Importin 13: a novel mediator of nuclear import and export.

Importin beta-related receptors mediate translocation through nuclear pore complexes. Co-operation with the RanGTPase system allows them to bind and subsequently release their substrates on opposite sides of the nuclear envelope, which in turn ensures a directed nucleocytoplasmic transport. Here we identify a novel family member from higher eukaryotes that functions primarily, but not exclusively, in import. It accounts for nuclear accumulation of the SUMO-1/sentrin-conjugating enzyme hUBC9 and mediates import of the RBM8 (Y14) protein, and is therefore referred to as importin 13 (Imp13). Unexpectedly, Imp13 also shows export activity towards the translation initiation factor eIF1A and is thus a case where a single importin beta-like receptor transports different substrates in opposite directions. However, Imp13 operates differently from typical exportins in that the binding of eIF1A to Imp13 is only regulated indirectly by RanGTP, and the cytoplasmic release of eIF1A from Imp13 is triggered by the loading of import substrates onto Imp13.

Sm protein-Sm site RNA interactions within the inner ring of the spliceosomal snRNP core structure.

Seven Sm proteins, E, F, G, D1, D2, D3 and B/B', assemble in a stepwise manner onto the single-stranded Sm site element (PuAU(4-6)GPu) of the U1, U2, U4 and U5 spliceosomal snRNAs, resulting in a doughnut-shaped core RNP structure. Here we show by UV cross-linking experiments using an Sm site RNA oligonucleotide (AAUUUUUGA) that several Sm proteins contact the Sm site RNA, with the most efficient cross-links observed for the G and B/B' proteins. Site-specific photo-cross-linking revealed that the G and B/B' proteins contact distinct uridines (in the first and third positions, respectively) in a highly position-specific manner. Amino acids involved in contacting the RNA are located at equivalent regions in both proteins, namely in loop L3 of the Sm1 motif, which has been predicted to jut into the hole of the Sm ring. Our results thus provide the first evidence that, within the core snRNP, multiple Sm protein-Sm site RNA contacts occur on the inner surface of the heptameric Sm protein ring.

A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs.

We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein (snRNP) particles from HeLa cells. It combines immunoprecipitation of the UV-irradiated particles under semi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cross-linking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA (snRNA) within purified U1 snRNPs and then confirmed the results by a large-scale preparation that allowed N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry of purified cross-linked peptide-oligonucleotide complexes. We identified Tyr(112) and Leu(175) within the RNA-binding domain of the U1 70K protein to be cross-linked to G(28) and U(30) in stem-loop I, respectively. We further applied our immunoprecipitation approach to HeLa U5 snRNP, as part of purified 25 S U4/U6.U5 tri-snRNPs. Cross-linking sites between the U5-specific 220-kDa protein (human homologue of Prp8p) and the U5 snRNA were located at multiple nucleotides within the highly conserved loop 1 and at one site in internal loop 1 of U5 snRNA. The cross-linking of four adjacent nucleotides indicates an extended interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein contact sites within native snRNP particles as well as other ribonucleoprotein particles.

Novel genes coding for lithotrophic sulfur oxidation of Paracoccus pantotrophus GB17.

