40-kDa Polypeptide Research Articles

Caenorhabditis elegansUNC-96 Is a New Component of M-Lines That Interacts with UNC-98 and Paramyosin and Is Required in Adult Muscle for Assembly and/or Maintenance of Thick Filaments

To gain further insight into the molecular architecture, assembly, and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans. By polarized light microscopy of body wall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent "needles." By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel approximately 47-kDa polypeptide that has no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance.

Repression of human γ-globin gene expression by a short isoform of the NF-E4 protein is associated with loss of NF-E2 and RNA polymerase II recruitment to the promoter

Binding of the stage selector protein (SSP) to the stage selector element (SSE) in the human gamma-globin promoter contributes to the preferential expression of the gamma-gene in fetal erythroid cells. The SSP contains the transcription factor CP2 and an erythroid-specific partner, NF-E4. The NF-E4 gene encodes a 22-kDa polypeptide employing a non-AUG initiation codon. Antisera specific to NF-E4 detects this species and an additional 14 kDa protein, which initiates from an internal methionine. Enforced expression of p14 NF-E4 in the K562 fetal/erythroid cell line, and in primary erythroid cord blood progenitors, results in repression of gamma-gene expression. Biochemical studies reveal that p14 NF-E4 interacts with CP2, resulting in diminished association of CP2 with the SSE in chromatin immunoprecipitation assays. p45 NF-E2 recruitment to the gamma-promoter is also lost, resulting in a reduction in RNA polymerase II and TBP binding and a fall in promoter transcriptional activity. This effect is specific, as enforced expression of a mutant form of p14 NF-E4, which fails to interact with CP2, also fails to repress gamma-gene expression in K562 cells. These findings provide one potential mechanism that could contribute to the autonomous silencing of the human gamma-genes in adult erythroid cells.

Purification of a 47-kDa calmodulin-binding polypeptide as an actin-binding protein from Neurospora crassa.

We have enriched a 47-kDa polypeptide (p47) from Neurospora crassa on the basis of its affinity to calmodulin. The p47 was purified to homogeneity by chromatography on a Mono S cation exchange column and evidence is presented that the polypeptide co-sediments specifically with F-actin. The intracellular distribution of p47 and actin was also examined using indirect double immunofluorescence staining of cells at different stages of development. Our results suggest that by altering the conformation binding site of actin to p47, calmodulin could play a regulatory role in the polarized hyphal growth of N. crassa.

Erratum to “Purification of a 47-kDa calmodulin-binding polypeptide as an actin-binding protein from Neurospora crassa”

Erratum to “Purification of a 47-kDa calmodulin-binding polypeptide as an actin-binding protein from Neurospora crassa”

Low-molecular-weight vitellogenin 1-like proteins are components of a UV-damaged-DNA binding activity highly expressed in zebrafish (Danio rerio) embryos

A strong UV-damaged-DNA binding activity had been detected in the extracts of zebrafish embryos at 12 hr after fertilization by gel shift assay (Hsu et al. 2002. Fish Physiol Biochem 25:41-51). We attempted to study the components of this binding activity and their importance in DNA damage recognition. Among the proteins extracted from gel retardation complexes, a 30- and a 35-kDa polypeptide binding preferentially to 6-4photoproducts (6-4PPs) generated by UV irradiation were identified by peptide mass fingerprinting (PMF) as homologs of zebrafish vitellogenin I (zfVg1), a 150-kDa metalloprotein known as the precursor of yolk proteins in embryos. zfVg1-like polypeptides ranging from 25 to 105 kDa were detected in 12- and 96-hr-old zebrafish extracts by immunoblot analysis. Immunoblot analysis of affinity-captured proteins confirmed the preferential binding of the 30-35-kDa polypeptides to the 6-4PP probe, while 96-hr-old larval extracts containing very low levels of these two factors failed to recognize 6-4PPs. The presence of zfVg1-like factors was important in maintaining the embryonic UV-binding activity, as inclusion of a monoclonal anti-zfVg1 antibody in reaction mixtures caused a concentration-dependent reduction in 6-4PP-specific binding. In contrast, DNA damage recognition was not disturbed at all by an anti-HSP 70 antibody. The formation of 6-4PP-binding complexes was abolished after the addition of the metal chelating agent 1,10-phenanthroline (OP) to zebrafish extracts and the loss of UV-binding capacity correlated with the disappearance of the 35-kDa factor in OP-treated extracts. Our results demonstrated the ability of low-molecular-weight zfVg1-like proteins in zebrafish embryos to bind UV-damaged DNA and the expression of this embryonic UV-binding activity was metal dependent. Whether zfVg1-like UV-binding proteins are involved in repairing damaged DNA in embryos or in processing helical structures similar to UV-distorted DNA needs further investigation.

