scholarly journals The Human Cytomegalovirus UL74 Gene Encodes the Third Component of the Glycoprotein H-Glycoprotein L-Containing Envelope Complex

1998 ◽  
Vol 72 (10) ◽  
pp. 8191-8197 ◽  
Author(s):  
Mary T. Huber ◽  
Teresa Compton
Keyword(s):  
Amino Acid ◽  
Human Cytomegalovirus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Protein Species ◽  
Gene Encoding ◽  
Peptide Backbone ◽  
Glycoprotein H ◽  
Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) gCIII envelope complex is composed of glycoprotein H (gH; gpUL75), glycoprotein L (gL; gpUL115), and a third, 125-kDa protein not related to gH or gL (M. T. Huber and T. Compton, J. Virol. 71:5391–5398, 1997; L. Li, J. A. Nelson, and W. J. Britt, J. Virol. 71:3090–3097, 1997). Glycosidase digestion analysis demonstrated that the 125-kDa protein was a glycoprotein containing ca. 60 kDa of N-linked oligosaccharides on a peptide backbone of 65 kDa or less. Based on these biochemical characteristics, two HCMV open reading frames, UL74 and TRL/IRL12, were identified as candidate genes for the 125-kDa glycoprotein. To identify the gene encoding the 125-kDa glycoprotein, we purified the gCIII complex, separated the components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected gH and the 125-kDa glycoprotein to amino acid microsequence analysis. Microsequencing of an internal peptide derived from purified 125-kDa glycoprotein yielded the amino acid sequence LYVGPTK. A FASTA search revealed an exact match of this sequence to amino acids 188 to 195 of the predicted product of the candidate gene UL74, which we have designated glycoprotein O (gO). Anti-gO antibodies reacted in immunoblots with a protein species migrating at ca. 100 to 125 kDa in lysates of HCMV-infected cells and with 100- and 125-kDa protein species in purified virions. Anti-gO antibodies also immunoprecipitated the gCIII complex and recognized the 125-kDa glycoprotein component of the gCIII complex. Positional homologs of the UL74 gene were found in other betaherpesviruses, and comparisons of the predicted products of the UL74 homolog genes demonstrated a number of conserved biochemical features.

scholarly journals Microbial Proline 4-Hydroxylase Screening and Gene Cloning

1999 ◽  
Vol 65 (9) ◽  
pp. 4028-4031 ◽  
Author(s):  
Takeshi Shibasaki ◽  
Hideo Mori ◽  
Shigeru Chiba ◽  
Akio Ozaki
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Genomic Library ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Original Strain ◽  
Degenerate Primers ◽  
E Coli ◽  
Dna Fragment

ABSTRACT Microbial proline 4-hydroxylases, which hydroxylate freel-proline totrans-4-hydroxy-l-proline, were screened in order to establish an industrial system for biotransformation of l-proline totrans-4-hydroxy-l-proline. Enzyme activities were detected in eight strains, including strains ofDactylosporangium spp. and Amycolatopsis spp. The Dactylosporangium sp. strain RH1 enzyme was partially purified 3,300-fold and was estimated to be a monomer polypeptide with an apparent molecular mass of 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degenerate primers based on the N-terminal amino acid sequence of the 31-kDa polypeptide were synthesized in order to amplify the corresponding 71-bp DNA fragment. A 5.5-kbp DNA fragment was isolated by using the 71-bp fragment labeled with digoxigenin as a probe for a genomic library ofDactylosporangium sp. strain RH1 constructed inEscherichia coli. One of the open reading frames found in the cloned DNA, which encoded a 272-amino-acid polypeptide (molecular mass, 29,715 daltons), was thought to be a proline 4-hydroxylase gene. The gene was expressed in E. coli as a fused protein with the N-terminal 34 amino acids of the β-galactosidase α-fragment. The E. coli recombinant exhibited proline 4-hydroxylase activity that was 13.6-fold higher than the activity in the original strain, Dactylosporangium sp. strain RH1. No homology was detected with other 2-oxoglutarate-dependent dioxygenases when databases were searched; however, the histidine motif conserved in 2-oxoglutarate-dependent dioxygenases was found in the gene.

scholarly journals Characterization of a Thermostable d-Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1

2003 ◽  
Vol 69 (2) ◽  
pp. 980-986 ◽  
Author(s):  
Dae Heoun Baek ◽  
Seok-Joon Kwon ◽  
Seung-Pyo Hong ◽  
Mi-Sun Kwak ◽  
Mi-Hwa Lee ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Optimum Temperature ◽  
Gene Encoding ◽  
Ph Range

ABSTRACT A gene encoding a new thermostable d-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards d-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards l-amino acid amides, d-amino acid-containing peptides, and NH2-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co2+ and Mn2+. The k cat/Km for d-alaninamide was measured as 544.4 ± 5.5 mM−1 min−1 at 50°C with 1 mM Co2+.

