Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 15 de 15
Filter
Add more filters










Publication year range
1.
Anal Biochem ; 600: 113699, 2020 07 01.
Article in English | MEDLINE | ID: mdl-32335063

ABSTRACT

Blood coagulation factor XIII-A (FXIII-A), a member of the transglutaminase enzyme family, is best known for its fibrin clot stabilizing function during blood coagulation. It possesses amine incorporating and protein crosslinking transamidase activities, but it is also able to cleave the previously formed isopeptide bond by its isopeptidase activity. Our aim was to develop a protein-based assay for better characterization of FXIII-A isopeptidase activity. The first attempt applying the crosslinked D-dimer of fibrin as a substrate was not successful because of poor reproducibility. Then, the principle of an earlier published anisotropy based activity assay was adapted for the measurement of FXIII-A isopeptidase activity. After crosslinking the fluorescently labelled α2-antiplasmin derived peptide and S100A4(GST) lysine donor protein, this protease-resistant γ-glutamyl-ε-lysine isopeptide bond containing protein-peptide product was applied as a substrate for FXIII-A. Using this substrate and detecting decreasing anisotropy, kinetic measurement of FXIII-A isopeptidase activity was achieved at high sensitivity even in a complex biological sample and in the presence of inhibitor.


Subject(s)
Carbon-Nitrogen Lyases/metabolism , Factor XIIIa/metabolism , Anisotropy , Carbon-Nitrogen Lyases/chemistry , Carbon-Nitrogen Lyases/isolation & purification , Factor XIIIa/chemistry , Fluorescence , Humans
2.
Methods Enzymol ; 606: 95-118, 2018.
Article in English | MEDLINE | ID: mdl-30097106

ABSTRACT

7-Carboxy-7-deazaguanine (CDG) is a common intermediate in the biosynthesis of 7-deazapurine-containing natural products. The biosynthesis of CDG from GTP requires three enzymes: GTP cyclohydrolase I, 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) synthase, and CDG synthase (QueE). QueE is a member of the radical S-adenosyl-l-methionine (SAM) superfamily and catalyzes the SAM-dependent radical-mediated ring contraction of CPH4 to generate CDG. This chapter focuses on methods to reconstitute the activity of QueE in vitro.


Subject(s)
Bacterial Proteins/isolation & purification , Biological Products/metabolism , Carbon-Nitrogen Lyases/isolation & purification , Purines/metabolism , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Biocatalysis , Carbon-Nitrogen Lyases/metabolism , Free Radicals/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , S-Adenosylmethionine/metabolism
3.
Biomed Khim ; 60(3): 332-7, 2014.
Article in Russian | MEDLINE | ID: mdl-25019395

ABSTRACT

From the highly purified but lowly active recombinant protein Destabilas-Lysozyme (Dest-Lys) by use cation-exchange column TSK CM 3-SW chromatography, it was separated non-active fraction IV, contained 90% of protein. Fractions I, II and III, represented proteins with lysozyme and isopeptidase activities. Their lysozyme activity correlates with the activity of natural Des-Lys. The ratio of the activities in fractions I - III is such, that maximal lysozyme activity is concentrated in fraction III, isopeptidase - in fraction I. It is discussed the possibility of Dest-Lys different functions regulation is depended on the formation of protein complex forms.


Subject(s)
Carbon-Nitrogen Lyases/isolation & purification , Endopeptidases/isolation & purification , Fibrinolytic Agents/isolation & purification , Hirudo medicinalis/chemistry , Muramidase/isolation & purification , Animals , Carbon-Nitrogen Lyases/chemistry , Carbon-Nitrogen Lyases/metabolism , Chromatography, Ion Exchange , Endopeptidases/chemistry , Endopeptidases/metabolism , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/metabolism , Hirudo medicinalis/enzymology , Kinetics , Muramidase/chemistry , Muramidase/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity
4.
Plant Physiol ; 164(2): 922-34, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24351687

ABSTRACT

The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1,520-bp complementary DNA (cDNA) for mimosinase from L. leucocephala and characterized the encoded enzyme for mimosine-degrading activity. The deduced amino acid sequence of the coding region of the cDNA was predicted to have a chloroplast transit peptide. The nucleotide sequence, excluding the sequence for the chloroplast transit peptide, was codon optimized and expressed in Escherichia coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found to be identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by electrospray ionization-tandem mass spectrometry. The enzyme activity requires pyridoxal 5'-phosphate but not α-keto acid; therefore, the enzyme is not an aminotransferase. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products. The dependence of the enzyme on pyridoxal 5'-phosphate and the production of 3H4P with the release of ammonia indicate that it is a carbon-nitrogen lyase. It was found to be highly efficient and specific in catalyzing mimosine degradation, with apparent Km and Vmax values of 1.16×10(-4) m and 5.05×10(-5) mol s(-1) mg(-1), respectively. The presence of other aromatic amino acids, including l-tyrosine, l-phenylalanine, and l-tryptophan, in the reaction did not show any competitive inhibition. The isolation of the mimosinase cDNA and the biochemical characterization of the recombinant enzyme will be useful in developing transgenic L. leucocephala with reduced mimosine content in the future.


