Purification, Characterization and N-Terminal Sequence of Phosphoserine Aminotransferase from the Green Alga Scenedesmus obliquus, Mutant C-2 A′

1994 ◽  
Vol 49 (1-2) ◽  
pp. 63-69 ◽  
Author(s):  
M. Stolz ◽  
D. Dörnemann
Keyword(s):  
Scenedesmus Obliquus ◽  
Gel Filtration ◽  
Terminal Sequence ◽  
Gene Encoding ◽  
Chromatographic Techniques ◽  
Phosphoserine Aminotransferase ◽  
Cell Homogenate ◽  
N Terminus ◽  
Molecular Masses ◽  
Soluble Supernatant

Phosphoserine aminotransferase (EC 2.6.1.52), an enzyme of the “phosphorylated pathway” leading to the formation of serine, was purified from Scenedesmus obliquus, mutant C-2 A′. Purification started from the soluble supernatant of a crude cell homogenate and included different affinity and DEAE chromatographic techniques, as well as gel filtration. The purified phosphoserine aminotransferase was enriched 1537-fold and identified to be a homodimer with subunit molecular masses of 40 kDa, each. The absorption spectrum is consistent with the presence of pyridoxal-5-phosphate as cofactor. From the purified enzyme 18 amino acids of the N-terminus could be determined, showing at least 67% homology with the serC gene encoding phosphoserine aminotransferases from bacterial organisms.

scholarly journals Structural comparison between the mitochondrial aralkyl-CoA and arylacetyl-CoA N-acyltransferases

1992 ◽  
Vol 288 (1) ◽  
pp. 315-317 ◽  
Author(s):  
M Kelley ◽  
D A Vessey
Keyword(s):  
Sequence Analysis ◽  
Gel Filtration ◽  
Slight Modification ◽  
Terminal Sequence ◽  
Structural Comparison ◽  
Bovine Kidney ◽  
Chemical Cleavage ◽  
Sds Page ◽  
N Terminus ◽  
Methionine Residues

The aralkyl and arylacetyl transferases were purified to homogeneity from bovine kidney by a slight modification of a previous procedure. The M(r) of the arylacetyl transferase was estimated to be 33,500 by SDS/PAGE and that of the aralkyl transferase to be 33,750 by a combination of SDS/PAGE and gel-filtration analysis. N-Terminal-sequence analysis indicated a blocked N-terminus for the arylacetyl transferase and gave the following sequence for the aralkyl transferase: M-F-L-L-Q-G-A-Q-M-L-Q-M-L-E-K. Amino acid analysis revealed differences in composition between the two enzymes. Most notable was the fact that the aralkyl transferase had more methionine and leucine. This difference could be partially accounted for by assuming that the methionine-and-leucine-rich N-terminus was missing from the arylacetyl transferase. Chemical cleavage of the two enzymes at methionine residues using CNBr gave rise to several peptides for each enzyme. N-Terminal-sequence analysis of the 8000-M(r) peptide from the arylacetyl transferase gave a sequence with 69% similarity to the 9000-M(r) peptide from the aralkyl transferase. This was taken to indicate a common origin for the two enzymes.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals Differential activation mechanisms of two isoforms of Gcr1 transcription factor generated from spliced and un-spliced transcripts in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Seungwoo Cha ◽  
Chang Pyo Hong ◽  
Hyun Ah Kang ◽  
Ji-Sook Hahn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Target Genes ◽  
Gene Encoding ◽  
Metabolic Switch ◽  
Differential Activation ◽  
N Terminus ◽  
Activation Mechanisms ◽  
In Trans ◽  
Separate Activation

Abstract Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1U and Gcr1S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1U or Gcr1S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1U, inhibits dimerization of Gcr1U and even acts in trans to inhibit Gcr1S dimerization. The Gcr1S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1U and Gcr1S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.

scholarly journals Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology

1979 ◽  
Vol 183 (3) ◽  
pp. 615-622 ◽  
Author(s):  
M A Kerr
Keyword(s):  
Sequence Homology ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Terminal Sequence ◽  
Complement Components ◽  
Factor B ◽  
Terminal Residue ◽  
Covalently Linked

