kD In Size Research Articles

Characterization of the Interaction of a Novel Stagonospora nodorum Host-Selective Toxin with a Wheat Susceptibility Gene

Recent work suggests that the Stagonospora nodorum-wheat pathosystem is controlled by host-selective toxins (HSTs; SnToxA, SnTox1, and SnTox2) that interact directly or indirectly with dominant host genes (Tsn1, Snn1, and Snn2) to induce disease. Here we describe and characterize a novel HST designated SnTox3, and the corresponding wheat sensitivity/susceptibility gene identified on chromosome arm 5BS, which we designated as Snn3. SnTox3 is a proteinaceous necrosis-inducing toxin between 10 and 30 kD in size. The S. nodorum isolates Sn1501 (SnToxA-, SnTox2+, and SnTox3+), SN15 (SnToxA+, SnTox2+, and SnTox3+), and SN15KO18, a strain of SN15 with a disrupted form of SnToxA, were evaluated on a population of wheat recombinant inbred lines. A compatible Snn3-SnTox3 interaction played a significant role in the development of disease caused by isolates Sn1501 and SN15KO18, with Snn2 being epistatic to Snn3. Snn3 was not significantly associated with disease caused by SN15 presumably due to the major effects observed for Snn2 and Tsn1, which were largely additive. This work introduces a fourth HST produced by S. nodorum and builds on the notion that the S. nodorum-wheat pathosystem is largely based on multiple host-toxin interactions that follow an inverse gene-for-gene scenario.

Expression, Distribution and Regulation of Phosphodiesterase 5

Phosphodiesterase 5 (PDE5) is one of eleven members of the mammalian phosphodiesterase family that hydrolyzes cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP). Best known as the target of the impotence drug sildenafil, PDE5 degrades cGMP in smooth muscle cells so as to maintain the contracted state of contractile organs such as the penis, blood vessels, uterus, and intestines. In addition, it regulates numerous other physiological processes such as neurogenesis and apoptosis. Like all other PDEs, PDE5 is dimeric; each subunit is approximately 100 kd in size and has two allosteric cGMP-binding sites and a catalytic domain. Protein kinase G (PKG)-mediated phosphorylation and allosteric cGMP binding upregulate PDE5 activity, while PP1 phosphatase-mediated dephosphorylation downregulates. Sildenafil and other selective inhibitors inhibit PDE5 by binding to the catalytic site. From two promoters a single PDE5A gene at human chromosome 4q26 encodes three alternatively spliced isoforms (PDE5A1-3) that differ in the N-terminus. The PDE5A promoter is located upstream of the three isoform-specific first exons (in the order of A1-A3-A2) and consists of a 139-bp core, a 308-bp upstream enhancer, and a 156-bp downstream enhancer. The weaker 182-bp PDE5A2 promoter is located between the A3- and A2-specific exons and contains an indispensable Sp1-binding sequence. Both promoters are responsive to cGMP or cAMP stimulation, and several studies have demonstrated regulation of PDE5 expression possibly through these promoters. Virtually all tissues and cell types express PDE5, with heart and cardiomyocytes being contentious. PDE5A1 and PDE5A2 are ubiquitous, but PDE5A3 is specific to smooth muscle.

Promoting Central Regeneration

In general, neurons in the mature vertebrate central nervous system fail to regenerate after injury. Intriguingly, however, activation of macrophages in the eye stimulates the regeneration of retinal ganglion cells (RGCs) so that their axons grow down the optic nerve beyond the site of injury (see Filbin). Yin et al. , who previously determined that a fraction of macrophage-secreted proteins less than 20 kD in size promoted axon regeneration, used mass spectrometry of a prominent 10- to 15-kD band and identified oncomodulin, a calcium-binding protein found in tumors. Although inactive on its own, oncomodulin potentiated the ability of mannose plus forskolin to stimulate axon outgrowth in cultured RGCs. Further, absorption of macrophage-conditioned medium (MCM) with anti-oncomodulin IgG eliminated its ability to promote axon growth. Oncomodulin showed specific high-affinity binding to RGCs, which depended on an increase in intracellular adenosine 3′,5′-monophosphate (cAMP, achieved through exposure to forskolin or the cAMP analog 8-bromoadenosine 3′,5′-monophosphate). Pharmacological analysis indicated that oncomodulin activity depended on calcium/calmodulin-dependent protein kinase II (CaMKII) and on gene transcription but not on various mitogen-activated protein kinase kinases or janus kinases. Delivery of oncomodulin- and 8-bromoadenosine 3′,5′-monophosphate-containing microspheres into the vitreous promoted optic nerve regeneration in vivo. Thus, oncomodulin appears to represent a previously unidentified macrophage-derived trophic factor capable of promoting axonal regeneration in at least some central neurons. Y. Yin, M. T. Henzl, B. Lorber, T. Nakazawa, T. T. Thomas, F. Jiang, R. Langer, L. I. Benowitz, Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells. Nat. Neurosci. 9 , 843-852 (2006). [PubMed] M. T. Filbin, How inflammation promotes regeneration. Nat. Neurosci. 9 , 715-717 (2006). [PubMed]

