PHYLOGENETIC ANALYSIS OFNa+/K+ATPase: INSIGHT INTO THE MECHANISM FOR THE GENESIS OF MULTI-ISOFORMS OF PROTEIN COMPLEX

2005 ◽  
Vol 13 (03) ◽  
pp. 299-312 ◽  
Author(s):  
FEI MA ◽  
HUIFANG HUANG ◽  
LUPING LIN ◽  
CHENGHAI XUE ◽  
JESSE LI-LING ◽  
...  
Keyword(s):  
Protein Complex ◽  
Phylogenetic Analyses ◽  
Amino Acid Sequences ◽  
Evolutionary Divergence ◽  
Β Subunit ◽  
Α Subunit ◽  
Duplication Events ◽  
Α And Β Subunits ◽  
Underlying Mechanisms ◽  
Insight Into

One most notable trend during the evolution is the substantial expansion of genomes along with dramatic expansion of protein diversity. It has been discovered that, whilst in prokaryotes subunits of many proteinases are encoded by single genes, these are mostly encoded by multi-genes in eukaryotes. To understand the mechanism for the genesis of multi-isoforms of protein complex, we have analyzed amino acid sequences of Na+/ K+ATPase from various species ranging from archaea to vertebrates. Phylogenetic relationship between the selected species was considered from the perspective of important functional domains of Na+/ K+ATPase including cation ATPase N termination, E1-E2 ATPase, hydrolase, and cation ATPase C termination of the α subunit, Na+/ K+ATPase of the β subunit, and ATP1G1_PLM_MAT8 of the γ subunit. Coincident trees, obtained through comparison of aforementioned domains of the α and β subunits, were used to examine the evolutionary divergence. By conservational and phylogenetic analyses, evolution of the Na+/ K+ATPase was outlined. Evidence was also found that essential domains of the Na+/ K+ATPase have been conserved during the evolution. These investigations seem to imply that various isoforms of α and β subunits of vertebral Na+/ K+ATPases have evolved from single ancestral α and β subunit genes through duplication events. In addition, our results seem to suggest a third fate for duplicated genes, e.g. the duplicate may have the same function as their ancestor gene. The results may also provide important clues to the underlying mechanisms of genesis of Na+/ K+ATPase multi-isoforms.

Heterogeneity of human luteinizing hormone. Detection and identification of α- and β-subunits in international reference preparations

1987 ◽  
Vol 114 (4) ◽  
pp. 572-576 ◽  
Author(s):  
L. A. van Ginkel ◽  
J. G. Loeber
Keyword(s):  
Luteinizing Hormone ◽  
Biologically Active ◽  
Β Subunit ◽  
Charge Heterogeneity ◽  
Α Subunit ◽  
Isoelectric Focussing ◽  
International Reference ◽  
Detection And Identification ◽  
Α And Β Subunits ◽  
Two Peaks

Abstract. The charge heterogeneity of three international reference preparations containing biologically active human luteinizing hormone (LH) or its free α-or β-subunits, respectively, was investigated with isoelectric focussing in sucrose density gradients. The immunoreactive profiles throughout the pH-gradient were assessed with radioimmunoassay (RIA) systems specific for intact, i.e. undissociated, LH or free α- or β-subunits. For the preparation of intact LH (MRC 68/40), two peaks were found at pH = 7.69 and 8.05; for the α-subunit preparation (NIBSC 78/554), the peak values were 4.76, 5.04, 5.94, 6.70, 6.96, 7.35, 8.02, 8.72 and 9.32; and for the β-subunit preparation (NIBSC 78/556), the values were 7.60, 8.40, 8.55 and 9.61. These results are in excellent agreement with those obtained for a highly purified LH preparation (NM 14) which was prepared by one of us and on which we have reported earlier. In addition, with the abovementioned techniques, the spontaneous dissociation of MRC 68/40 into subunits upon incubation at 37°C in phosphate buffer was clearly demonstrated by the increased immunoreactivity in the profiles assessed with both RIA-subunit systems. It is concluded that charge heterogeneity of LHi, α- and β-subunits, as observed for different preparations, is confined to a limited population of forms. Differences between preparations are only quantitative. A single preparation, therefore, can be used as a general model for the study of human luteinizing hormone.

scholarly journals Amino acid sequence of the α subunit and computer modelling of the α and β subunits of echicetin from the venom of Echis carinatus (saw-scaled viper)