An acetylated 120-kDa lysosomal transmembrane protein is absent from mucopolysaccharidosis IIIC fibroblasts: A candidate molecule for MPS IIIC

Genetic deficiency of the lysosomal enzyme, acetyl-CoA: α-glucosaminide N-acetyltransferase ( N-acetyltransferase), which catalyses the transmembrane acetylation of heparan sulfate results in severe neurodegenerative disease, mucopolysaccharidosis IIIC. N-Acetyltransferase has never been characterized structurally and its gene has never been identified. We combined traditional methods of enzyme purification with organellar proteomics, isolating lysosomal membranes from mouse liver using differential centrifugation and osmolysis, followed by detergent extraction and purification of N-acetyltransferase by liquid chromatography. Partially purified enzyme had a molecular mass of 240 kDa and p I of 7.4 by gel filtration and chromatofocusing. Its specific activity varied with protein concentration typical of oligomeric enzymes or multienzyme complexes. Incubation of N-acetyltransferase with acetyl[ 14C]CoA in the absence of the acceptor of the acetyl group resulted in radioactive labeling of a 120-kDa polypeptide, suggesting that it represents a subunit containing the enzyme active site. Furthermore, following acetyl[ 14C]-labeling, the 120-kDa protein was present in the lysosomal membranes purified from the normal human skin fibroblasts but absent in those from the skin fibroblasts of MPS IIIC patients.

Interaction with the Na,K-ATPase and Tissue Distribution of FXYD5 (Related to Ion Channel)

FXYD5 (related to ion channel, dysadherin) is a member of the FXYD family of single span type I membrane proteins. Five members of this group have been shown to interact with the Na,K-ATPase and to modulate its properties. However, FXYD5 is structurally different from other family members and has been suggested to play a role in regulating E-cadherin and promoting metastasis (Ino, Y., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 365-370). The goal of this study was to determine whether FXYD5 can modulate the Na,K-ATPase activity, establish its cellular and tissue distribution, and characterize its biochemical properties. Anti-FXYD5 antibodies detected a 24-kDa polypeptide that was preferentially expressed in kidney, intestine, spleen, and lung. In kidney, FXYD5 resides in the basolateral membrane of the connecting tubule, the collecting tubule, and the intercalated cells of the collecting duct. However, there is also labeling of the apical membrane in long thin limb of Henle's loop. FXYD5 was effectively immunoprecipitated by antibodies to the alpha subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates alpha. Co-expressing FXYD5 with the alpha1 and beta1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabain-blockable outward current or as ouabain-sensitive (86)Rb(+) uptake. Thus, as found with other FXYD proteins, FXYD5 interacts with the Na,K-ATPase and modulates its properties.

Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.)

Aeromonas hydrophila causes disease under stress conditions or in concert with infection by other pathogens in goldfish. Sero-diagnostic and/or immunoprophylactic tools against Aeromonas infection in goldfish are not available so far. The present study was undertaken to fractionate and characterise the outer membrane proteins (OMP) of A. hydrophila and to identify suitable immunoreactive components. A total of 10 fractions were generated from crude OMP antigens upon gel permeation and subsequent ion-exchange chromatography. One of the fractionated antigens (GPID2), primarily a 57-kDa polypeptide, showed maximum sero-reactivity, even higher than the crude OMP. Suitability of GPID2 antigen for use in diagnostic preparations was assessed by dip-stick ELISA. In vitro goldfish lymphoproliferative ability of fractionated antigen, GPIID2 (primarily a 23-kDa polypeptide) was observed to be higher than all the fractionated antigens as well as crude OMP. It can be concluded that the 57 kDa and 23 kDa polypeptides of the OMP of A. hydrophila, possessing high immunoreactivity, should be given due attention while preparing immunodiagnostic and immunoprophylatic tools against Aeromonas infections in goldfish.