scholarly journals Identification of Mur, an Atypical Peptidoglycan Hydrolase Derived from Leuconostoc citreum

2001 ◽  
Vol 67 (2) ◽  
pp. 858-864 ◽  
Author(s):  
Recep Cibik ◽  
Patrick Tailliez ◽  
Philippe Langella ◽  
Marie-Pierre Chapot-Chartier
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chimeric Protein ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Calculated Molecular Mass ◽  
Cell Wall Binding ◽  
Putative Signal Peptide ◽  
Leuconostoc Citreum

ABSTRACT A gene encoding a protein homologous to known bacterialN-acetyl-muramidases has been cloned fromLeuconostoc citreum by a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases, L. citreum Mur protein does not contain amino acid repeats involved in cell wall binding. The purifiedL. citreum Mur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion of L. citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis. Expression of the Mur-cA fusion protein was able to complement an acmA mutation inL. lactis; normal cell separation after cell division was restored by Mur-cA expression.

scholarly journals Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

2001 ◽  
Vol 67 (6) ◽  
pp. 2445-2452 ◽  
Author(s):  
Yuji Kannan ◽  
Yuichi Koga ◽  
Yohei Inoue ◽  
Mitsuru Haruki ◽  
Masahiro Takagi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Heat Inactivation ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Hyperthermophilic Archaeon

ABSTRACT The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensissubtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding,T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly−82 to Gly316), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests thatT. kodakaraensis subtilisin exists in a monomeric form.T. kodakaraensis subtilisin hydrolyzed the synthetic substrateN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca2+ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.

scholarly journals Infection of Macrophage-Like THP-1 Cells withMycobacterium avium Results in a Decrease in Their Ability to Phosphorylate Nucleolin

2000 ◽  
Vol 68 (6) ◽  
pp. 3121-3128 ◽  
Author(s):  
Rodolfo C. Garcia ◽  
Elena Banfi ◽  
Maria G. Pittis
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Host Response ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequencing ◽  
Intact Cells ◽  
Infected Cells

ABSTRACT This study of the phosphorylation ability of macrophage-like cells upon infection with Mycobacterium avium was undertaken to establish potential targets of the interference with host response mechanisms. Cytosolic and membrane fractions from noninfected and infected cells were incubated with [γ-32P]ATP, in the presence of Mg2+ and the absence of Ca2+, and the patterns of phosphoproteins synthesized were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lower levels of a 110-kDa phosphoprotein were observed in association with cytosolic fractions from mycobacterium-infected cells compared to noninfected cells or cells treated with lipopolysaccharide or having ingestedEscherichia coli or killed M. avium. The 110-kDa phosphoprotein was present in the soluble fraction (230,000 ×g supernatant) after the kinase incubation, from where it was partially purified and identified as phosphonucleolin by amino acid sequencing. The decrease in nucleolin phosphorylation observed was not related to changes in the cytosolic or membrane levels of this protein, and was detected also in the cytosolic fraction of32P-labeled intact cells.

scholarly journals Evidence for the Presence of an F-Type ATP Synthase Involved in Sulfate Respiration in Desulfovibrio vulgaris

2000 ◽  
Vol 182 (8) ◽  
pp. 2200-2206 ◽  
Author(s):  
Kiyoshi Ozawa ◽  
Takanori Meikari ◽  
Ken Motohashi ◽  
Masasuke Yoshida ◽  
Hideo Akutsu
Keyword(s):  
Amino Acid ◽  
Atp Synthase ◽  
Cytoplasmic Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Level ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Desulfovibrio Vulgaris ◽  
F Cells

ABSTRACT Using a library of genomic DNA from Desulfovibrio vulgaris Miyazaki F, a strict anaerobe, and two synthetic deoxyoligonucleotide probes designed for F-type ATPases, the genes for open reading frames (ORFs) 1 to 5 were cloned and sequenced. The predicted protein sequences of the gene products indicate that they are composed of 172, 488, 294, 471, and 134 amino acids, respectively, and that they share considerable identity at the amino acid level with δ, α, γ, β, and ɛ subunits found in other F-type ATPases, respectively. Furthermore, a component carrying ATPase activity was partially purified from the cytoplasmic membrane fraction of theD. vulgaris Miyazaki F cells. The N-terminal amino acid sequences of three major polypeptides separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis were identical to those of the products predicted by the sequences of ORF-2, ORF-3, and ORF-4, suggesting that an F-type ATPase is functioning in the D. vulgaris Miyazaki F cytoplasmic membrane. The amount of the F-type ATPase produced in the D. vulgaris Miyazaki F cells is similar to that in the Escherichia coli cells cultured aerobically. It indicates that the enzyme works as an ATP synthase in the D. vulgaris Miyazaki F cells in connection with sulfate respiration.