1997 ◽  
Vol 323 (2) ◽  
pp. 533-537 ◽  
Author(s):  
János POLGÁR ◽  
Edith M. MAGNENAT ◽  
Manuel C. PEITSCH ◽  
Timothy N. C. WELLS ◽  
Mansoor S. A. SAQI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Computer Modelling ◽  
Platelet Glycoprotein ◽  
Β Subunit ◽  
Α Subunit ◽  
Echis Carinatus ◽  
Α And Β Subunits ◽  
Lectin Protein ◽  
Von Willebrand

Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the β subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin α and β subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the α subunit and computer models of the α and β subunits. The sequence of α echicetin is highly similar to the α and β chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated α or β subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor–ristocetin or α-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin β subunit might have been due to partial reduction of the protein.

Somatic immunoglobulin sequence divergence and its implications for studies of evolutionary divergence

Genome ◽  
1988 ◽  
Vol 30 (3) ◽  
pp. 341-346
Author(s):  
G. Brian Golding
Keyword(s):  
Amino Acid ◽  
High Frequency ◽  
Somatic Mutations ◽  
Sequence Divergence ◽  
Amino Acid Sequences ◽  
Evolutionary Divergence ◽  
Immunoglobulin Genes ◽  
Actual Knowledge ◽  
Evolutionary Pattern ◽  
Insight Into

The divergence of immunoglobulin genes due to somatic mutation provides a natural example of DNA sequence divergence. This divergence was examined to gain insight into the processes of evolution and the determinants of the variance-to-mean ratio of sequence divergence. Normally, this ratio is found to be larger than expected (1.0 under Poisson assumptions) for the evolutionary divergence or most genes. Although not significantly less than one, all seven groups of immunoglobulin amino acid sequences have ratios smaller than expected, contrary to the evolutionary pattern generally observed. The substitutions in the immunoglobulin genes appear to be highly nonrandom and an excess of parallel changes (the major nonrandom feature of these mutations) is shown to cause smaller ratios. Because convergent or parallel mutations are often observed in the evolutionary divergence of genes, this suggests that forces causing the large observed ratios may actually have to be more powerful than previously expected. Further, since selection is one of the likely causes of parallel mutations, it should be noted that selection could significantly decrease the variance-to-mean ratio. The high frequency of parallel mutations and their resulting effects, as observed in the immunoglobulin genes, suggest that only poor inferences of sequence divergence can be made without actual knowledge of the ancestral sequence.Key words: molecular evolution, parallel mutations, neutral allele theory, sequence divergence, immunoglobulin, somatic mutations.

scholarly journals The α- and β-subunits of the Human UDP-N-acetylglucosamine:Lysosomal Enzyme Phosphotransferase Are Encoded by a Single cDNA

2005 ◽  
Vol 280 (43) ◽  
pp. 36141-36149 ◽  
Author(s):  
Mariko Kudo ◽  
Ming Bao ◽  
Anil D'Souza ◽  
Fu Ying ◽  
Huaqin Pan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Amino Acid Sequences ◽  
Subunit Structure ◽  
Bac Clone ◽  
Significant Similarity ◽  
Α Subunit ◽  
Γ Subunit ◽  
Mannose 6 Phosphate ◽  
Α And Β Subunits

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an α2β2γ2-subunit structure was proposed. Although cDNA encoding the γ-subunit has been described, cDNAs for the α- and β-subunits have not. Using partial amino acid sequences from the bovine α- and β-subunits, we have isolated a human cDNA that encodes both the α- and β-subunits. Both subunits contain a single predicted membrane-spanning domain. The α- and β-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the α/β-subunits-precursor cDNA with or without the γ-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the α-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.

scholarly journals IpdAB, a virulence factor in Mycobacterium tuberculosis, is a cholesterol ring-cleaving hydrolase

2018 ◽  
Vol 115 (15) ◽  
pp. E3378-E3387 ◽  
Author(s):  
Adam M. Crowe ◽  
Sean D. Workman ◽  
Nobuhiko Watanabe ◽  
Liam J. Worrall ◽  
Natalie C. J. Strynadka ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Important Determinant ◽  
Phylogenetic Analyses ◽  
High Specificity ◽  
Α Subunit ◽  
Mechanistic Insight ◽  
Degrading Bacteria ◽  
Coa Thioesters ◽  
Hydrolysis Of ◽  
Insight Into