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.

scholarly journals Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis

1994 ◽  
Vol 297 (2) ◽  
pp. 351-357 ◽  
Author(s):  
A Lemassu ◽  
M Daffé
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Mimicry ◽  
Degradation Products ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Structural Features ◽  
Two Dimensional ◽  
Phagocytic Cells ◽  
Molecular Masses ◽  
Branched Structure

The cell envelope which surrounds pathogenic mycobacteria is postulated to be a defence barrier against phagocytic cells and its outermost constituents have a tendency to accumulate in the culture medium. The present work demonstrates that the exocellular material of Mycobacterium tuberculosis contains large amounts of polysaccharides with only traces, if any at all, of lipids. Three types of polysaccharides were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents. They consisted of D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 123, 13 and 4 kDa respectively. Their predominant structural features were determined by the characterization of the per-O-methyl derivatives of enzymic, acetolysis and Smith-degradation products and by 1H- and 13C-n.m.r. spectroscopy of the purified polysaccharides, using mono- and two-dimensional homonuclear chemical-shift correlated spectroscopy and two-dimensional heteronuclear (1H/13C) spectroscopy. The glucan which represented up to 90% of the polysaccharides was composed of repeating units of five or six-->4-alpha-D-Glcp-1--> residues and a -->4-alpha-D-Glcp substituted at position 6 with an alpha-D-Glcp, indicating a glycogen-like highly branched structure not related to the so-called polysaccharide-II previously identified in tuberculin. The arabinomannan consisted of a mannan segment composed of a -->6-alpha-D-Man-1--> core substituted at some positions 2 with an alpha-D-Manp. The arabinan termini of the arabinomannan were found to be extensively capped with mannosyl residues. The possibility that these polysaccharides contribute to the persistence of the tubercle bacillus in the macrophage by molecular mimicry is discussed.

Evaluation of dialysis treatment in uremic patients by gel filtration of serum

1990 ◽  
Vol 36 (11) ◽  
pp. 1906-1910 ◽  
Author(s):  
J Osada ◽  
T Gea ◽  
C Sanz ◽  
I Millan ◽  
J Botella
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Other ◽  
Control Group ◽  
Dialysis Treatment ◽  
Additional Information ◽  
Molecular Masses ◽  
Two Peaks

Abstract A group of substances of molecular masses between 300 and 1500 Da have been found to be toxic metabolites in patients with uremia. We determined the concentration in serum of these molecules in the following groups of patients: two hemodialyzed groups (one with cuprophane and the other with polyacrylonitrile dialyzers), one group treated with continuous ambulatory peritoneal dialysis, one group of nondialyzed azotemic patients, and one control group of healthy persons. Ultrafiltrates of the subjects' sera were fractionated on Sephadex G-15 followed by ion-exchange chromatography. Eluates were monitored by absorbance at 254 and 206 nm. Partially characterized peaks P1 and P2, obtained by gel filtration, correlated with the concentration of creatinine in serum; their concentrations were significantly (P less than 0.01) larger in hemodialyzed groups than in peritoneal dialyzed or in nondialyzed azotemic patients. After ion-exchange chromatography, two peaks (P'5 and P'6) correlated with serum creatinine and also were larger in hemodialyzed patients than in the other groups. Apparently, adequate discrimination is obtained by gel-filtration analysis and further analysis by ion-exchange chromatography does not provide additional information in most of the affected patients.

Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease

2012 ◽  
Vol 446 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Anders Tryggvesson ◽  
Frida M. Ståhlberg ◽  
Axel Mogk ◽  
Kornelius Zeth ◽  
Adrian K. Clarke
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Terminal Sequence ◽  
Core Complex ◽  
Clp Protease ◽  
E Coli ◽  
Loop Region ◽  
N Terminus ◽  
Specific Association ◽  
The Stability

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC–ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

scholarly journals Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics ofIgnicoccusSpecies

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Lydia J. Kreuter ◽  
Andrea Weinfurtner ◽  
Alexander Ziegler ◽  
Julia Weigl ◽  
Jan Hoffmann ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Cellular Membrane ◽  
Content Type ◽  
The Pacific ◽  
Native Electrophoresis ◽  
Molecular Masses ◽  
The Pacific Ocean

