Inorganic polyphosphate (polyP), a linear polymer of hundreds of orthophosphate (Pi) residues linked by high-energy, phosphoanhydride bonds, has been identified and measured in a variety of mammalian cell lines and tissues by unambiguous enzyme methods. Subpicomole amounts of polyP (0.5 pmol/100 micrograms of protein) were determined by its conversion to ATP by Escherichia coli polyphosphate kinase and, alternatively, to Pi by Saccharomyces cerevisiae exopolyphosphatase. Levels of 25 to 120 microM (in terms of Pi residues), in chains 50 to 800 residues long, were found in rodent tissues (brain, heart, kidneys, liver, and lungs) and in subcellular fractions (nuclei, mitochondria, plasma membranes, and microsomes). PolyP in brain was predominantly near 800 residues and found at similar levels pre- and postnatally. Conversion of Pi into polyP by cell lines of fibroblasts, T-cells, kidney, and adrenal cells attained levels in excess of 10 pmol per mg of cell protein per h. Synthesis of polyP from Pi in the medium bypasses intracellular Pi and ATP pools suggesting the direct involvement of membrane component(s). In confluent PC12 (adrenal pheochromocytoma) cells, polyP turnover was virtually complete in an hour, whereas in fibroblasts there was little turnover in four hours. The ubiquity of polyP and variations in its size, location, and metabolism are indicative of a multiplicity of functions for this polymer in mammalian systems.

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Whereas exopolyphosphatases have been purified from yeast and a variety of bacteria, this is the first report characterizing endopolyphosphatases that act on long chain inorganic polyphosphate (polyP). The activity from Saccharomyces cerevisiae, localized in vacuoles, has been purified to homogeneity from a strain that possesses vacuolar proteases. The endopolyphosphatase is a dimer of 35-kDa subunits. Distributive action on polyP 750 produces shorter chains to a limit of about polyP 60 , as well as the more abundant release of polyP 3 ; the K m for polyP 750 is 185 nM. Endopolyphosphatases have been identified in a wide variety of sources, except for most eubacteria tested. The activity has been partially purified from rat and bovine brain where its abundance is about 10 times higher than in other tissues but less than 1 ⁄10 that of yeast; the limit product of digestion of the partially purified brain enzyme is polyP 3 .Inorganic polyphosphates (polyP) 1 are linear polymers of orthophosphate residues linked by high energy phosphoanhydride bonds. Likely prevalent in prebiotic evolution (1) polyP has been found in all organisms ranging from bacteria to mammals (2). The ubiquitous occurrence of polyP suggests multiple roles depending on the species, cell, subcellular localization, and physiological state.Our approach toward understanding the functions of polyP has been to identify and isolate the enzymes that synthesize and degrade polyP. Such enzymes have been identified in a variety of microorganisms. PolyP kinase, the enzyme that catalyzes the reversible transfer of the terminal phosphate from ATP to synthesize polyP, has been purified to homogeneity in several bacteria (3)(4)(5). Exopolyphosphatases that catalyze the hydrolysis of terminal phosphates from polyP have been purified from Escherichia coli (6), Corynebacterium xerosis (7), and Saccharomyces cerevisiae (8,9). Phosphotransferases that transfer a phosphate from polyP to AMP (10), NAD (11), glucose (12), and 1,3-diphosphoglycerate (13) have also been described. Endopolyphosphatases (PPN) also called polyP depolymerases or polyphosphorylases catalyze the non-processive cleavage of polyP to release intermediate-size chains during the course of the reaction. PPN are the least studied of the polyP-metabolizing enzymes and have been reported in species of Penicillium and Aspergillus and in S. cerevisiae (14 -16).During the course of investigating polyP metabolism in a variety of cells, we discovered PPN activities in organisms from archae to mammals but little or none among eubacteria. In this report, we describe the purification and characteristics of PPN from S. cerevisiae, where it is most abundant, using a mutant strain in which a major exopolyPase activity has been deleted (17). EXPERIMENTAL PROCEDURESCells and Tissues-S. cerevisiae CRX (17) was grown in YPD medium (1% yeast extract, 2% tryptone, 2% glucose) at 30°C to an A 600 of 14.5. The harvested cells were resuspended in an equal volume of 50 mM Tris-HCl, pH 7.5, 10% sucrose, frozen in liquid ni...

High throughput sequencing of a mouse keratinocyte library was used to identify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors. We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen. Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF superfamily. Two hydrophobic regions, corresponding to a putative signal sequence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putative N-linked glycosylation sites. Epigen shows 24 -37% identity to members of the EGF superfamily including EGF, transforming growth factor ␣, and Epiregulin. Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver. Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor ␣ in several assays. In epithelial cells, Epigen stimulated the phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter. Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1. Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epithelial cells, may constitute a novel molecular target for wound-healing therapy.

Annexin II is a calcium and phospholipid binding protein and a substrate for protein-tyrosine kinases. Recent investigations have revealed involvement of annexin II in DNA synthesis and cell proliferation. Increased levels of annexin II are observed in cancer cells and tissues. To investigate the expression of annexin II in pancreatic adenocarcinoma cells and primary tumors, we measured the levels of annexin II mRNA and protein in normal human pancreas, five established human pancreatic adenocarcinoma cell lines, three primary pancreatic cancers and one metastatic tumor. All five cell lines examined had 5- to 15-fold higher levels of annexin II as compared to normal pancreas. Significant elevations (2- to 8-fold) of annexin II expression were observed in the three primary pancreatic tumors and one metastatic tumor examined. Immunocytochemical analysis indicates that the increased expression of annexin II is limited to proliferating ductular adenocarcinoma, and annexin II expression co-localizes with cells that express PCNA. In normal pancreas, annexin II expression is seen in ductal and ductular cells and no expression is seen in acinar or islet cells. We conclude from these findings that annexin II has a role in cell proliferation and its regulation is altered in pancreatic cancer.