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...

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Mutants of the extreme thermophile Thermus flavus in the pyrimidine biosynthetic pathway (Pyr ؊ ) were isolated by resistance to 5-fluoroorotic acid. The pyrE gene, which codes for the orotate phosphoribosyltransferase, was cloned by recombination with one of the isolated Pyr ؊ T. flavus mutant strains. It was subcloned by complementation of an Escherichia coli pyrE mutant strain and was sequenced. The deduced polypeptide sequence extends over 183 amino acids. Several independent Pyr ؊ mutations were mapped within the pyrE locus by recombination with fragments of the cloned gene.