (which detected apical APs) reduced APs activity and activated quiescent excised chloride channels. These results suggest that CFTR channels may be regulated by membrane‐bound phosphatases.

Comparative studies on the mechanism of activation of the two human trypsinogens.

The activation of human trypsinogens 1 and 2 by porcine enterokinase at pH 5.6 shows that the two human zymogens are equivalent substrates for this enzyme and that both proteins are activated faster than the cationic bovine trypsinogen. At pH 8.0 and in the presence of 20 mM calcium the two human trypsinogens are activated by either human trypsin at the same rate but the affinity of both trypsins is higher for trypsinogen 1 than for trypsinogen 2. Two Ca2+ binding sites are identified in the two human zymogens and their pK(Ca2+) values determined. For trypsinogen 1 the values are respectively of 2.8 and 3.3 for the primary and secondary Ca2+ binding sites, and for trypsinogen 2 of 3.4 and 2.7. These values are markedly different from those obtained for bovine cationic trypsinogen, especially in the case of trypsinogen 1. These results point out a different degree of saturation of the calcium binding sites of the 2 human zymogens that must exist in physiological conditions, suggesting different biological activities of the two trypsinogens.

Uptake of plasmid/glycosylated polymer complexes and gene transfer efficiency in differentiated airway epithelial cells.

We have studied gene transfer efficiency of glycosylated polylysines and glycosylated polyethylenimines as vectors in immortalized differentiated airway gland serous cells and primary cultures of human airway surface epithelial cells.

EFFECT OF LACTOFERRIN ON THE GROWTH OF A HUMAN COLON ADENOCARCINOMA CELL LINE- COMPARISON WITH TRANSFERRIN

SummaryLactoferrin 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.

Immunohistochemical localization of intestinal phospholipase A2 in rat paneth cells

SummaryUsing the peroxidase-anti-peroxidase (PAP) technique with a specific rabbit anti-swine intestinal-phospholipase-A2 serum, the immunoreactivity of this phospholipase A2 was localized in rat-intestinal Paneth cells. The specific rabbit anti-swine intestinal-phospholipase-A2 serum did not stain the rat-pancreatic acinar cells which were stained by a specific rabbit anti-swine pancreatic-phospholipase-A2 serum. Specific rabbit anti-swine pancreatic-phospholipase-A2 serum did not stain rat-intestinal Paneth cells. Therefore, there is no cross-immunoreactivity between pancreatic and intestinal phospholipases.

Proteolysis of human trypsinogen 1. Pathogenic implication in chronic pancreatitis

SDS electrophoresis on polyacrylamide gels of purified trypsinogen 1 has shown the occurrence of a proteolysis in some molecules during long storage at -20 degrees C. This proteolyzed trypsinogen gives a positive reaction with an antiserum directed against the precipitate protein, major protein of about 14 000 molecular weight extracted from precipitates present in the pancreatic juice of patients with chronic pancreatitis. The autoactivation of proteolyzed trypsinogen 1 liberates a polypeptide of 14 000 molecular weight which is immunologically identical to the precipitate protein. These results show that the major protein present in pancreatic precipitates (and pancreatic stones) of patients with chronic pancreatitis is a degradation product of trypsinogen 1 liberated by a proteolysis which necessarily requires a premature zymogen activation in the disease.

The two human trypsinogens: catalytic properties of the corresponding trypsins.

The catalytic properties of the two human trypsins obtained from purified trypsinogens have been studied. The catalytic rate constant kcat and the pK of the ionisable residue implicated in the active site have been determined with Bz-Arg-OEt. The hydrolysis of Tos-Arg-OMe by human trypsins does not follow the simple Michaelis-Menten scheme and indicates a difference in the conformational flexibility of the active site-regions of the two enzymes. Both enzyme are readily autolyzed and calcium ion plays a fundamental role in stabilizing trypsin activity. However trypsin 2 self-digests more rapidly than trypsin 1. These results are a prerequisite to the elucidation of the fate of pancreatic enzymes in human digestive tract.