Genomic organization of three novel toxins from the scorpion Buthus martensi Karsch that are active on potassium channels

The cDNA and genomic DNA of three novel toxins from the scorpion Buthus martensi Karsch that are active on K+ channels, designated BmKTX (where KTX is kaliotoxin), BmTX1 and BmTX2, were cloned and sequenced. On the basis of their known amino acid sequences, gene-specific primers for 3ʹ and 5ʹ rapid amplification of cDNA ends (RACE) were designed and synthesized. By overlapping the two partial cDNA sequences obtained by 3ʹ and 5ʹ RACE, their full-length cDNA sequences were completed. BmKTX encodes a signal peptide of 22 amino acid residues and a mature toxin of 38 residues, whereas BmTX1 and BmTX2 encode signal peptides of 20 and 21 residues respectively and a mature toxin of 38 residues for each. Their cDNA-deduced amino acid sequences were totally consistent with those determined except that the C-terminus of BmKTX had an additional Gly residue, which was removed during post-translational processing and was indispensable for the amidation of its C-terminal Lys residue. In addition, the first deduced amino acid for both BmTX1 and BmTX2 is Gln instead of pyro-Glu in the reported toxins, which obviously also undergoes post-translational processing. The genomic DNA species of these three toxins were also amplified by PCR, then cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptides at position -6 for BmKTX and at position -5 for both BmTX1 and BmTX2 upstream of the mature toxins, and consisted of 87, 87 and 80 bp respectively.

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Cloning of rodent cDNA encoding the poly(ADP-ribose) polymerase catalytic domain and analysis of mRNA levels during the cell cycle

Biochemistry and Cell Biology ◽

10.1139/o89-097 ◽

1989 ◽

Vol 67 (9) ◽

pp. 653-660 ◽

Cited By ~ 21

Author(s):

J. Thibodeau ◽

G. Gradwohl ◽

C. Dumas ◽

S. Clairoux-Moreau ◽

G. Brunet ◽

...

Keyword(s):

Cell Cycle ◽

Amino Acid ◽

Genomic Dna ◽

Catalytic Domain ◽

Binding Domain ◽

Amino Acid Sequences ◽

Phosphorylation Sites ◽

Mrna Levels ◽

C Terminus ◽

Strong Homology

We have isolated a partial 2.0 kb cDNA (pRATC) encoding the entire 489 amino acids of the NAD binding domain located at the C terminus of the rat poly(ADP-ribose) polymerase. pRATC sequences were analysed and compared with the human mRNA. Our analysis reveals a remarkable homology between the rat and human nucleotide and amino acid sequences. Although a few minor amino acid changes were detected, we have found that the total number of possible phosphorylation sites remained constant in the NAD binding domain of both enzymes. We have also found that a 102 amino acid sequence, containing the putative nucleotide binding site Gly-Lys-Gly (position 378), is perfectly conserved between the rat and human sequences. Strong homology was also detected between pRATC and genomic DNA isolated from various vertebrates. In addition, we have analysed the levels of poly(ADP-ribose) polymerase mRNA throughout the cell cycle. Our results show that the levels of mRNA culminate in the G1 phase. We have also found that the increase in enzymatic activity observed in rats following treatment with phenobarbital did not correspond to an increase in the mRNA levels.Key words: poly(ADP-ribose) polymerase.

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Intersubunit interaction and catalytic activity of catechol 2,3-dioxygenases

Biochemical Journal ◽

10.1042/bj20021657 ◽

2003 ◽

Vol 371 (2) ◽

pp. 557-564 ◽

Cited By ~ 14

Author(s):

Akiko OKUTA ◽

Kouhei OHNISHI ◽

Sakiko YAGAME ◽

Shigeaki HARAYAMA

Keyword(s):

Amino Acid ◽

Quaternary Structure ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Large Protein ◽