Arabidopsis peroxisomes possess functionally redundant membrane and matrix isoforms of monodehydroascorbate reductase

The H2O2 byproduct of fatty acid catabolism in plant peroxisomes is removed in part by a membrane-associated antioxidant system that involves both an ascorbate peroxidase and a monodehydroascorbate reductase (MDAR). Despite descriptions of 32-kDa MDAR polypeptides in pea and castor peroxisomal membranes and cDNA sequences for several 'cytosolic' MDARs, the genetic and protein factors responsible for peroxisomal MDAR function have yet to be elucidated. Of the six MDAR polypeptides in the Arabidopsis proteome, named AtMDAR1 to AtMDAR6 in this study, 47-kDa AtMDAR1 and 54-kDa AtMDAR4 possess amino acid sequences that resemble matrix (PTS1) and membrane peroxisomal targeting signals, respectively. Epitope-tagged versions of these two MDARs and a pea 47-kDa MDAR (PsMDAR) sorted in vivo directly from the cytosol to peroxisomes in Arabidopsis and BY-2 suspension cells, whereas AtMDAR2 and AtMDAR3 accumulated in the cytosol. The PTS1-dependent sorting of AtMDAR1 and PsMDAR to peroxisomes was incomplete (inefficient?), but was improved for PsMDAR after changing its PTS1 sequence from -SKI to the canonical tripeptide -SKL. A C-terminal transmembrane domain and basic cluster of AtMDAR4 were necessary and sufficient for targeting directly to peroxisomes. MDAR activity in isolated Arabidopsis peroxisomes was distributed among both water-soluble matrix and KCl-insoluble membrane subfractions that contained respectively 47- and 54-kDa MDAR polypeptides. Notably, a 32-kDa MDAR was not identified. Combined with membrane association and topological orientation findings, these results indicate that ascorbate recycling in Arabidopsis (and probably other plant) peroxisomes is coordinated through functionally redundant MDARs that reside in the membrane and the matrix of the organelle.

Transcription-dependent Association of Multiple Positive Transcription Elongation Factor Units to a HEXIM Multimer

The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb.HEXIM1.7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb.HEXIM1.7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.

Structure and Properties of the C-terminal Domain of Insulin-like Growth Factor-binding Protein-1 Isolated from Human Amniotic Fluid

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Angstroms resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe(2+) ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules.

A digestive β-glucosidase from the silkworm, Bombyx mori: cDNA cloning, expression and enzymatic characterization

A digestive β-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori β-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori β-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori β-glucosidase to be a single gene. Northern blot analysis of B. mori β-glucosidase gene confirmed larval midgut-specific expression. The B. mori β-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori β-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant β-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori β-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant β-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per μg of recombinant B. mori β-glucosidase. The purified recombinant B. mori β-glucosidase showed the highest activity at 35 °C and pH 6.0, and were stable at 50 °C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori β-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.

FLESH SOFTENING AND PHOSPHORYLATION OF SOLUBLE POLYPEPTIDES IN RELATION TO ETHYLENE PRODUCTION IN PRUNUS PERSICA FRUITS WITH DIFFERENT RIPENING PATTERNS

