The cDNA encoding a novel isoform of human trypsinogen was identified. The isoelectric points of the proenzyme and active forms calculated from the deduced amino acid sequence are consistent with those of mesotrypsin(ogen), known to be an inhibitor-resistant trypsin isoform. The cDNA attached with a bacterial signal peptide sequence was expressed in Escherichia coli. The recombinant proenzyme purified from periplasm showed enterokinase-dependent activation similar to a major isoform of human trypsinogen. The enzyme was far less inhibited by trypsin inhibitors such as soybean trypsin inhibitor, aprotinin, or pancreatic secretory trypsin inhibitor than the control trypsin. A gel filtration assay showed that the enzyme and aprotinin did not form a stable complex. It is noteworthy that the amino acid at position 198, which is in close vicinity to the active Ser, is Arg while those of other major trypsins are all Gly. It is concluded that the cloned cDNA encodes human mesotrypsinogen, a unique isoform of trypsinogen with inhibitor resistance.Pancreatic trypsin is a key enzyme, which leads to activation of a number of pancreatic digestive proenzymes including chymotrypsinogens, procarboxypeptidases, proelastases, and prophospholipase A 2 as well as trypsinogens themselves. Three different trypsinogens, which show unique isoelectric points (pI), have been described in human pancreatic juice using twodimensional isoelectric focusing/SDS-PAGE.

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A family of homologous genes is shown to encode GP-2, the major glycosylphosphatidylinositol (GPI)-linked glycoprotein of pancreatic zymogen granule membranes, and Tamm-Horsfall protein (THP), a GPI-linked glycoprotein associated with apical vesicles in kidney thick ascending limb of Henle (TALH) cells. The C-terminal regions of GP-2 (Asp5s-Phe-5-) and THP (Asp'75-IHis'4") from rat show 53% identity, 86% similarity, and 26 conserved cysteine residues including one epidermal growth factor motif. The unique N-terminal domain of rat THP (unique-THP, shows four conserved epidermal growth factor motifs, three in tandem and one in reverse orientation. GP-2 homologues are observed in a wide variety of epithelial cells, several of which contain highly regulated secretory processes. GP-2 released from zymogen granule membranes with phosphatidylinositol phospholipase C reacts with anti-cross-reactive determinant antibody (anti-CRD), confirming the GPI nature of the pancreatic homologue. In contrast, GP-2 and THP, released endogenously from pancreas and kidney, respectively, do not react with anti-cross-reactive determinant antibody, suggesting alternative enzymatic mechanisms for their physiological release. Globular domains of GP-2 and THP, but not albumin,show pH-and ion-dependent self-association in vitro. The GP-2/THP family appears to represent a newly discovered class of GPI-anchored proteins, which may utilize pH-and ion-dependent self-association mechanisms for establishing membrane (micro)domains targeted to intracellular secretory compartments.A growing number of hydrophilic proteins have recently been shown to be linked to membranes via a glycosylphosphatidylinositol (GPI) anchor (1, 2). Demonstrating diverse activities in mammalian cells, including cell adhesion, T-cell activation, and hydrolytic activities, GPI-anchored proteins are normally found attached to the outer surface of the plasma membrane. In polarized Madin-Darby canine kidney cells in culture, endogenous GPI-anchored and phosphatidylinositol phospholipase C (PI-PLC)-releasable proteins appeared to be restricted to the apical plasma membrane (3). In some cell lines, the surface expression and release of GPIlinked proteins is modulated by serum starvation or insulin (4). However, it is not clear why these proteins are tethered to membranes by GPI-lipid anchors or what role might be served by their potential release from the membrane (1). We have recently cloned and characterized rat (5) and dog GP-2 (6), the major glycoprotein of zymogen granule membranes (ZGMs) in the exocrine pancreas, and demonstrated that the membrane form of GP-2, targeted to apical plasma membranes via regulated exocytosis, is attached to the ectoleaflet of granule membranes by a GPI anchor. After granule assembly, we have also shown that GP-2 is released to the granule content fraction by a pH-dependent anchorcleavage activity associated with granule membranes (6). However, the function of GP-2 in ZGMs has remained obscure and its release from granule membranes into...

Pyridine nucleotide coenzymes are involved in >500 enzyme reactions and are biosynthesized from the amino acid L-tryptophan (L-Trp) as well as the vitamin niacin. Hence, "true" niacin-deficient animals cannot be "created" using nutritional techniques. We wanted to establish a truly niacin-deficient model animal using a protocol that did not involve manipulating dietary L-Trp. We generated mice that are missing the quinolinic acid (QA) phosphoribosyltransferase (QPRT) gene. QPRT activity was not detected in qprt(-/-)mice. The qprt(+/+), qprt(+/-), or qprt(-/-) mice (8 wk old) were fed a complete diet containing 30 mg nicotinic acid (NiA) and 2.3 g L-Trp/kg diet or an NiA-free diet containing 2.3 g L-Trp/kg diet for 23 d. When qprt(-/-)mice were fed a complete diet, food intake and body weight gain did not differ from those of the qprt(+/+) and qprt(+/-) mice. On the contrary, in the qprt(-/-) mice fed the NiA-free diet, food intake and body weight were reduced to 60% (P < 0.01) and 70% (P < 0.05) of the corresponding values for the qprt(-/-) mice fed the complete diet at d 23, respectively. The nutritional levels of niacin, such as blood and liver NAD concentrations, were also lower in the qprt(-/-) mice than in the qprt(+/+) and the qprt(+/-) mice. Urinary excretion of QA was greater in the qprt(-/-) mice than in the qprt(+/+) and qprt(+/-) mice (P < 0.01). These data suggest that we generated truly niacin-deficient mice.