Mycobacterium tuberculosis (Mtb) grows on host-derived cholesterol during infection. IpdAB, found in all steroid-degrading bacteria and a determinant of pathogenicity, has been implicated in the hydrolysis of the last steroid ring. Phylogenetic analyses revealed that IpdAB orthologs form a clade of CoA transferases (CoTs). In a coupled assay with a thiolase, IpdAB transformed the cholesterol catabolite (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA) and CoASH to 4-methyl-5-oxo-octanedioyl-CoA (MOODA-CoA) and acetyl-CoA with high specificity (kcat/Km = 5.8 ± 0.8 × 104 M−1⋅s−1). The structure of MOODA-CoA was consistent with IpdAB hydrolyzing COCHEA-CoA to a β-keto-thioester, a thiolase substrate. Contrary to characterized CoTs, IpdAB exhibited no activity toward small CoA thioesters. Further, IpdAB lacks the catalytic glutamate residue that is conserved in the β-subunit of characterized CoTs and a glutamyl-CoA intermediate was not trapped during turnover. By contrast, Glu105A, conserved in the α-subunit of IpdAB, was essential for catalysis. A crystal structure of the IpdAB·COCHEA-CoA complex, solved to 1.4 Å, revealed that Glu105A is positioned to act as a catalytic base. Upon titration with COCHEA-CoA, the E105AA variant accumulated a yellow-colored species (λmax = 310 nm; Kd = 0.4 ± 0.2 μM) typical of β-keto enolates. In the presence of D2O, IpdAB catalyzed the deuteration of COCHEA-CoA adjacent to the hydroxylation site at rates consistent with kcat. Based on these data and additional IpdAB variants, we propose a retro-Claisen condensation-like mechanism for the IpdAB-mediated hydrolysis of COCHEA-CoA. This study expands the range of known reactions catalyzed by the CoT superfamily and provides mechanistic insight into an important determinant of Mtb pathogenesis.

Interaction of α- and β-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase β-subunit

2000 ◽  
Vol 278 (4) ◽  
pp. C727-C738 ◽  
Author(s):  
Curtis T. Okamoto ◽  
Dar C. Chow ◽  
And John G. Forte
Keyword(s):  
Cell Surface ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Live Cells ◽  
Β Subunit ◽  
Stringent Requirement ◽  
Α Subunit ◽  
Α And Β Subunits ◽  
Darby Canine Kidney

The assembly of the β-subunit of the gastric H-K-ATPase (HKβ) with the α-subunit of the H-K-ATPase or the Na-K-ATPase (NaKα) was characterized with two anti-HKβ monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, in H-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cells transfected with HKβ, MAb 2/2E6 was observed to bind to HKβ only when interactions between α- and β-subunits were disrupted by various denaturants. The epitope for MAb 2/2E6 was mapped to the tetrapeptide S226LHY229 of the extracellular domain of HKβ. The epitope for MAb 2G11 was mapped to the eight NH2-terminal amino acids of the cytoplasmic domain of HKβ. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HKβ with α-subunits of the endogenous cell surface NaKα, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HKβ. In HKβ-transfected LLC-PK1 cells, significant immunofluorescent labeling of HKβ at the cell surface could be detected without postfixation denaturation or in live cells, although a fraction of transfected HKβ could also be coimmunoprecipitated with NaKα. Thus assembly of HKβ with NaKα does not appear to be a stringent requirement for cell surface delivery of HKβ in LLC-PK1 cells but may be required in MDCK cells. In addition, endogenous posttranslational regulatory mechanisms to prevent hybrid α-β heterodimer assembly appear to be compromised in transfected cultured renal epithelial cells. Finally, the extracellular epitope for assembly-sensitive MAb 2/2E6 may represent a region of HKβ that is associated with α-β interaction.

scholarly journals Characterization of Imjin Virus, a Newly Isolated Hantavirus from the Ussuri White-Toothed Shrew (Crocidura lasiura)

2009 ◽  
Vol 83 (12) ◽  
pp. 6184-6191 ◽  
Author(s):  
Jin-Won Song ◽  
Hae Ji Kang ◽  
Se Hun Gu ◽  
Sung Sil Moon ◽  
Shannon N. Bennett ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Amino Acid Sequences ◽  
Evolutionary Divergence ◽  
Female Ratio ◽  
Male Predominance ◽  
Demilitarized Zone ◽  
Cross Neutralization ◽  
The Republic ◽  
Male To Female