Androgen Regulation of JM-27 is Associated With the Diseased Prostate

Despite intense research efforts, the etiology of prostatic hyperplasia associated with both benign prostatic hyperplasia (BPH) and prostate cancer remains poorly understood. Our previous studies using array technology identified JM-27 as a transcript that is dramatically up-regulated in the prostates of patients with symptomatic BPH and in normal, adjacent prostatic regions of patients with prostate cancer. In the present study, using an extended sample set, we show a correlation between the messenger RNA and protein expression of JM-27. To investigate the possible functions of this gene, its expression in the rat prostate was examined by immunoblot analysis using a polyclonal antibody specific to human JM-27. This antibody reacts with 2 rat polypeptides of 17 kd and 27 kd in size. Whereas the 27-kd form of the JM-27 protein found in human prostate is selectively expressed in the dorsolateral lobes of the rat prostate, the 17-kd form is expressed only in the ventral lobe. Expression of both forms of this protein appears to be androgen-regulated. There is a time-dependent decrease in expression of the protein products in the ventral and dorsolateral lobes of the rat prostate after castration. Administration of exogenous testosterone in castrated animals maintains protein expression in both lobes. Androgens are believed to play a central role in prostate growth and development, and therefore, it is tempting to speculate that JM-27, an androgen-regulated gene, may be involved in prostatic growth regulation. Further studies are underway to evaluate such a function for JM-27 in prostatic diseases.

Functional Analysis of Methylthioribose Kinase Genes in Plants

Through a biochemical and a genetic approach, we have identified several plant genes encoding methylthioribose (MTR) kinase, an enzyme involved in recycling of methionine through the methylthioadenosine (MTA) cycle. OsMTK1, an MTR kinase from rice (Oryza sativa), is 48.6 kD in size and shows cooperative kinetics with a V(max) of 4.9 pmol/min and a K0.5 of 16.8 microm. MTR kinase genes are the first genes to be identified from the MTA cycle in plants. Insertional mutagenesis of the unique AtMTK gene in Arabidopsis (Arabidopsis thaliana) resulted in an inability of plants to grow on MTA as a supplemental sulfur source. MTK knock-out plants were not impaired in growth under standard conditions, indicating that the MTA cycle is a nonessential metabolic pathway in Arabidopsis when sulfur levels are replete. In rice, OsMTK genes were strongly up-regulated in shoots and roots when plants were exposed to sulfur starvation. Gene expression was largely unaffected by lack of nitrogen or iron in the nutrient solution, indicating that OsMTK regulation was linked specifically to sulfur metabolism.

P‐43 IgG antibodies against sarcoptic mite antigens in dogs cross‐reacting with house dust and storage mite antigens

Sera from dogs suffering from scabies were used to evaluate possible antigenic cross‐reactivity with proteins from house dust or storage mites. Polyacrylamide gel electrophoresis on gradient gels was used to create size‐dependent protein bands of Sarcoptes scabiei ssp. vulpis, Dermatophagoides farinae, D. pteronyssinus, Acarus siro, Lepidoglyphus destructor and Tyrophagus putrescentiae. Anti‐canine IgG antibodies conjugated with alkaline phosphatase were used for immunostaining. Different patterns for Sarcoptes could be seen with strong bands repetitively observed of approximately 22, 112, 116, 132 and 200 kD in size. The band at approximately 22 kD seems likely to have cross‐reactivity with a protein of the same size in A. siro. The one at approximately 200 kD might share antigenic activity with bands of D. farinae and, to a lesser extent, D. pteronyssinus. Funding: Laupeneck AG.