C Terminus ◽

Large Protein Family ◽

Quaternary Structures ◽

Hybrid Enzymes ◽

Hybrid Forms

Catechol 2,3-dioxygenases (C23Os; EC 1.3.11.2) form a large protein family that is divided into several subgroups. Amino acid sequences of C23Os belonging to subgroup I.2.A and those belonging to I.2.B are found to be approx. 50% identical. When the central parts of the C23O sequences belonging to I.2.B were fused with the N-terminal and C-terminal sequences of I.2.A C23O, the hybrid enzymes were not active. To understand why these hybrid C23Os were inactive, hybrids between XylEP (C23O found in a Pseudomonas strain; subgroup I.2.A) and XylES (C23O found in a Sphingomonas strain; subgroup I.2.B) were constructed. HB3-C23O consisted mostly of the XylES sequence, except that its C-terminal end was derived from XylEP. While HB3-C23O was not active, HB4-C23O, carrying shorter C-terminal XylEP sequences than HB3-C23O, was active. This observation indicated that certain amino acid residues at the C-terminus were crucial for C23O activity in the hybrid forms of enzymes between XylEP and XylES. According to the crystal structure of XylEP, the C-terminal region is involved in the formation of a quaternary structure. Amino acid differences between HB3-C23O and HB4-C23O included the specific β-strand for oligomerization. Thus the quaternary structures of active C23Os, XylES, XylEP and HB4-C23O, as well as that of inactive HB3-C23O, were examined. Active enzymes XylES, XylEP and HB4-C23O were homotetrameric, while HB3-C23O existed only as a monomer. We concluded that hybrids of subgroups I.2.A and I.2.B were often inactive because of a defect in their oligomerization.

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The membrane domain of the human erythrocyte anion transport protein. Epitope mapping of a monoclonal antibody defines the location of a cytoplasmic loop near the C-terminus of the protein

Biochemical Journal ◽

10.1042/bj2720265 ◽

1990 ◽

Vol 272 (1) ◽

pp. 265-268 ◽

Cited By ~ 16

Author(s):

S D Wainwright ◽

W J Mawby ◽

M J A Tanner

Keyword(s):

Amino Acid ◽

Human Erythrocyte ◽

Protein Band ◽

Anion Transport ◽

Transport Protein ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

C Terminus ◽

Murine Monoclonal Antibodies ◽

Cytoplasmic Side

We have used synthetic peptides to study the location of the amino acid sequences in the human erythrocyte anion transport protein (band 3) which are recognized by four murine monoclonal antibodies, BRIC 130, 132, 154 and 155. These antibodies are known to react with epitopes in the protein which are on the cytoplasmic side of the membrane. The results suggest that the amino acid residues important for the reaction of BRIC 130 and BRIC 154/155 are located within amino acids 899-908 and 895-901 respectively in the cytoplasmic tail of the protein. The BRIC 132 epitope is located within amino acid residues 813-824. This is part of a surface loop in the protein which probably extends from residue 814 to residue 832 and is located on the cytoplasmic side of the membrane. These results provide direct evidence for the topographical location of a sequence in a poorly understood region of the protein.

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The distribution of physical, chemical and conformational properties in signal and nascent peptides

Biochemical Journal ◽

10.1042/bj2690691 ◽

1990 ◽

Vol 269 (3) ◽

pp. 691-696 ◽

Cited By ~ 23

Author(s):

M Prabhakaran

Keyword(s):