In peach fruit, it is desirable to individuate simple biochemical/physiological parameters, genetically determined and useful for marker-assisted selection to obtain improved genotypes for flesh firmness. The accumulation of proteins involved in cell wall re-organisation, such as endo-polygalacturonases (endo-PGs) and expansins, has been studied in fruits of two genotypes with either Non-Melting(NMF) or Melting Flesh (MF) texture, and in fruits of three 'Slow-Ripening' ('SR': altered ripening pattern) genotypes. In the flesh cell walls of MF fruits, the amounts of an endo-PG-like polypeptide were relevant and increased with ripening, while it was produced in extremely low amounts in NMF fruits. A similar pattern was observed for expansins. NMF fruits produced more ethylene than the MF ones. The Ca 2+ -dependent phosphorylation of a soluble polypeptide (Mw ≃ 52 kDa) decreased with ripening in NMF fruits and remained constant in the MF ones. In 'SR' genotypes, phosphorylation of this polypeptide was detectable only in ethylene-producing fruits, which softened their flesh and expressed the endo-PG-like polypeptide. The data suggest that phosphorylation of the 52-kDa polypeptide may be involved in the modulation of ethylene signalling and flesh softening.

Identification of a New Variant of PDE1A Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterase Expressed in Mouse Sperm1

In mature sperm, cAMP plays an important role as a second messenger regulating functions that include capacitation, the acrosome reaction, motility, and, in some cases, chemosensing. We have cloned from mouse testis a novel calmodulin-stimulated cyclic nucleotide phosphodiesterase 1A isoform, Pde1a_v7 (mmPDE1A7), which arises from an alternative transcription start in the cyclic nucleotide phosphodiesterase 1A gene. The open reading frame is predicted to encode a polypeptide with a molecular mass of 52 kDa. Two further variants of this form, which contain two additional new exons, arise from alternative splicing. Analysis of testis cDNA by real-time polymerase chain reaction (PCR) indicates that the Pde1A_v7 transcript variant is the most abundant. The PDE1A_v7 protein uniquely lacks the first amino-terminal calmodulin-binding domain, but does possess an inhibitory domain and a second calmodulin-binding site shared with other variants. In vitro translation of the corresponding Pde1a_v7 cDNA produced a 52-kDa polypeptide having cyclic nucleotide hydrolytic activity, which was stimulated threefold by calcium-bound calmodulin. Immunoprecipitation of cyclic nucleotide phosphodiesterase 1 activity from detergent extracts of mouse sperm revealed a major protein of the size expected for PDE1A_v7, and the immunocytochemical staining for cyclic nucleotide phosphodiesterase 1A in mouse sperm showed intense immunoreactivity in the tail only. These observations, along with the PCR data, strongly suggest that this new variant PDE1A_v7 is the major form of cyclic nucleotide phosphodiesterase 1A expressed in mature sperm and is therefore likely to play an important role in cyclic nucleotide regulation of mature sperm function.

The 5′-leader sequence of sugar beet mitochondrial atp6 encodes a novel polypeptide that is characteristic of Owen cytoplasmic male sterility

Cytoplasmic male sterility (CMS) is a mitochondrially encoded trait, which is characterized by a failure of plants to produce viable pollen. We have investigated the protein profile of mitochondria from sugar beet plants with normal (fertile) or CMS cytoplasm, and observed that a 35-kDa polypeptide is expressed in Owen CMS plants but not in normal plants. The variant 35-kDa polypeptide was found in CMS mitochondria placed in five different nuclear backgrounds. Interestingly, this polypeptide proved to be antigenically related to a 387-codon ORF (preSatp6) that is fused in-frame with the downstream atp6. The presequence extension of the atp6 ORF is commonly found in higher plants, but whether or not it is normally expressed has hitherto remained unclear. Our study is thus the first to demonstrate that the atp6 presequence is actually translated in mitochondria. We also observed that preSATP6 is a mitochondrial membrane protein that assembles into a homogeneous 200-kDa protein complex. In organello translation experiments in the presence of protease inhibitors showed a reduction in the abundance of mature preSATP6 with time, suggesting that the mature preSATP6 may be derived by proteolytic processing of a translation product of the preSatp6/Satp6 ORF.