Androgen Regulation of JM‐27 Is Associated With the Diseased Prostate

Despite intense research efforts, the etiology of prostatic hyperplasia associated with both benign prostatic hyperplasia (BPH) and prostate cancer remains poorly understood. Our previous studies using array technology identified JM-27 as a transcript that is dramatically up-regulated in the prostates of patients with symptomatic BPH and in normal, adjacent prostatic regions of patients with prostate cancer. In the present study, using an extended sample set, we show a correlation between the messenger RNA and protein expression of JM-27. To investigate the possible functions of this gene, its expression in the rat prostate was examined by immunoblot analysis using a polyclonal antibody specific to human JM-27. This antibody reacts with 2 rat polypeptides of 17 kd and 27 kd in size. Whereas the 27-kd form of the JM-27 protein found in human prostate is selectively expressed in the dorsolateral lobes of the rat prostate, the 17-kd form is expressed only in the ventral lobe. Expression of both forms of this protein appears to be androgen-regulated. There is a time-dependent decrease in expression of the protein products in the ventral and dorsolateral lobes of the rat prostate after castration. Administration of exogenous testosterone in castrated animals maintains protein expression in both lobes. Androgens are believed to play a central role in prostate growth and development, and therefore, it is tempting to speculate that JM-27, an androgen-regulated gene, may be involved in prostatic growth regulation. Further studies are underway to evaluate such a function for JM-27 in prostatic diseases.

Purification and Characterization of Lectin from Seeds of Delonix regia

A lectin from the crude extract of seeds of Delonix regia (DRL) has been purified by ammonium sulphate fractionation followed by specific adsorption on Sephadex G-50 column and subsequent displacement with 100 mM D-glucose. The purified lectin (yield 1.41 mg g−1 dry seed) is a hetero-tetramer of 156 kD in size, consisting of four polypeptides (Mr of 32, 36, 42 and 46 kD) as detected on SDS-PAGE. It is a thermostable protein and remains active between pH 2.0–11.0. The lectin agglutinated erythrocytes of human and other primates. The hemagglutinating activity was not affected by cations and chelating agents. Of the 23 different sugars tested for specificity, maximum inhibition of the hemagglutination was shown by D-glucose. The immunological crossreactions of DRL with monospecific antibodies against SBA, Con A, PNA, DBA and PHA-E indicate that DRL is very closely related to Concanavalin A.

Expression and function of periostin-isoforms in bone.

Periostin was originally identified in MC3T3-E1 osteoblast-like cells. We have identified an isoform of periostin referred to as periostin-like-factor (PLF). It is homologous to other proteins such as fasciclin I (fas I), MPB70, betaIG-H3, and Algal-CAMs. All of these proteins are implicated in regulating cell adhesion. PLF and the other isoforms of periostin differ in their C-terminal sequences. PLF and periostin differ in two specific regions, between 673 and 699 amino acids (aa) and 785-812 aa. Periostin isoforms are expressed in vivo and in vitro during the stages of osteoblast differentiation and maturation. Their mRNAs are present in pre-osteoblast cells as detected by in situ hybridization, and the proteins are between 86 and 93 kD in size as determined by Western blot analysis. Antisense oligonucleotides and antibodies directed against the isoforms of periostin were used to block the activity of these proteins. In both cases, the levels of osteoblast-specific-differentiation markers were markedly reduced suggesting a role for these proteins in osteoblast differentiation.

Antigenecity of domestic soy bean species and comparison of reactivity with serum specific IgE

Rationale Due to the regional difference, many studies have been made to see the allergenicity of various foods commonly consumed in each area. In this study, we attempted to investigate the variability in antigenicity and allergenicity of soybean protein, one of main food allergen in children, by comparing 15 species of domestic soybeans in Korea. Methods Protein was extracted after removing fat by hexane from 15 species of soybean which were provided by the Rural Development Administration of Korea. Protein was separated by SDS-PAGE and transferred to PVDF. Western blot was performed using polyclonal anti-soy antibody and specific IgE to soy in pooled sera. Sera were obtained from 4 atopic dermatitis patients, of which level was above 49.1 U/mL measured by CAP assay. Results More than 20 protein bands were separated variably by SDS-PAGE from 10 kD to 100 kD in size according to the species, and 37 kD protein was not detected in 3 species. Polyclonal anti-soy antibody reacted to the variable proteins from each species. In one species, strong reactivity with 14 kD and 16 kD protein was found. Various proteins were bound to specific IgE in pooled sera. Among them, reaction with 14 kD protein was detected only in one species. Conclusions Our result showed various antigenicity and allergenicity in each species of Korean domestic soybeans. It suggests that this variability should be considered in food allergy research.

Relationship between platelet imidazoline receptor-binding peptides and candidate imidazoline-1 receptor, IRAS.

