scholarly journals The Polarity of an Amino Acid at Position 1891 of Severe Fever with Thrombocytopenia Syndrome Virus L Protein Is Critical for the Polymerase Activity

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 33
Author(s):  
Kisho Noda ◽  
Yoshimi Tsuda ◽  
Fumiya Kozawa ◽  
Manabu Igarashi ◽  
Kenta Shimizu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cytopathic Effect ◽  
Amino Acid Position ◽  
Polymerase Activity ◽  
Protein Activity ◽  
L Protein ◽  
A Cell ◽  
Genome Transcription ◽  
Severe Fever

Severe fever with thrombocytopenia syndrome virus subclone B7 shows strong plaque formation and cytopathic effect induction compared with other subclones and the parental strain YG1. Compared to YG1 and the other subclones, only B7 possesses a single substitution in the L protein at the amino acid position 1891, in which N is changed to K (N1891K). In this study, we evaluate the effects of this mutation on L protein activity via a cell-based minigenome assay. Substitutions of N with basic amino acids (K or R) enhanced polymerase activity, while substitutions with an acidic amino acid (E) decreased this activity. Mutation to other neutral amino acids showed no significant effect on activity. These results suggest that the characteristic of the amino acid at position 1891 of the L protein are critical for its function, especially with respect to the charge status. Our data indicate that this C-terminal domain of the L protein may be crucial to its functions in genome transcription and viral replication.

scholarly journals Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

1986 ◽  
Vol 6 (10) ◽  
pp. 3470-3480 ◽  
Author(s):  
E Moran ◽  
B Zerler ◽  
T M Harrison ◽  
M B Mathews
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Point Mutations ◽  
Apparent Molecular Weight ◽  
Amino Acid Position ◽  
Cysteine Residue ◽  
Transformation Function ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

Isolation and partial characterization of amphibian tyrosine oxidase polysomes

Development ◽  
1970 ◽  
Vol 24 (1) ◽  
pp. 109-118
Author(s):  
E. L. Triplett ◽  
R. Herzog ◽  
L. P. Russell
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Enzymic Activity ◽  
Antigenic Determinants ◽  
Free System ◽  
A Cell ◽  
Generating System ◽  
Soluble Components

A population of polysomes isolated from frogskinis capable of supporting protein synthesis in a cell-free system containing an energy generating system, ‘soluble components’, and amino acids. These polysomes catalyse the oxidation of DOPA after gentle trypsinization, and they also have antigenic determinants attributable to tyrosine oxidase. Skin polysomes sedimented in 10–30 % sucrose gradients contain tyrosine oxidase peaks of enzymic activity at the bottom and top of the tube and in the 250 S regions. A peak of tyrosine oxidase antigenic acitvity is found in the 250–350S region of the gradient. Polysomes resolved on the gradient retain the ability to support protein synthesis in a cellfree system. All 250–350S particles capable of supporting the incorporation of [14C]amino acid into tyrosine oxidase are precipitable with tyrosine oxidase antibodies. It is probable that 250–350S tyrosine oxidase antibody precipitates contain only polysomes for this protein.

scholarly journals Incorporation of 3H from δ-(l-α-amino (4,5-3H)adipyl)-l-cysteinyl-d-(4,4-3H)valine into isopenicillin N

1979 ◽  
Vol 184 (2) ◽  
pp. 421-426 ◽  
Author(s):  
J O'Sullivan ◽  
R C Bleaney ◽  
J A Huddleston ◽  
E P Abraham
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Adipic Acid ◽  
Experimental Error ◽  
Acid Oxidase ◽  
Cephalosporium Acremonium ◽  
Amino Acid Oxidase ◽  
Free System ◽  
A Cell ◽  
Isopenicillin N

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.

scholarly journals Characterization of a Bifidobacterium longum BORI Dipeptidase Belonging to the U34 Family

2007 ◽  
Vol 73 (17) ◽  
pp. 5598-5606 ◽  
Author(s):  
Jeong Min Seo ◽  
Geun Eog Ji ◽  
Sang Hee Cho ◽  
Myung Soo Park ◽  
Hyong Joo Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mass ◽  
Bifidobacterium Longum ◽  
Deae Cellulose ◽  
Cell Extract ◽  
Coding Region ◽  
Tryptic Fragment ◽  
C Terminus ◽  
A Cell

ABSTRACT A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50°C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a β-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.