Subject(s)
Biocatalysis , Carbon-Nitrogen Lyases/metabolism , Fabaceae/enzymology , Mimosine/metabolism , Arabidopsis/enzymology , Carbon-Nitrogen Lyases/isolation & purification , Catalytic Domain , Chromatography, High Pressure Liquid , Cloning, Molecular , Codon/genetics , Conserved Sequence , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Escherichia coli/metabolism , Heat-Shock Response , Kinetics , Lyases/metabolism , Mass Spectrometry , Mimosine/chemistry , Models, Biological , Open Reading Frames/genetics , Phylogeny , Pyridones/chemistry , Pyridones/metabolism , Recombinant Proteins/metabolism , Reference Standards , Substrate Specificity , Temperature
5.
Chem Biodivers ; 7(4): 860-70, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20397221

ABSTRACT

Strictosidine synthase (STR1) catalyzes the stereoselective formation of 3alpha(S)-strictosidine from tryptamine and secologanin. Strictosidine is the key intermediate in the biosynthesis of 2,000 plant monoterpenoid indole alkaloids, and it is a key precursor of enzyme-mediated synthesis of alkaloids. An improved expression system is described which leads to optimized His(6)-STR1 synthesis in Escherichia coli. Optimal production of STR1 was achieved by determining the impact of co-expression of chaperones pG-Tf2 and pG-LJE8. The amount and activity of STR1 was doubled in the presence of chaperone pG-Tf2 alone. His(6)-STR1 immobilized on Ni-NTA can be used for enzymatic synthesis of strictosidines on a preparative scale. With the newly co-expressed His(6)-STR1, novel 3alpha(S)-12-azastrictosidine was obtained by enzymatic catalysis of 7-azatryptamine and secologanin. The results obtained are of significant importance for application to chemo-enzymatic approaches leading to diversification of alkaloids with novel improved structures.


Subject(s)
Alkaloids/chemistry , Carbon-Nitrogen Lyases/genetics , Carbon-Nitrogen Lyases/metabolism , Biocatalysis , Carbon-Nitrogen Lyases/isolation & purification , Catharanthus/enzymology , Histidine/genetics , Iridoid Glucosides , Iridoids/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Oligopeptides/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Tryptamines/chemistry , Vinca Alkaloids/biosynthesis , Vinca Alkaloids/chemistry
6.
Appl Biochem Biotechnol ; 162(4): 966-74, 2010 Oct.
Article in English | MEDLINE | ID: mdl-19795222

ABSTRACT

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH 7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0-10.5. The optimal temperature was found to be near 40 degrees C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca2+. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K (m) value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Carbon-Nitrogen Lyases/chemistry , Carbon-Nitrogen Lyases/isolation & purification , Stenotrophomonas/enzymology , Bacterial Proteins/metabolism , Carbon-Nitrogen Lyases/metabolism , Isoelectric Point , Kinetics , Molecular Weight , Stenotrophomonas/chemistry , Substrate Specificity
7.
Appl Environ Microbiol ; 74(20): 6280-8, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18708510

ABSTRACT

Cyanase catalyzes the decomposition of cyanate into CO(2) and ammonium, with carbamate as an unstable intermediate. The cyanase of Pseudomonas pseudoalcaligenes CECT5344 was negatively regulated by ammonium and positively regulated by cyanate, cyanide, and some cyanometallic complexes. Cyanase activity was not detected in cell extracts from cells grown with ammonium, even in the presence of cyanate. Nevertheless, a low level of cyanase activity was detected in nitrogen-starved cells. The cyn gene cluster of P. pseudoalcaligenes CECT5344 was cloned and analyzed. The cynA, cynB, and cynD genes encode an ABC-type transporter, the cynS gene codes for the cyanase, and the cynF gene encodes a novel sigma(54)-dependent transcriptional regulator which is not present in other bacterial cyn gene clusters. The CynS protein was expressed in Escherichia coli and purified by following a simple and rapid protocol. The P. pseudoalcaligenes cyanase showed an optimal pH of 8.5 degrees C and a temperature of 65 degrees C. An insertion mutation was generated in the cynS gene. The resulting mutant was unable to use cyanate as the sole nitrogen source but showed the same resistance to cyanate as the wild-type strain. These results, in conjunction with the induction pattern of the enzymatic activity, suggest that the enzyme has an assimilatory function. Although the induction of cyanase activity in cyanide-degrading cells suggests that some cyanate may be generated from cyanide, the cynS mutant was not affected in its ability to degrade cyanide, which unambiguously indicates that cyanate is not a central metabolite in cyanide assimilation.