The gene region coding for lithotrophic sulfur oxidation of Paracoccus pantotrophus GB17 is located on a 13-kb insert of plasmid pEG12. Upstream of the previously described six open reading frames (ORFs) soxABCDEF with a partial sequence of soxA and soxF (C. Wodara, F. Bardischewsky, and C. G. Friedrich, J. Bacteriol. 179:5014-5023, 1997), 4,350 bp were sequenced. The sequence completed soxA, and uncovered six new ORFs upstream of soxA, designated ORF1, ORF2, and ORF3, and soxXYZ. ORF1 could encode a 275-amino-acid polypeptide of 29,332 Da with a 61 to 63% similarity to LysR transcriptional regulators. ORF2 could encode a 245-amino-acid polypeptide of 26,022 Da with the potential to form six transmembrane helices and with a 48 to 51% similarity to proteins involved in redox transport in cytochrome c biogenesis. ORF3 could encode a periplasmic polypeptide of 186 amino acids of 20,638 Da with a similarity to thioredoxin-like proteins and with a putative signal peptide of 21 amino acids. Purified SoxXA, SoxYZ, and SoxB are essential for thiosulfate or sulfite-dependent cytochrome c reduction in vitro. N-terminal and internal amino acid sequences identified SoxX, SoxY, SoxZ, and SoxA to be coded by the respective genes. The molecular masses of the mature proteins determined by electrospray ionization spectroscopy (SoxX, 14,834 Da; SoxY, 11,094 Da; SoxZ, 11,717 Da; and SoxA, 30,452 Da) were identical or close to those deduced from the nucleotide sequence with differences for the covalent heme moieties. SoxXA represents a novel type of periplasmic c-type cytochromes, with SoxX as a monoheme and SoxA as a hybrid diheme cytochrome c. SoxYZ is an as-yet-unprecedented soluble protein. SoxY has a putative signal peptide with a twin arginine motif and possibly cotransports SoxZ to the periplasm. SoxYZ neither contains a metal nor a complex redox center, as proposed for proteins likely to be transported via the Tat system.

Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes.

Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A.

Overexpression of the proteasome subunits LMP2, LMP7, and MECL-1, but not PA28 alpha/beta, enhances the presentation of an immunodominant lymphocytic choriomeningitis virus T cell epitope.

The proteasome is a large protease complex that generates most of the peptide ligands of MHC class I molecules either in their final form or in the form of N-terminally extended precursors. Upon the stimulation of cells with IFN-gamma, three constitutively expressed subunits of the 20S proteasome are replaced by the inducible subunits LMP2 (low-molecular mass polypeptide 2), LMP7, and MECL-1 (multicatalytic endopeptidase complex-like-1) to form so-called immunoproteasomes. We show in this study that overexpression of these three subunits in triple transfectants led to a marked enhancement in the H-2Ld-restricted presentation of the immunodominant nonameric epitope NP118, which is derived from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. Overexpression of the alpha and beta subunits of the IFN-gamma-inducible proteasome regulator PA28, in contrast, did not have a comparable effect. In vitro, immunoproteasomes as compared with constitutive proteasomes generated higher amounts of 11- and 12-mer fragments containing the NP118 epitope. These are likely to be cytosolic precursors of NP118, as a proline anchor residue in the second position of NP118 may interfere with TAP-mediated transport of the nonameric epitope itself. In conclusion, we provide evidence that up-regulation of the three inducible subunits, LMP2, LMP7, and MECL-1, can result in a marked improvement of Ag presentation and that, depending on the epitope, PA28 and immunoproteasomes may differentially affect Ag processing.

The selective proteasome inhibitors lactacystin and epoxomicin can be used to either up- or down-regulate antigen presentation at nontoxic doses.

The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5-1 microM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89-168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5-1 microM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.

Characterization of a new type of sulfite dehydrogenase from Paracoccus pantotrophus GB17.