A candidate human imidazoline-1 receptor, designated imidazoline receptor antisera-selected (IRAS) protein, was cloned based on immunoreactivity with antiserum against a purified imidazoline receptor binding peptide (IRBP antiserum). Human IRAS is 167 kD in size, different from 33- to 85-kD IRBP bands previously linked to the human platelet I(1) receptor. To explore the possible relationship between IRAS and these smaller proteins, seven different epitope-specific antisera against IRAS were raised in rabbits for comparison with IRBP antiserum. Focus was on antiserum(227-241), corresponding to amino acids No. 227 to 241 in IRAS, because this antiserum was found uniquely able to immunoprecipitate non-denatured 85-kD and 170-kD forms of IRAS from a human megakaryoblastoma cell line (MEG01), a model of platelet-producing cells. Human platelets lacked the 170-kD form of IRAS, but 33-kD and 85-kD bands were detectable and seemed to be possible fragments of full-length IRAS. The intensity of the 85-kD band detected by antiserum(227-241) was significantly correlated (r = 0.62, P = 0.04) with the intensity of the 33-kD band across 11 human platelet samples. A positive correlation between the intensities of the 33-kD and 85-kD bands is consistent with both being fragments of IRAS.

Physiology and biophysics of hemi-gap-junctional channels expressed in Xenopus oocytes.

Gap junctional channels are intercellular channels that allow the passage of ions and other small molecules less than about 1 kD in size between neighbouring cells. They are composed of two oligomeric protein subunits called connexons or hemichannels which reside in the plasma membrane of closely opposed cells. Several recent studies suggest that unpaired connexons may be active on the non-junctional plasma membrane of cells. Here I present a short overview of the properties of connexon channels expressed in Xenopus oocytes.

Signaling in plants by intercellular RNA and protein movement.

Plants and animals have had about 1.6 billion years—the time that has passed since they diverged from their last unicellular ancestor—to evolve different mechanisms for solving unique problems of development and intercellular signaling. As an example, plant cells are separated from each other by a substantial extracellular matrix, the cell wall. Although the cell wall is not impervious, it is likely to hinder cell-to-cell communication. Moreover, because plant cells cannot migrate during development, location, instead of lineage, is the primary determinant of cell fate in plants, making communication over both long and short distances essential for the coordination of plant growth. It appears that plants have significantly overcome the downside of having a cell wall by forming channels between cells to allow the transit of signaling and other important molecules. Plants may even be considered as supracellular organisms, in that whole tissues are symplastically connected. The channels that connect plant cells are called plasmodesmata (PD), and their investigation offers tantalizing clues as to how plant cells may use them to communicate with each other and to coordinate development. Although once seen as constricted channels through which only molecules <1 kD in size could pass (Terry and Robards 1987; Burnell 1988), PD have recently been shown to be far more dynamic and allow passage of both proteins and nucleic acids. Other findings, such as the observation that transcription factors can move between cells, and that RNA movement may be behind a range of long-distance signals in plants, raise further questions regarding a regulatory role of PD in development. Because of the importance of PD, we first summarize what we know about them, and then go on to discuss intercellular movement of proteins and RNA in general.

The B lymphocyte adaptor molecule of 32 kD (Bam32) regulates B cell antigen receptor signaling and cell survival.

The B lymphocyte–associated adaptor protein 32 kD in size (Bam32) is expressed at high levels in germinal center (GC) B cells. It has an NH2-terminal src homology 2 (SH2) domain which binds phospholipase C (PLC)γ2, and a COOH-terminal pleckstrin homology (PH) domain. Thus, Bam32 may function to integrate protein tyrosine kinase (PTK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways in B cells. To further define the role Bam32 plays in B cells, we generated Bam32-deficient DT40 cells. These Bam32−/− cells exhibited lower levels of B cell antigen receptor (BCR)-induced calcium mobilization with modest decreases in tyrosine phosphorylation of phospholipase C (PLC)γ2. Moreover, BCR-induced activation of extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways was impaired in Bam32−/− cells but not the activation of Akt-related pathways. Activation of downstream transcription factors such as nuclear factor of activated T cells (NF-AT) and nuclear factor of κ binding (NF-κB) was also impaired in Bam32−/− cells. Furthermore, Bam32−/− cells were more susceptible to BCR-induced death. Taken together, these findings suggest that Bam32 functions to regulate BCR-induced signaling and cell survival most likely in germinal centers.

Gap junctions: structure and function (Review).

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to approximately 1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.