scholarly journals Discovery of a Second Form of Tripartite Complex Containing gH-gL of Human Herpesvirus 6 and Observations on CD46

2004 ◽  
Vol 78 (9) ◽  
pp. 4609-4616 ◽  
Author(s):  
Yasuko Mori ◽  
Pilailuk Akkapaiboon ◽  
Sayoko Yonemoto ◽  
Masato Koike ◽  
Masaya Takemoto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Cytomegalovirus ◽  
Human Herpesvirus ◽  
Viral Envelope ◽  
Amino Terminus ◽  
Protein Species ◽  
Human Herpesvirus 6 ◽  
Glycoprotein H ◽  
Infected Cells ◽  
Herpesvirus 6

ABSTRACT The human herpesvirus 6 (HHV-6) glycoprotein H (gH)-glycoprotein L (gL) complex associates with glycoprotein Q (gQ) (Y. Mori, P. Akkapaiboon, X. Yang, and K. Yamanishi, J. Virol. 77:2452-2458, 2003), and the gH-gL-gQ complex interacts with human CD46 (Y. Mori, X. Yang, P. Akkapaiboon, T. Okuno, and K. Yamanishi, J. Virol. 77:4992-4999, 2003). Here, we show that the HHV-6 U47 gene, which is a positional homolog of the human cytomegalovirus glycoprotein O (gO) gene, encodes a third component of the HHV-6 gH-gL-containing envelope complex. A monoclonal antibody (MAb) against the amino terminus of HHV-6 gO reacted in immunoblots with protein species migrating at 120 to 130 kDa and 74 to 80 kDa in lysates of HHV-6-infected cells and with a 74- to 80-kDa protein species in purified virions. The 80-kDa form of gO was coimmunoprecipitated with an anti-gH MAb, but an anti-gQ MAb, which coimmunoprecipitated gH, did not coprecipitate gO. Furthermore, the gH-gL-gO complex did not bind to human CD46, indicating that the complex was not a ligand for CD46. These findings suggested that the viral envelope contains at least two kinds of tripartite complexes, gH-gL-gQ and gH-gL-gO, and that the gH-gL-gO complex may play a role different from that of gH-gL-gQ during viral infection. This is the first report of two kinds of gH-gL complexes on the viral envelope in a member of the herpesvirus family.

scholarly journals SO-LAAO, a Novel l-Amino Acid Oxidase That Enables Streptococcus oligofermentans To Outcompete Streptococcus mutans by Generating H2O2 from Peptone

2008 ◽  
Vol 190 (13) ◽  
pp. 4716-4721 ◽  
Author(s):  
Huichun Tong ◽  
Wei Chen ◽  
Wenyuan Shi ◽  
Fengxia Qi ◽  
Xiuzhu Dong
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Streptococcus Mutans ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Binding Motif ◽  
Acid Oxidase ◽  
Lactate Oxidase ◽  
Amino Acid Oxidase

ABSTRACT We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H2O2) through lactate oxidase activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone-rich plates. Further studies demonstrated that the H2O2 produced on peptone by S. oligofermentans was mainly derived from seven l-amino acids, i.e., l-aspartic acid, l-tryptophan, l-lysine, l-isoleucine, l-arginine, l-asparagine, and l-glutamine, indicating the possible existence of l-amino acid oxidase (LAAO) that can produce H2O2 from l-amino acids. Through searching the S. oligofermentans genome for open reading frames with a conserved flavin adenine dinucleotide binding motif that exists in the known LAAOs, including those of snake venom, fungi, and bacteria, a putative LAAO gene, assigned as aaoS o , was cloned and overexpressed in Escherichia coli. The purified protein, SO-LAAO, showed a molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and catalyzed H2O2 formation from the seven l-amino acids determined above, thus confirming its LAAO activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known LAAOs in both substrate profile and sequence, suggesting that it could represent a novel LAAO. An aaoS o mutant of S. oligofermentans did lose H2O2 formation from the seven l-amino acids, further verifying its function as an LAAO. Furthermore, the inhibition by S. oligofermentans of S. mutans in a peptone-rich mixed-species biofilm was greatly reduced for the aaoS o mutant, indicating the gene's importance in interspecies competition.

scholarly journals The molybdenum–iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ‘mapping’

1976 ◽  
Vol 155 (2) ◽  
pp. 383-389 ◽  
Author(s):  
C Kennedy ◽  
R R. Eady ◽  
E Kondorosi ◽  
D K Rekosh
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Sodium Dodecyl Sulphate ◽  
Azotobacter Vinelandii ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Content ◽  
Rhizobium Japonicum ◽  
Fe Protein

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide ‘maps’ after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

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