ABSTRACT Until recently, the single known exception to the rodent-hantavirus association was Thottapalayam virus (TPMV), a long-unclassified virus isolated from the Asian house shrew (Suncus murinus). Robust gene amplification techniques have now uncovered several genetically distinct hantaviruses from shrews in widely separated geographic regions. Here, we report the characterization of a newly identified hantavirus, designated Imjin virus (MJNV), isolated from the lung tissues of Ussuri white-toothed shrews of the species Crocidura lasiura (order Soricomorpha, family Soricidae, subfamily Crocidurinae) captured near the demilitarized zone in the Republic of Korea during 2004 and 2005. Seasonal trapping revealed the highest prevalence of MJNV infection during the autumn, with evidence of infected shrews' clustering in distinct foci. Also, marked male predominance among anti-MJNV immunoglobulin G antibody-positive Ussuri shrews was found, whereas the male-to-female ratio among seronegative Ussuri shrews was near 1. Plaque reduction neutralization tests showed no cross neutralization for MJNV and rodent-borne hantaviruses but one-way cross neutralization for MJNV and TPMV. The nucleotide and deduced amino acid sequences for the different MJNV genomic segments revealed nearly the same calculated distances from hantaviruses harbored by rodents in the subfamilies Murinae, Arvicolinae, Neotominae, and Sigmodontinae. Phylogenetic analyses of full-length S, M, and L segment sequences demonstrated that MJNV shared a common ancestry with TPMV and remained in a distinct out-group, suggesting early evolutionary divergence. Studies are in progress to determine if MJNV is pathogenic for humans.

scholarly journals Reconstitution, morphology and crystallization of a fatty acid β-oxidation multienzyme complex from Pseudomonas fragi

1997 ◽  
Vol 328 (3) ◽  
pp. 815-820 ◽  
Author(s):  
Momoyo ISHIKAWA ◽  
Yuriko MIKAMI ◽  
Jiro USUKURA ◽  
Hiroshi IWASAKI ◽  
Hideo SHINAGAWA ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Gene Product ◽  
Multienzyme Complex ◽  
Β Subunit ◽  
Pseudomonas Fragi ◽  
Α Subunit ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Α And Β Subunits ◽  
Product Forms ◽  
Enoyl Coa Hydratase

The fatty acid β-oxidation multienzyme complex from Pseudomonas fragi, HDT, exhibits predominantly the three enzymic activities of 2-enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The HDT complex is encoded by the faoAB operon, consisting of the faoA and faoB genes that encode two individual constituents, the α-subunit and the β-subunit. We have constructed Escherichia coli overexpression systems for the faoAB gene product (coexpression of the α- and β-subunits), the α-subunit alone and the β-subunit alone, and have purified the three respective products. Gel-filtration analysis revealed that the faoAB gene product forms a heterotetrameric structure, α2β2, identical with the native HDT oligomeric state from P. fragi, whereas the α-subunit and β-subunit individually form dimers. Electron microscopy demonstrated that each protein morphologically adopts the above oligomeric structures. The HDT complex, reconstituted in vitro from the isolated α- and β-subunits, exhibits the three original enzymic activities and yields the same crystal as those from the native enzyme. CD measurements indicated that the α- and β-dimers hardly alter their global conformations upon the formation of the HDT complex. Interestingly, the β-dimer alone does not exhibit 3-oxoacyl-CoA thiolase activity, whereas the α-dimer alone exhibits both the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. These results suggest that the contact between the α- and β-subunits is essential for the thiolase activity. We have identified several structurally important proteolytic sites within each subunit, which are protected in the intact heterotetrameric molecule. These findings allow the possible location of the interface between the two subunits, which should be crucial for the exhibition of thiolase activity.

Human pituitary thyrotropin. The primary structure of the α and β subunits

1977 ◽  
Vol 55 (7) ◽  
pp. 755-760 ◽  
Author(s):  
M. R. Sairam ◽  
Choh Hao Li
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Primary Structure ◽  
Carbohydrate Moiety ◽  
Amino Acid Residues ◽  
Β Subunit ◽  
Human Pituitary ◽  
Α Subunit ◽  
Α And Β Subunits

The amino acid sequence of the α and β subunits of human pituitary thyrotropin has been proposed based on the data derived from the tryptic and chymotryptic peptides. The α subunit consists of 89 amino acid residues with two carbohydrate moieties, presumably linked to asparagines in position 49 and 75, and its amino acid sequence is identical to that of lutropin α. The β subunit has 112 residues with a single carbohydrate moiety attached to asparagine at position 23. Comparison of the primary structure of human thyrotropin β with that of the bovine hormone reveals that only 11 residue positions have been found to be different.

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