Amino Acid ◽

Sequence Data ◽

Graphical Analysis ◽

Amino Acid Sequences ◽

Signal Peptides ◽

Hydrophobic Core ◽

Amino Acid Residues ◽

Conformational Properties ◽

The Individual ◽

Translocated Proteins

Signal peptides play a major role in an as-yet-undefined way in the translocation of proteins across membranes. The sequential arrangement of the chemical, physical and conformational properties of the signal and nascent amino acid sequences of the translocated proteins has been compiled and analysed in the present study. The sequence data of 126 signal peptides of length between 18 and 21 residues form the basis of this study. The statistical distribution of the following properties was studied hydrophobicity, Mr, bulkiness, chromatographic index and preference for adopting alpha-helical, β-sheet and turn structures. The contribution of each property to the sequence arrangement was derived. A hydrophobic core sequence was found in all signal peptides investigated. The structural arrangement of the cleavage site was also clearly revealed by this study. Most of the physical properties of the individual sequences correlated (correlation coefficient approximately 0.4) very well with the average distribution. The preferred occupancy of amino acid residues in the signal and nascent sequences was also calculated and correlated with their property distribution. The periodic behaviour of the signal and nascent chains was revealed by calculating their hydrophobic moments for various repetitive conformations. A graphical analysis of average hydrophobic moments versus average hydrophobicity of peptides revealed the transmembrane characteristics of signal peptides and globular characteristics of the nascent peptides.

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Computational Analysis of Therapeutic Enzyme Uricase from Different Source Organisms

Current Proteomics ◽

10.2174/1570164616666190617165107 ◽

2020 ◽

Vol 17 (1) ◽

pp. 59-77

Author(s):

Anand Kumar Nelapati ◽

JagadeeshBabu PonnanEttiyappan

Keyword(s):

Uric Acid ◽

Amino Acid ◽

Sequence Alignment ◽

Multiple Sequence Alignment ◽

Protein Sequences ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Multiple Sequence ◽

Physiochemical Properties ◽

Pharmaceutical Industries

Background:Hyperuricemia and gout are the conditions, which is a response of accumulation of uric acid in the blood and urine. Uric acid is the product of purine metabolic pathway in humans. Uricase is a therapeutic enzyme that can enzymatically reduces the concentration of uric acid in serum and urine into more a soluble allantoin. Uricases are widely available in several sources like bacteria, fungi, yeast, plants and animals.Objective:The present study is aimed at elucidating the structure and physiochemical properties of uricase by insilico analysis.Methods:A total number of sixty amino acid sequences of uricase belongs to different sources were obtained from NCBI and different analysis like Multiple Sequence Alignment (MSA), homology search, phylogenetic relation, motif search, domain architecture and physiochemical properties including pI, EC, Ai, Ii, and were performed.Results:Multiple sequence alignment of all the selected protein sequences has exhibited distinct difference between bacterial, fungal, plant and animal sources based on the position-specific existence of conserved amino acid residues. The maximum homology of all the selected protein sequences is between 51-388. In singular category, homology is between 16-337 for bacterial uricase, 14-339 for fungal uricase, 12-317 for plants uricase, and 37-361 for animals uricase. The phylogenetic tree constructed based on the amino acid sequences disclosed clusters indicating that uricase is from different source. The physiochemical features revealed that the uricase amino acid residues are in between 300- 338 with a molecular weight as 33-39kDa and theoretical pI ranging from 4.95-8.88. The amino acid composition results showed that valine amino acid has a high average frequency of 8.79 percentage compared to different amino acids in all analyzed species.Conclusion:In the area of bioinformatics field, this work might be informative and a stepping-stone to other researchers to get an idea about the physicochemical features, evolutionary history and structural motifs of uricase that can be widely used in biotechnological and pharmaceutical industries. Therefore, the proposed in silico analysis can be considered for protein engineering work, as well as for gout therapy.

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Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium

Biochemical Journal ◽

10.1042/bj2510691 ◽

1988 ◽

Vol 251 (3) ◽

pp. 691-699 ◽

Cited By ~ 106

Author(s):

R W Olafson ◽

W D McCubbin ◽

C M Kay

Keyword(s):

Amino Acid ◽

Metal Binding ◽

Helical Structure ◽

Basic Amino Acid ◽

Cysteine Residues ◽

Amino Acid Residues ◽

C Terminus ◽

Empirical Relations ◽

Heavy Metal Binding ◽

Synechococcus Sp

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

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Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽

10.3390/cells10030715 ◽

2021 ◽

Vol 10 (3) ◽

pp. 715

Author(s):

Tamara Tomanić ◽

Claire Martin ◽

Holly Stefen ◽

Esmeralda Parić ◽

Peter Gunning ◽

...