The Presumptive Magnetosome Protein Mms16 Is a Poly(3-Hydroxybutyrate) Granule-Bound Protein (Phasin) in Magnetospirillum gryphiswaldense

The Mms16 protein has been previously found to be associated with isolated magnetosomes from two Magnetospirillum strains. A function of this protein as a magnetosome-specific GTPase involved in the formation of intracellular magnetosome membrane vesicles was suggested. Here we present a study of the Mms16 protein from Magnetospirillum gryphiswaldense to clarify its function. Insertion-duplication mutagenesis of the mms16 gene did not affect the formation of magnetosome particles but resulted in the loss of the ability of M. gryphiswaldense cell extracts to activate poly(3-hydroxybutyrate) (PHB) depolymerization in vitro, which was coincident with loss of the most abundant 16-kDa polypeptide from preparations of PHB granule-bound proteins. The mms16 mutation could be functionally complemented by enhanced yellow fluorescent protein (EYFP) fused to ApdA, which is a PHB granule-bound protein (phasin) in Rhodospirillum rubrum sharing 55% identity to Mms16. Fusions of Mms16 and ApdA to enhanced green fluorescent protein (EGFP) or EYFP were colocalized in vivo with the PHB granules but not with the magnetosome particles after conjugative transfer to M. gryphiswaldense. Although the Mms16-EGFP fusion protein became detectable by Western analysis in all cell fractions upon cell disruption, it was predominantly associated with isolated PHB granules. Contrary to previous suggestions, our results argue against an essential role of Mms16 in magnetosome formation, and the previously observed magnetosome localization is likely an artifact due to unspecific adsorption during preparation. Instead, we conclude that Mms16 in vivo is a PHB granule-bound protein (phasin) and acts in vitro as an activator of PHB hydrolysis by R. rubrum PHB depolymerase PhaZ1. Accordingly, we suggest renaming the Mms16 protein of Magnetospirillum species to ApdA, as in R. rubrum.

Characterization of the Protein Dimerization Domain Responsible for Assembly of Functional Selenodeiodinases

Thyroid hormone metabolism is catalyzed by a small family of selenoenzymes. Type I deiodinase (D1) is the best characterized family member and is an integral membrane protein composed of two 27-kDa subunits that assemble to a functional holoenzyme after translation. To characterize the protein domain(s) responsible for this post-translational assembly event, we used deletion/truncation analysis coupled with immune depletion assays to map the dimerization domain of D1. The results of our studies show that a highly conserved sequence of 16 amino acids in the C-terminal half of the D1 subunit, -D148FL-YI-EAH-DGW163-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDD(GFP)) to examine holoenzyme assembly of other family members. Overexpression of either the DDD(GFP) or an inert D1 subunit (M4) into SeD2 (accession number U53505)-expressing C6 cells specifically led to the loss of >90% of the catalytic activity. Catalytically inactive D2 heterodimers composed of SeD2: DDD(GFP) subunits were rescued by specific immune precipitation with anti-SeD2 IgG, suggesting that SeD2 requires two functional subunits to assemble a catalytically active holoenzyme. These findings identify and characterize the essential dimerization domain responsible for post-translational assembly of selenodeiodinases and show that family members can intermingle through this highly conserved protein domain.

Molecular cloning and characterization of a peroxiredoxin gene from the mole cricket, Gryllotalpa orientalis

We report the cloning, expression and characterization of a cDNA encoding the antioxidant enzyme peroxiredoxin (Prx) from the mole cricket, Gryllotalpa orientalis. The G. orientalis Prx (GoPrx) cDNA contains an open reading frame of 660 bp encoding 220 amino acid residues and possesses one cysteine residue that is characteristic of the 1-Cys subgroup of the peroxiredoxin family. The deduced amino acid sequence of the GoPrx cDNA showed 69% identity to Drosophila melanogaster DPx-2540, 50% to D. melanogaster DPx-6005, and 47% to Glossina morsitans morsitans Prx. Phylogenetic analysis further confirmed a closer relationship of the deduced amino acid sequences of the GoPrx gene to the DPx-2540 within the 1-Cys Prx cluster. The cDNA encoding GoPrx was expressed as a 27-kDa polypeptide in baculovirus-infected insect Sf9 cells. The purified recombinant GoPrx was shown to reduce H 2O 2 in the presence of electrons donated by dithiothreitol, but did not show the activity in the presence of thioredoxin as electron donor. Northern blot analysis revealed the presence of GoPrx transcripts in all tissues examined. When H 2O 2 was injected into the body cavity of G. orientalis adult, GoPrx mRNA expression was up-regulated in the fat body tissues. Furthermore, the expression levels of GoPrx mRNA in the fat body were particularly high when G. orientalis adult was exposed at low (4 °C) and high (37 °C) temperatures, suggesting that the GoPrx seems to play a protective role against oxidative stress caused by temperature shock.