ABSTRACTIn this study, the ATP synthase ofIgnicoccus hospitaliswas purified, characterized, and structurally compared to the respective enzymes of the otherIgnicoccusspecies, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genusIgnicoccuscomprises three described species, i.e.,I. hospitalisandIgnicoccus islandicusfrom hot marine sediments near Iceland andIgnicoccus pacificusfrom a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC).I. hospitalisis the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeonNanoarchaeum equitans.I. hospitalisgrows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome ofI. hospitalisencodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximalin vitroactivity of theI. hospitalisenzyme was measured around pH 6, the optimal stability of the A1AOcomplex seemed to be at pH 9. Interestingly, the soluble A1subcomplexes of the differentIgnicoccusspecies exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCETheCrenarchaeotarepresent one of the major phyla within theArchaeadomain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of theCrenarchaeotauntil now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subjectI. hospitalishas a particular importance among crenarchaeotes, since it is the only known host ofN. equitans. The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.

Kinetic Characteristics, Substrate Specificity and Catalytic Properties of Phosphoserine Aminotransferase from the Green Alga Scenedesmus obliquus, Mutant C-2A

1995 ◽  
Vol 50 (9-10) ◽  
pp. 630-637 ◽  
Author(s):  
M. Stolz ◽  
D. Dörnemann
Keyword(s):  
Catalytic Properties ◽  
Scenedesmus Obliquus ◽  
Initial Reaction ◽  
Kinetic Characteristics ◽  
Forward Reaction ◽  
Phosphoserine Aminotransferase ◽  
Substrate Analogues ◽  
Ping Pong ◽  
The One ◽  
Competitive Substrate

Abstract Phosphoserine am inotransferase (EC 2.6.1.52) has been purified from Scenedesmus obliquus. mutant C -2A′, as reported previously (Stolz and Dörnemann, 1994). The current studies on its catalytic properties, involving initial reaction velocities as a function of the phosphoserine concentration at various fixed concentrations of 2-oxoglutarate as aminoacceptor, indicate a bi-bi ping pong mechanism . The application of a variety of substrate analogues of phosphoserine revealed no significant metabolisation of these com pounds and thus a considerable specificity of the enzyme. 4,5-dioxovalerate with glutamate as am inodonor is effective as competitive substrate to phosphohydroxypyruvate in the forward reaction and yields 5-am inolevulinate. 4.5-Dioxovalerate and glutam ate-1-semialdehyde can both serve as competitive aminoacceptor in the reverse reaction with phosphoserine and as substrate with 2-oxoglutarate as am inoacceptor. Comparison of the phosphoserine transamination with the transamination of 4,5-dioxovalerate revealed for both reactions a pH -optim um of 6 .8 -7 .0 in Mes/Bis-Trisbuffer. How ever, the Km-values and the Vmax for phosphoserine and 2-oxoglutarate on the one side, and 4,5-dioxovalerate and glutamate on the other were found to differ by orders of magnitude

Purification and characterization of a lectin from Xanthomonas campestris NCIM 5028

1996 ◽  
Vol 42 (6) ◽  
pp. 609-612 ◽  
Author(s):  
Bhagyashree Joshi ◽  
Jayant M. Khire ◽  
Hephzibah SivaRaman ◽  
M. Islam Khan
Keyword(s):  
Molecular Mass ◽  
Host Plant ◽  
Xanthomonas Campestris ◽  
Simple Procedure ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Molecular Masses ◽  
Lectin Purification

A lectin was isolated from culture filtrates of Xanthomonas campestris NCIM 5028, by a simple procedure of hydrophobic chromatography on phenyl-Sepharose after ammonium sulphate precipitation. The lectin was a heterodimer, with subunit molecular masses of 30 000 and 28 000. Gel filtration on S-300 column, calibrated with markers, showed its molecular mass to be approximately 70 000. Its isoelectric point was 7.2. The agglutination of the rabbit erythrocytes by the lectin was inhibited by fetuin glycopeptides and host plant (Brassica oleracea) extracts.Key words: Xanthomonas campestris, lectin, purification.

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