scholarly journals Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein

1994 ◽  
Vol 124 (6) ◽  
pp. 949-961 ◽  
Author(s):  
LA Jesaitis ◽  
DA Goodenough
Keyword(s):  
Amino Acid ◽  
Tumor Suppressor ◽  
Amino Acid Sequence ◽  
Tight Junction ◽  
Mdck Cell ◽  
Sequence Information ◽  
Unique Region ◽  
Cell Extracts ◽  
Junction Protein ◽  
Discs Large

ZO-1 is a 210-225-kD peripheral membrane protein associated with cytoplasmic surfaces of the zonula occludens or tight junction. A 160-kD polypeptide, designated ZO-2, was found to coimmunoprecipitate with ZO-1 from MDCK cell extracts prepared under conditions which preserve protein associations (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88: 3460-3464). We have isolated ZO-2 from MDCK cell monolayers by bulk coimmunoprecipitation with ZO-1 followed by electroelution from preparative SDS-PAGE gel slices. Amino acid sequence information obtained from a ZO-2 tryptic fragment was used to isolate a partial cDNA clone from an MDCK library. The deduced amino acid sequence revealed that canine ZO-2 contains a region that is very similar to sequences in human and mouse ZO-1. This region includes both a 90-amino acid repeat domain of unknown function and guanylate kinase-like domains which are shared among members of the family of proteins that includes ZO-1, erythrocyte p55, the product of the lethal(1)discs-large-1 (dlg) gene of Drosophila, and a synapse-associated protein from rat brain, PSD-95/SAP90. The dlg gene product has been shown to act as a tumor suppressor in the imaginal disc of the Drosophila larva, although the functions of other family members have not yet been defined. A polyclonal antiserum was raised against a unique region of ZO-2 and found to exclusively label the cytoplasmic surfaces of tight junctions in MDCK plasma membrane preparations, indicating that ZO-2 is a tight junction-associated protein. Immunohistochemical staining of frozen sections of whole tissue demonstrated that ZO-2 localized to the region of the tight junction in a number of epithelia, including liver, intestine, kidney, testis, and arterial endothelium, suggesting that this protein is a ubiquitous component of the tight junction. Double-label immunofluorescence microscopy performed on cryosections of heart, a nonepithelial tissue, revealed the presence of ZO-1 but no ZO-2 staining at the fascia adherens, a specialized junction of cardiac myocytes which has previously been shown to contain ZO-1 (Itoh, M., S. Yonemura, A. Nagafuchi, S. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). Thus it appears that ZO-2 is not a component of the fascia adherens, and that unlike ZO-1, this protein is restricted to the epithelial tight junction.

scholarly journals ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

1998 ◽  
Vol 141 (1) ◽  
pp. 199-208 ◽  
Author(s):  
Julie Haskins ◽  
Lijie Gu ◽  
Erika S. Wittchen ◽  
Jennifer Hibbard ◽  
Bruce R. Stevenson
Keyword(s):  
Amino Acid ◽  
Tight Junction ◽  
Molecular Mass ◽  
Mdck Cells ◽  
Cdna Libraries ◽  
Pdz Domains ◽  
Large Tumor ◽  
Junction Protein ◽  
Discs Large

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ∼130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.

scholarly journals Molecular Identification of Family 38 α-Mannosidase of Bacillus sp. Strain GL1, Responsible for Complete Depolymerization of Xanthan

2002 ◽  
Vol 68 (6) ◽  
pp. 2731-2736 ◽  
Author(s):  
Hirokazu Nankai ◽  
Wataru Hashimoto ◽  
Kousaku Murata
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Extract ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Sequence Information ◽  
Reading Frame ◽  
A Cell ◽  
Terminal Amino ◽  
Amino Acid Sequence Information

ABSTRACT When cells of Bacillus sp. strain GL1 were grown in a medium containing xanthan as a carbon source, α-mannosidase exhibiting activity toward p-nitrophenyl-α-d-mannopyranoside (pNP-α-d-Man) was produced intracellularly. The 350-kDa α-mannosidase purified from a cell extract of the bacterium was a trimer comprising three identical subunits, each with a molecular mass of 110 kDa. The enzyme hydrolyzed pNP-α-d-Man (Km = 0.49 mM) and d-mannosyl-(α-1,3)-d-glucose most efficiently at pH 7.5 to 9.0, indicating that the enzyme catalyzes the last step of the xanthan depolymerization pathway of Bacillus sp. strain GL1. The gene for α-mannosidase cloned most by using N-terminal amino acid sequence information contained an open reading frame (3,144 bp) capable of coding for a polypeptide with a molecular weight of 119,239. The deduced amino acid sequence showed homology with the amino acid sequences of α-mannosidases belonging to glycoside hydrolase family 38.

scholarly journals A Mono-2-Ethylhexyl Phthalate Hydrolase from a Gordonia sp. That Is Able To Dissimilate Di-2-Ethylhexyl Phthalate

2006 ◽  
Vol 72 (4) ◽  
pp. 2394-2399 ◽  
Author(s):  
Tuguhiro Nishioka ◽  
Makoto Iwata ◽  
Takuya Imaoka ◽  
Maiko Mutoh ◽  
Yoshihiro Egashira ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Contaminated Soil ◽  
Primary Structure ◽  
Independent Group ◽  
Serine Hydrolases ◽  
Cell Extracts ◽  
Significant Homology ◽  
Serine Esterases ◽  
Ethylhexyl Phthalate

ABSTRACT Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The Km and V max values for mono-2-ethylhexyl phthalate were 26.9 ± 4.3 μM and 18.1 ± 0.9 μmol/min · mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.

scholarly journals The gdhB Gene of Pseudomonas aeruginosaEncodes an Arginine-Inducible NAD+-Dependent Glutamate Dehydrogenase Which Is Subject to Allosteric Regulation

2001 ◽  
Vol 183 (2) ◽  
pp. 490-499 ◽  
Author(s):  
Chung-Dar Lu ◽  
Ahmed T. Abdelal
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Glutamate Dehydrogenase ◽  
Legionella Pneumophila ◽  
Caulobacter Crescentus ◽  
Sodium Dodecyl ◽  
Sequence Information ◽  
Saturation Curve ◽  
Moderate Degree ◽  
Amino Terminal

ABSTRACT The NAD+-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encodinggdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, andCaulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) fromSaccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.

scholarly journals Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

2002 ◽  
Vol 184 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
Keietsu Abe ◽  
Fumito Ohnishi ◽  
Kyoko Yagi ◽  
Tasuku Nakajima ◽  
Takeshi Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Expression Vector ◽  
Alcaligenes Faecalis ◽  
E Coli ◽  
Cell Extracts ◽  
Membrane Spanning ◽  
Metabolic Cycle

ABSTRACT Tetragenococcus halophila D10 catalyzes the decarboxylation of l-aspartate with nearly stoichiometric release of l-alanine and CO2. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an l-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter → aspD → aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known l-aspartate-β-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of l-aspartate-β-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

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