Peptidsynthese im zellfreien System unter dem Einfluß von Hefe-Ribonucleinsäure

1968 ◽  
Vol 23 (1) ◽  
pp. 50-58 ◽  
Author(s):  
W. Duntze ◽  
W. Atzpodien ◽  
B. Ulrich ◽  
H. Holzer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ribosomal Rna ◽  
Gel Filtration ◽  
Amino Acid Incorporation ◽  
Free System ◽  
Total Rna ◽  
Acid Incorporation ◽  
Yeast Ribosomes ◽  
A Cell

Total RNA from yeast stimulates the incorporation of 14C-amino acids into material insoluble in hot trichloroacetic acid in a cell-free E. coli system. In a sucrose gradient the stimulating RNA fraction sediments together with the 28 S fraction of ribosomal RNA. RNA from isolated yeast ribosomes preincubated with RNase was active in amino acid incorporation as well.Preincubation of ribosomal RNA at 70° resulted in an increased incorporation activity of the RNA. However, attempts to separate an active messenger fraction from total RNA as well as from 28 S RNA by heating were unsuccessful. The presented data indicate that ribosomal RNA itself is active in cell-free amino acid incorporation.By hydrolysis of the incorporation products it could be shown that the 14C-amino acids used in the cell free system were incorporated into peptides. The bulk of the radioactive peptides had a molecular weight below 2000 as estimated by Sephadex gel filtration.

scholarly journals Aichi Virus 2A Protein Is Involved in Viral RNA Replication

2008 ◽  
Vol 82 (19) ◽  
pp. 9765-9769 ◽  
Author(s):  
Jun Sasaki ◽  
Koki Taniguchi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Replication ◽  
Vero Cells ◽  
Cellular Protein ◽  
The Other ◽  
Aichi Virus ◽  
Positive Strand ◽  
Free Translation ◽  
A Cell

ABSTRACT The Aichi virus 2A protein is not a protease, unlike many other picornavirus 2A proteins, and it is related to a cellular protein, H-rev107. Here, we examined the replication properties of two 2A mutants in Vero cells and a cell-free translation/replication system. In one mutant, amino acids 36 to 126 were replaced with an unrelated amino acid sequence. In the other mutant, the NC motif conserved in the H-rev107 family of proteins was changed to alanine residues. The two mutations abolished virus replication in cells. The mutations affected both negative- and positive-strand synthesis, the defect in positive-strand synthesis being more severe than that in negative-strand synthesis.

scholarly journals Amino Acids 785, 787 of the Na+/H+ Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation

2021 ◽  
Vol 22 (21) ◽  
pp. 11349
Author(s):  
Xiuju Li ◽  
Tommy Tu ◽  
Sicheng Quan ◽  
Francisco J. Quintero ◽  
Richard Fahlman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Apparent Molecular Weight ◽  
Slight Reduction ◽  
Membrane Domain ◽  
Protein Activity ◽  
Extracellular Sodium ◽  
Vitro Protein

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser785 and Ser787, that were similar in location and context to two amino acids of the Arabidopsis Na+/H+ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783–790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.

Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes

1986 ◽  
Vol 6 (10) ◽  
pp. 3470-3480
Author(s):  
E Moran ◽  
B Zerler ◽  
T M Harrison ◽  
M B Mathews
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Point Mutations ◽  
Apparent Molecular Weight ◽  
Amino Acid Position ◽  
Cysteine Residue ◽  
Transformation Function ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

scholarly journals Five new mutations in the uroporphyrinogen decarboxylase gene identified in families with cutaneous porphyria

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3589-3600 ◽  
Author(s):  
JF McManus ◽  
CG Begley ◽  
S Sassa ◽  
S Ratnaike
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Stop Codon ◽  
Porphyria Cutanea Tarda ◽  
Premature Stop Codon ◽  
Amino Acid Position ◽  
Uroporphyrinogen Decarboxylase ◽  
The Impact ◽  
Exon 10 ◽  
New Mutations

We describe five new mutations in the uroporphyrinogen decarboxylase (UROD) gene. All mutations were observed in conjunction with decreased erythrocyte UROD and clinical familial porphyria cutanea tarda (fPCT), (four families) or hepatoerythropoietic porphyria (HEP), (one family). The fPCT mutations included three point mutations that resulted in amino acid substitutions: a lysine to glutamine at amino acid position 253 (exon 7); a glycine to arginine at position 318 (exon 10); an isoleucine to threonine at position 334 (exon 10). The lysine to glutamine at amino acid position 253 was found in conjunction with a single C nucleotide deletion in exon 8 on the same allele of the UROD gene in the same family. This deletion resulted in a shift in the reading frame and the introduction of a premature stop codon 8 amino acids downstream. In the fourth family, a 31-bp deletion (nucleotides 828–858: exon 8) of the coding region, resulted in a frameshift and the introduction of a stop codon 19 amino acids downstream. A point mutation was observed in an individual diagnosed with HEP, resulting in an alanine to glycine change at amino acid position 80 and was present on both alleles. All mutations were confirmed in at least one other family member. The impact of these mutations on the function of the UROD protein was examined using in vitro protein expression and with activity assessed using pentacarboxylic acid porphyrinogen I as a substrate for UROD. Although three mutations reduced UROD activity to < 15% of normal, one resulted in a UROD protein with 50% functional activity and the other had near normal activity. These results indicate that many different genetic lesions of the UROD gene are associated with fPCT.

scholarly journals Caenorhabditis elegans LET-767 is able to metabolize androgens and estrogens and likely shares common ancestor with human types 3 and 12 17β-hydroxysteroid dehydrogenases

2007 ◽  
Vol 195 (2) ◽  
pp. 271-279 ◽  
Author(s):  
Serge Desnoyers ◽  
Pierre-Gilles Blanchard ◽  
Jean-François St-Laurent ◽  
Steve N Gagnon ◽  
David L Baillie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Common Ancestor ◽  
Amino Acid Position ◽  
Substrate Selectivity ◽  
Hydroxysteroid Dehydrogenases ◽  
High Conservation ◽  
Function Relationship ◽  
Relationship Of

Mutations that inactivate LET-767 are shown to affect growth, reproduction, and development in Caenorhabditis elegans. Sequence analysis indicates that LET-767 shares the highest homology with human types 3 and 12 17β-hydroxysteroid dehydrogenases (17β-HSD3 and 12). Using LET-767 transiently transfected into human embryonic kidney-293 cells, we have found that the enzyme catalyzes the transformation of both 4-androstenedione into testosterone and estrone into estradiol, similar to that of mouse 17β-HSD12 but different from human and primate enzymes that catalyze the transformation of estrone into estradiol. Previously, we have shown that amino acid F234 in human 17β-HSD12 is responsible for the selectivity of the enzyme toward estrogens. To assess whether this amino acid position 234 in LET-767 could play a role in androgen–estrogen selectivity, we have changed the methionine M234 in LET-767 into F. The results show that the M234F change causes the loss of the ability to transform androstenedione into testosterone, while conserving the ability to transform estrone into estradiol, thus confirming the role of amino acid position 234 in substrate selectivity. To further analyze the structure–function relationship of this enzyme, we have changed the three amino acids corresponding to lethal mutations in let-767 gene. The data show that these mutations strongly affect the ability of LET-767 to convert estrone in to estradiol and abolish its ability to transform androstenedione into testosterone. The high conservation of the active site and amino acids responsible for enzymatic activity and substrate selectivity strongly suggests that LET-767 shares a common ancestor with human 17β-HSD3 and 12.

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