The periplasmic sulfite dehydrogenase of Paracoccus pantotrophus GB17 was purified to homogeneity by a four-step procedure from cells grown lithoautotrophically with thiosulfate. The molecular mass of native sulfite dehydrogenase was 190 kDa as determined by native gradient PAGE. SDS-PAGE showed sulfite dehydrogenase to comprise two subunits with molecular masses of 47 kDa and 50 kDa, suggesting an alpha2beta2 structure. The N-terminal amino acid sequence and immunochemical analysis using SoxC-specific antibodies identified the 47-kDa protein as the soxC gene product. SoxD-specific antibodies identified the 50-kDa protein as SoxD. Based on the molecular masses deduced from the nucleotide sequence for mature SoxC (43,442 Da) and SoxD (37,637 Da) sulfite dehydrogenase contained 1.30 mol molybdenum/mol alpha2beta2 sulfite dehydrogenase. The iron content was 3.17 mol/mol alpha2beta2 sulfite dehydrogenase, and 3.53 mol heme/mol alpha2beta2 sulfite dehydrogenase was determined by pyridine hemochrome analysis. These data are consistent with the two heme-binding domains (CxxCH), characteristic for c-type cytochromes, deduced from the soxD nucleotide sequence. Electrospray ionization revealed two masses for SoxC of 43,503 and 43,897 Da. The difference in molecular mass was attributed to the molybdenum cofactor of SoxC. For SoxD a mass of 38,815 Da was determined; this accounted for the polypeptide and two covalently bound hemes. Reconstitution of the catalytic activity of sulfite dehydrogenase required additional fractions; these eluted from Q Sepharose at 0.05, 0.25, and 0.30 M NaCl. The K(m) of sulfite dehydrogenase for sulfite was 7.0 microM and for cytochrome c 19 microM. Sulfite dehydrogenase activity was inhibited by sulfate and phosphate. The structural and catalytic properties make sulfite dehydrogenase from P. denitrificans GB17 distinct from sulfite oxidases of other prokaryotic or eukaryotic sources.

Mammalian Sec61 is associated with Sec62 and Sec63.

In yeast, efficient protein transport across the endoplasmic reticulum (ER) membrane may occur co-translationally or post-translationally. The latter process is mediated by a membrane protein complex that consists of the Sec61p complex and the Sec62p-Sec63p subcomplex. In contrast, in mammalian cells protein translocation is almost exclusively co-translational. This transport depends on the Sec61 complex, which is homologous to the yeast Sec61p complex and has been identified in mammals as a ribosome-bound pore-forming membrane protein complex. We report here the existence of ribosome-free mammalian Sec61 complexes that associate with two ubiquitous proteins of the ER membrane. According to primary sequence analysis both proteins display homology to the yeast proteins Sec62p and Sec63p and are therefore named Sec62 and Sec63, respectively. The probable function of the mammalian Sec61-Sec62-Sec63 complex is discussed with respect to its abundance in ER membranes, which, in contrast to yeast ER membranes, apparently lack efficient post-translational translocation activity.

Phosphorylation of Oct-2 at sites located in the POU domain induces differential down-regulation of Oct-2 DNA-binding ability.

We compared the effects of phosphorylation of Oct-2 protein on its binding to the consensus octamer sequence (ATGCAAAT) and two non-canonical sequences present in human (AAGCAAAT) and murine (AAACAAAT) promoters of the BLR1 (Burkitts' lymphoma receptor 1) gene encoding chemokine receptor CXCR5 (CXC-chemokine receptor 5). The latter cis-acting elements represent low-affinity recognition sequences for the octamer transcription factors. Okadaic acid was found to induce hyperphosphorylation of Oct-2 specifically in cells of lymphoid lineage. Potentially phosphorylated amino acid residues localized to the POU-specific domain of Oct-2. Whereas binding of Oct-2 to the octamer site from the human BLR1 promoter or to the consensus octamer sequence was unaffected by phosphorylation of this factor, a strong reduction of Oct-2 binding to the octamer site from the murine BLR1 promoter was observed. This finding correlates well with the down-regulation of expression of the BLR1 gene in murine splenic cells but not in lymphoid cells of human origin treated with okadaic acid. These data support the hypothesis that phosphorylation of Oct-2 may be a mechanism by which activities of the promoters containing non-canonical octamer sequences are differentially regulated in response to extracellular stimuli.

Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target possessing similarities to AHNAK.