Thermophilic phospholipase A2 in the cytosolic fraction from the archaeon Pyrococcus horikoshii

AbstractHyperthermophilic archaeon Pyrococcus horikoshii produced phospholipase A2 in a cytosolic fraction. The enzyme displayed optimal activity at 90°C and pH 7 and preferentially hydrolyzed sn‐2 fatty acids in the following order: linoleoyl&gt; oleoyl&gt;arachidoyl phosphatidylcholine. Phospholipase A2 had similar activities toward l‐α‐1‐palmitoyl‐2‐arachidoyl derivatives of phosphatidylcholine and phosphatidylethanolamine. Phospholipase A2 activity was unaffected by the addition of 0.0001–1 mM calcium, but was inhibited slightly by the addition of 2–10 mM calcium. The activity was enhanced more than 5–18‐fold in the presence of 3–20% (vol/vol) glycerol. The activity was unaffected by the addition of 1–5 mM EDTA or 0.01–20 mM dithiothreitol. A caldarchaetidic acid derivative having a molecular weight of 1544 disappeared upon incubation of the cytosolic fraction with total lipid. The phospholipase A2 was difficult to purify by general chromatography because it existed as an aggregate. Electrophoresis was carried out using 10–15% polyacrylamide gels containing sodium dodecyl sulfate (SDS‐PAGE). No activity of phospholipase A2 activity was observed in the absence of proteins less than 19 kD in size, as fractionated by SDS‐PAGE.

Elliptocytosis in patients with C-terminal domain mutations of protein 4.1 correlates with encoded messenger RNA levels rather than with alterations in primary protein structure

Early biochemical studies defined 4 functional domains of the erythroid protein 4.1 (4.1R). From amino-terminal to carboxy-terminal, these are 30 kd, 16 kd, 10 kd, and 22/24 kd in size. Although the functional properties of both the 30-kd and the 10-kd domain have been demonstrated in red cells, no functional activities have been assigned to either the 16-kd or the 22/24-kd domain in these cells. We here describe new mutations in the sequence encoding the C-terminal 22/24-kd domain that are associated with hereditary elliptocytosis. An unusually mild phenotype observed in heterozygous and homozygous members of 1 family suggested heterogeneity in the pattern of expression of 4.1R deficiency. Using a variety of protein and messenger RNA (mRNA) quantification strategies, we showed that, regardless of the alteration in the C-terminal primary sequence, when the protein is produced, it assembles at the cell membrane. In addition, we found that alterations in red cell morphologic features and membrane function correlate with the amount of membrane-associated protein-and therefore with the amount of mRNA accumulated-rather than with the primary structure of the variant proteins. These data suggest that an intact sequence at exons 19 through 21 encoding part of the C-terminal 22/24-kd region is not required for proper protein 4.1R assembly in mature red cells. (Blood. 2000;95:1834-1841)

Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells.

Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [(35)S]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H. pylori but not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylori CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

Further Characterization and Detectionof Indian Citrus Ringspot Virus

Indian citrus ringspot virus (ICRSV) from a Kinnow mandarin orchard at the IARI, New Delhi, India (isolate K1) has flexuous particles 650 nm long, with clearly visible cross- banding. It can be mechanically transmitted to and multiplied in Phaseolus vulgaris cv. Saxa. The genome consists of one ssRNA of about 7.5 kb and the coat protein (CP) is 34 kd in size. The virus has been purified and an antiserum prepared suitable for DAS ELISA. Partial sequencing of the genome, revealing the CP ORF near the 3' end and further sequences upstream, indicates that ICRSV, while morphologically resembling a capillovirus, has no homology with this genus. Absence of serological relationship confirms this. Short amino acid motifs in the CP sequence reveal limited similarities with the genera Potexvirus, Carlavirus, Foveavirus and Allexivirus , but no strong similarity to any one of these, so that the virus does not fall into any known genus. RNA and cDNA probes that detect K1 are available, and also primers suitable for RT-PCR. EM decora- tion tests with an antiserum to K1 revealed the presence, in a Kagzi Kalan lime-lemon hybrid of a virus (KK) with similar morphology but differing consistently from K1 by one serological step. Moreover, in the original Kinnow orchard, we detected a further virus (K2), again with similar morphology, serologically related to K1 but differing by five steps. K2 now in turn appears, in pre- liminary results, to be a mixture containing yet a further morphologically similar virus, K3. Thus, Indian citrus ringspot disease may be caused by a series of viruses, and while diagnostic methods are now available for K1, these may not immediately solve the problem of field diagnosis.

The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase.

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.