Keyword(s):

Amino Acid ◽

Hippocampal Neurons ◽

Molecular Mechanisms ◽

Growth Cones ◽

Amino Acid Residues ◽

Terminal Amino Acid ◽

C Terminus ◽

Primary Mouse ◽

Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

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Bile-pigment formation from different leghaemoglobins. Methine-bridge specificity of coupled oxidation

Biochemical Journal ◽

10.1042/bj1760359 ◽

1978 ◽

Vol 176 (2) ◽

pp. 359-364 ◽

Cited By ~ 12

Author(s):

Päivi Lehtovaara ◽

Ulla Perttilä

Keyword(s):

Amino Acid ◽

Polypeptide Chain ◽

Broad Bean ◽

Amino Acid Sequences ◽

Second Step ◽

Bile Pigment ◽

Site Specificity ◽

Amino Acid Residues ◽

Reaction Step ◽

Pigment Formation

The coupled oxidation of leghaemoglobins with O2 and ascorbate yielded oxyleghaemoglobin in the first reaction step, and the second step was the degradation of haem characterized by an A675 increase. Leghaemoglobins were degraded to biliverdin isomers specifically, depending on the structure of the protein. The main leghaemoglobin components of Glycine (soya bean) and Phaseolus (kidney bean) were degraded to biliverdin mixtures containing about 50% of the β-form, about 30% of the α-form and about 20% of the δ-isomer, whereas the leghaemoglobin I components of Vicia (broad bean) and Pisum (pea) were degraded almost exclusively to the β-isomer, with traces of the α-isomer. The amino acid sequences of Glycine and Phaseolus leghaemoglobins resemble each other, as do those of Vicia and Pisum. The site specificity of bile-pigment formation from leghaemoglobins can be tentatively explained by specific differences in the amino acid sequences at those regions of the polypeptide chain that are in the vicinity of the appropriate methine bridges. The ligand-binding site in different leghaemoglobins may be outlined on the basis of the present results, supposing that the haem is degraded when a reduction product of haem-bound O2 reacts with a methine bridge of the haem, and that the bridge specificity is regulated by hindering amino acid residues that determine the location of the bound O2. The residue phenylalanine-CD1 appears to be further away from the haem plane or in a markedly more flexible position in leghaemoglobins than in mammalian globins. The haem-bound oxygen atom B, in Fe–O(A)–O(B), seems to be free to rotate in all directions except that of the γ-bridge in Glycine and Phaseolus leghaemoglobins, but its position in Vicia and Pisum leghaemoglobin I might be restricted to the direction of the β-methine bridge.

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Molecular cloning of two human liver 3 α-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder

Biochemical Journal ◽

10.1042/bj2990545 ◽

1994 ◽

Vol 299 (2) ◽

pp. 545-552 ◽

Cited By ~ 63

Author(s):

Y Deyashiki ◽

A Ogasawara ◽

T Nakayama ◽

M Nakanishi ◽

Y Miyabe ◽

...

Keyword(s):

Bile Acid ◽

Amino Acid ◽

Human Liver ◽

Polyclonal Antibodies ◽

Amino Acid Sequences ◽

Cdna Clones ◽

Amino Acid Residues ◽

Human Bile ◽

Coding Regions ◽

Computer Based

Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5′-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively.

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The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase

Biochemical Journal ◽

10.1042/bj1330805 ◽

1973 ◽

Vol 133 (4) ◽

pp. 805-819 ◽

Cited By ~ 12

Author(s):

Francesco Bossa ◽

Donatella Barra ◽

Massimo Carloni ◽

Paolo Fasella ◽

Francesca Riva ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Composition ◽

Acid Composition ◽

Primary Structure ◽

Aspartate Aminotransferase ◽

Heart Muscle ◽

Science And Technology ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Lending Library

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

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