A novel calcium channel-associated protein of approximately 700 kDa has been identified in mammalian cardiomyocytes that undergoes substantial cAMP-dependent protein kinase (PKA) phosphorylation. It was therefore designated as phosphoprotein 700 (pp700). The pp700 interacts specifically with the beta(2) subunit of cardiac L-type calcium channels as revealed by coprecipitation experiments using affinity-purified antibodies against different calcium channel subunits. It is surprising that amino acid sequence analysis of pig pp700 revealed homology to AHNAK-encoded protein, which was originally identified in human cell lines of neural crest origin as 700-kDa phosphoprotein. Cardiac AHNAK expression was assessed on mRNA level by reverse transcriptase-polymerase chain reaction. Sequence-directed antibodies raised against human AHNAK recognized pp700 in immunoblotting and immunoprecipitation experiments, confirming the homology between both proteins. Anti-AHNAK antibodies labeled preferentially the plasma membrane of cardiomyocytes in cryosections of rat cardiac tissue and isolated cardiomyocytes. Sarcolemmal pp700/AHNAK localization was not influenced by stimulation of either the PKA or the protein kinase C pathway. In back-phosphorylation studies with cardiac biopsies, we identified distinct pp700 pools. The membrane-associated fraction of pp700 underwent substantial in vivo phosphorylation on beta-adrenergic receptor stimulation by isoproterenol, whereas the cytoplasmic fraction of pp700 was not accessible to endogenous PKA. It is important that in vivo phosphorylation occurred in that pp700 fraction which coprecipitated with the calcium channel beta subunit. We hypothesize that both phosphorylation of pp700 and its coupling to the beta subunit play a physiological role in cardiac beta-adrenergic signal transduction. Haase, H., Podzuweit, T., Lutsch, G., Hohaus, A., Kostka, S., Lindschau, C., Kott, M., Kraft, R., Morano, I. Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target that has similarities to AHNAK.

MAPKAP kinase 2 phosphorylates serum response factor in vitro and in vivo.

Several growth factor- and calcium-regulated kinases such as pp90(rsk) or CaM kinase IV can phosphorylate the transcription factor serum response factor (SRF) at serine 103 (Ser-103). However, it is unknown whether stress-regulated kinases can also phosphorylate SRF. We show that treatment of cells with anisomycin, arsenite, sodium fluoride, or tetrafluoroaluminate induces phosphorylation of SRF at Ser-103 in both HeLa and NIH3T3 cells. This phosphorylation is dependent on the kinase p38/SAPK2 and correlates with the activation of MAPKAP kinase 2 (MK2). MK2 phosphorylates SRF in vitro at Ser-103 with similar efficiency as the small heat shock protein Hsp25 and significantly better than CREB. Comparison of wild type murine fibroblasts with those derived from MK2-deficient mice (Mk(-/-)) reveals MK2 as the major SRF kinase induced by arsenite. These results demonstrate that SRF is targeted by several signal transduction pathways within cells and establishes SRF as a nuclear target for MAPKAP kinase 2.

Sequence information within proteasomal prosequences mediates efficient integration of beta-subunits into the 20 S proteasome complex.

The maturation of proteases is governed by prosequences. During the biogenesis of the highly oligomeric eukaryotic 20 S proteasome five different prosequence-containing subunits have to be integrated and processed either by autocatalysis or by neighbouring subunits. To analyse the functional impact of proteasomal prosequences during complex formation, the propeptide of the facultative subunit beta1i/LMP2 was truncated to nine amino acid residues or completely deleted. Additionally, the charged residues within the truncated beta1i/LMP2 version were replaced by neutral residues. While deletion did not affect subunit incorporation, the presence of charged residues within the truncated version of the LMP2 propeptide diminished incorporation efficiency, an effect that was restored upon replacement of the charged amino acids against neutral components. During immunoproteasome formation, incorporation and processing of inducible proteasome beta-subunits are cooperative processes. We demonstrate a linear correlation of the levels of beta2i/MECL1 and beta1i/LMP2 within 20 S proteasomes, suggesting a physical interaction to be the molecular basis for the biased incorporation of both subunits. In the absence of beta5i/LMP7, precursor complexes containing unprocessed beta1i/LMP2 accumulated. The contribution of beta5i/LMP7 on the cooperative formation of a homogeneous population of immunoproteasome is therefore most likely based on an acceleration of the beta1i/LMP2 and potentially of beta2i/MECL1 processing kinetics.

Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by caspase 3.

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified proteins that are involved in anti-IgM antibody-mediated apoptosis using a subclone of the human Burkitt lymphoma cell line BL60. Apoptosis-associated proteins were detected by high resolution two-dimensional gel electrophoresis on a micropreparative scale. Comparison of the high resolution two-dimensional gel electrophoresis protein patterns from apoptotic and non-apoptotic cells showed differences in approximately 80 spots including protein modifications. Analysis of the predominantly altered proteins was performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. Analysis was significantly improved by using new micropreparative high resolution two-dimensional gels employing high protein concentrations. The following 12 apoptosis-associated proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A1, hnRNP C1/C2, FUSE-binding protein, dUTPase, lymphocyte-specific protein LSP1, UV excision repair protein RAD23 homologue B (HHR23B), 60 S acidic ribosomal protein P0 (L10E), heterochromatin protein 1 homologue alpha (HP1alpha), nucleolin, lamin, neutral calponin, and actin. Fragmentation of actin, hnRNP A1, hnRNP C1/C2, 60 S acidic ribosomal protein P0, lamin, and nucleolin could be inhibited by benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, a selective irreversible inhibitor of CPP32 (caspase 3).

Regeneration of implanted splenic tissue in the rat: re-innervation is host age-dependent and necessary for tissue development.

The loss of spleen may lead to fatal bacterial infections. To prevent this, splenic autotransplantation has been performed in humans and experimental animals. However, there is still controversy about the protective function of this procedure. Since innervation plays an important role in splenic function, we investigated whether splenic regenerates are re-innervated, and whether this depends on the donor and host age. Splenic tissue (30 mg) was implanted into the greater omentum of either young (2 days) or old (12 months) rats, from either young or old syngeneic animals. After 3 months of regeneration, the weight of the regenerates was determined, PGP+ nerve fibers were revealed by immunohistology, and subdivided into nerve fibers of sympathetic (TH+, NPY+) or sensory (SP+, CGRP+) origin. In addition, proliferating (Ki-67 proliferation antigen+) and apoptotic cells (TUNEL technique+) were likewise investigated. No innervation of splenic regenerates was observed after implantation into old hosts, correlating with poorly developed splenic compartments. In contrast, almost normal re-innervation occurred in young hosts after implantation of both young and old splenic tissue. These regenerates showed well-developed splenic compartments and a normal number and tissue distribution of proliferating and apoptotic cells. However, after the implantation of young tissue, the final size of splenic regenerates was three times larger (140 +/- 30 vs. 40 +/- 10 mg). Thus, re-innervation of splenic implants is necessary for their subsequent development. It is determined by host age, whereas the final size of the splenic regenerates is regulated by donor age-dependent factors. This model is useful for studying both the process leading to initial innervation and the consequences of this innervation.

Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor.

NLS proteins are transported into the nucleus by the importin alpha/beta heterodimer. Importin alpha binds the NLS, while importin beta mediates translocation through the nuclear pore complex. After translocation, RanGTP, whose predicted concentration is high in the nucleus and low in the cytoplasm, binds importin beta and displaces importin alpha. Importin alpha must then be returned to the cytoplasm, leaving the NLS protein behind. Here, we report that the previously identified CAS protein mediates importin alpha re-export. CAS binds strongly to importin alpha only in the presence of RanGTP, forming an importin alpha/CAS/RanGTP complex. Importin alpha is released from this complex in the cytoplasm by the combined action of RanBP1 and RanGAP1. CAS binds preferentially to NLS-free importin alpha, explaining why import substrates stay in the nucleus.