Protein–protein interactions of a Kvβ subunit in the cochlea

Accessory subunits associated with voltage-gated potassium (Kv) channels can influence the biophysical properties and promote the surface expression of channel-forming alpha-subunits. Previously, we cloned several alpha-subunits and a beta-subunit from a cDNA library of the chicken cochlea. In the present study, we raised an antibody against the N-terminus of chicken Kvbeta1.1 (cKvbeta1.1) and characterized the Kvbeta-related polypeptide in cochlear tissues and heterologous cells. The anti-cKvbeta1.1 antibody recognizes a 45-kDa polypeptide in chick cochlear extracts as well as in Chinese hamster ovary (CHO) cells transfected with cKvbeta1.1. The accessory subunit was localized to the ganglion cells of the chick cochlea using immunohistochemistry and in situ hybridization. Coimmunoprecipitation studies show that Kvbeta1.1 interacts with Shaker channel members Kvalpha1.2 and 1.3, both of which colocalize with beta to the cochlear ganglion cells. Additionally, coimmunoprecipitation studies show that Kvalpha1.2 and 1.3 interact with each other, suggesting that these ion channels are formed by heteromultimers. In comparison, Kvbeta did not coprecipitate with a member of the Shal subfamily. The presence of Kvbeta in the cochlea suggests that this subunit contributes to the modulation of auditory signals in the ganglion cells, presumably by regulating properties of inactivation as well as surface expression of Kvalpha channels.

Purification and characterization of a fructosyltransferase from onion bulbs and its key role in the synthesis of fructo‐oligosaccharides in vivo

A fructosyltransferase that transfers the terminal (2 --> 1)-beta-linked D-fructosyl group of fructo-oligosaccharides (1(F)(1-beta-D-fructofuranosyl)(n) sucrose, n >/= 1) to HO-6 of the glucosyl residue and HO-1 of the fructosyl residue of similar saccharides (1(F)(1-beta-D-fructofuranosyl)(m) sucrose, m >/= 0) has been purified from an extract of the bulbs of onion (Allium cepa). Successive column chromatography using DEAE-Sepharose CL-6B, Toyopearl HW65, Toyopearl HW55, DEAE-Sepharose CL-6B (2nd time), Sephadex G-100, Concanavalin A Sepharose, and Toyopearl HW-65 (2nd time) were applied for protein purification. The general properties of the enzyme, were as follows: molecular masses of 66 kDa (gel filtration chromatography), and of 52 kDa and 25 kDa (SDS-PAGE); optimum pH of c. 5.68, stable at 20-40 degrees C for 15 min; stable in a range of pH 5.30-6.31 at 30 degrees C for 30 min, inhibited by Hg(2+), Ag(+), p-chloromercuribenzoic acid (p-CMB) and sodium dodecyl sulfate (SDS), activated by sodium deoxycholate, Triton X-100 and Tween-80. The amino acid sequence of the N-terminus moiety of the 52-kDa polypeptide was ADNEFPWTNDMLAWQRCGFHFRTVRNYMNDPSGPMYYKGWYHLFYQHNKDFAYXG and the amino acid sequence from the N-terminus of the 25-kDa polypeptide was ADVGYXCSTSGGAATRGTLGPFGLL VLANQDLTENTATYFYVSKGTDGALRTHFCQDET. The enzyme tentatively classified as fructan: fructan 6(G)-fructosyltransferase (6G-FFT). The enzyme is proposed to play an important role in the synthesis of inulin and inulinneo-series fructo-oligosaccharides in onion bulbs.