Analysis of mammalian 20S proteasome biogenesis: the maturation of beta-subunits is an ordered two-step mechanism involving autocatalysis.

Maturation of eukaryotic 20S proteasomes involves the processing of beta-subunits by limited proteolysis. To study the processing mechanism we analysed different point mutations of the beta-subunit LMP2 in transfected human T2 cells. Here we show that the presence of the intact Gly-1Thr1 consensus motif and Lys33 are essential for correct processing. Mutation of Thr1, the active site residue in mature subunits, or of Lys33, results in complete inhibition of processing at the consensus site. In addition, proprotein processing in vitro of wild-type LMP2, incorporated in immature 16S precursor complexes, can be blocked by a proteasome-specific inhibitor. While the processing of inhibitor-treated wild-type proprotein was completely prevented, the site-directed mutagenesis of LMP2 results in processing intermediates carrying an extension of 8-10 residues preceding Thr1, suggesting an additional cleavage event within the prosequence. Furthermore, exchange of mammalian prosequences interferes with processing efficiency and suggests subunit specificity. Based on our data we propose a model for self-activation of proteasomal beta-subunits in which residue Thr1 serves as nucleophile and Lys33 as proton donor/acceptor. We provide evidence that subunit processing of mammalian beta-subunits proceeds via a novel ordered two-step mechanism involving autocatalysis.

Importin provides a link between nuclear protein import and U snRNA export.

Importin-alpha mediates nuclear protein import by binding nuclear localization signals and importin-beta. We find approximately 30% of SRP1p, the yeast importin-alpha, in a nuclear complex with the Saccharomyces cerevisiae nuclear cap-binding protein complex (CBC). Similarly, a large fraction of Xenopus CBC is associated with importin-alpha in the nucleus. CBC promotes nuclear export of capped U snRNAs and shuttles between nucleus and cytoplasm. The CBC-importin-alpha complex binds specifically to capped RNA, suggesting that CBC might shuttle while bound to importin-alpha. Strikingly, importin-beta binding displaces the RNA from the CBC-importin-alpha complex. Thus, the commitment of CBC for nuclear reentry triggers the release of the export substrate into the cytoplasm. We provide evidence for a mechanism that ensures that importin-mediated RNA release is a specifically cytoplasmic event.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase from tomato (Lycopersicon esculentum cv. Lukullus)--purification, biochemical properties and behaviour during stress.

Dinucleoside 5',5"'-P1,P4-tetraphosphate hydrolase (EC 3.6.1.17) has been purified to homogeneity from tomato (Lycopersicon esculentum) cells grown in suspension. The purification procedure comprised ammonium sulphate fractionation following five standard chromatography steps and a final chromatography on Ap4A-Sepharose. The homogeneous hydrolase has a molecular mass of 20 kDa and an isoelectric point of 4.5. The enzyme hydrolyses diadenosine tetraphosphate (Ap4A) asymmetrically to AMP and ATP. Among other naturally occurring dinucleoside oligophosphates, Ap5A and Ap6A are substrates whereas Ap3A is not. Of various phosphonate analogues tested, the Ap5A analogue, AppCH2pCH2ppA, was not cleaved and the Ap3A analogue, ApCH2CH2ppA, was a very poor substrate. Enzyme activity is stimulated by 5 mM Mg2+ and inhibited by fluoride anion; I50 = 6.25 microM. The K(m) value for Ap4A is 0.8 microM. The enzyme exhibits a broad pH optimum from pH 6.5 to 9.0. In order to analyze the protein at the molecular level an internal peptide sequence from the homogeneous enzyme was identified. Within the sequence of 17 amino acids a kinase II motif as a general part of a conserved sequence of nucleotide binding sites was found. Against the internal peptide sequence a polyclonal antiserum was raised. By investigating the intracellular level of Ap4A hydrolase under different kinds of environmental stress, no changes occurred in response to heat shock. But, heavy metal stress and phosphate deprivation lead to a decrease in Ap4A hydrolase.