Subject(s)
Carbon-Nitrogen Lyases/genetics , Carbon-Nitrogen Lyases/metabolism , Cyanides/metabolism , Pseudomonas pseudoalcaligenes/enzymology , ATP-Binding Cassette Transporters/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon-Nitrogen Lyases/isolation & purification , Cloning, Molecular , Cyanates/metabolism , Enzyme Stability , Escherichia coli/genetics , Gene Deletion , Gene Expression , Gene Expression Regulation, Bacterial , Gene Order , Genes, Bacterial , Hydrogen-Ion Concentration , Molecular Sequence Data , Multigene Family , Mutagenesis, Insertional , Pseudomonas pseudoalcaligenes/genetics , Pseudomonas pseudoalcaligenes/metabolism , Quaternary Ammonium Compounds/metabolism , Sequence Alignment , Sequence Analysis, DNA , Temperature , Transcription Factors/genetics
8.
Biochim Biophys Acta ; 1774(12): 1563-70, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17964868

ABSTRACT

Thiohalophilus thiocyanoxidans is a first halophilic sulfur-oxidizing chemolithoautotrophic bacterium capable of growth with thiocyanate as an electron donor at salinity up to 4 M NaCl. The cells, grown with thiocyanate, but not with thiosulfate, contained an enzyme complex hydrolyzing thiocyanate to sulfide and ammonia under anaerobic conditions with carbonyl sulfide as an intermediate. Despite the fact of utilization of the <>, high cyanase activity was also detected in thiocyanate-induced cells. Three-stage column chromotography resulted in a highly purified thiocyanate-hydrolyzing protein with an apparent molecular mass of 140 kDa that consists of three subunits with masses 17, 19 and 29 kDa. The enzyme is a Co,Fe-containing protein resembling on its function and subunit composition the enzyme thiocyanate hydrolase from the Betaproteobacterium Thiobacillus thioparus. Cyanase, copurified with thiocyanate hydrolase, is a bisubstrate multisubunit enzyme with an apparent subunit molecular mass of 14 kDa. A possible role of cyanase in thiocyanate degradation by T. thiocyanoxidans is discussed.


Subject(s)
Chemoautotrophic Growth/physiology , Hydrolases/metabolism , Hydrolases/physiology , Sulfur-Reducing Bacteria/enzymology , Thiocyanates/metabolism , Carbon-Nitrogen Lyases/isolation & purification , Gammaproteobacteria/enzymology , Gammaproteobacteria/growth & development , Hydrolases/isolation & purification , Hydrolysis , Kinetics , Oxidation-Reduction , Sulfur-Reducing Bacteria/growth & development
9.
Biodegradation ; 15(4): 229-39, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15473552

ABSTRACT

Following biodegradation tests according to the OECD guidelines for testing of chemicals 301F different degradation rates were observed for the three stereoisomers of iminodisuccinate (IDS). A strain was isolated from activated sludge, which used two of three isomers, R,S-IDS and S,S-IDS, as sole source of carbon, nitrogen, and energy. The isolated strain was identified by 16S-rDNA and referred to as Ralstonia sp. SLRS7. An IDS-degrading lyase was isolated from the cell-free extract. The enzyme was purified by three chromatographic steps, which included anion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. The lyase catalysed the non-hydrolytic cleavage of IDS without requirement of any cofactors. Cleavage of S,S-IDS led to the formation of fumaric acid and L-aspartic acid. Interestingly R,S-IDS yielded only D-aspartic acid besides fumaric acid. R,R-IDS was not transformed. Thus, the IDS-degrading enzyme is a carbon-nitrogen lyase attacking only the asymmetric carbon atom exhibiting the S-configuration. Besides S,S-IDS and R,S-IDS cleavage, the lyase catalysed also the transformation of certain S,S-IDS metal complexes, namely Ca(2+)-, Mg(2+)- and Mn(2+)-IDS. The maximum enzyme activity was found at pH 8.0-8.5 and 35 degrees C. SDS-PAGE analysis revealed a single 57-kDa protein band. The native enzyme was estimated to be around 240 kDa indicating a homotetramer enzyme.


Subject(s)
Amino Acids/chemistry , Carbon-Nitrogen Lyases/isolation & purification , Succinates/chemistry , Amino Acids/metabolism , Biodegradation, Environmental , Carbon-Nitrogen Lyases/metabolism , Chromatography, Gel , Chromatography, Ion Exchange , Culture Media , Edetic Acid/chemistry , Ethylenediamines/chemistry , Molecular Structure , Molecular Weight , Ralstonia/enzymology , Ralstonia/isolation & purification , Sewage/microbiology , Stereoisomerism , Substrate Specificity , Succinates/metabolism
10.
Biochim Biophys Acta ; 1702(1): 121-4, 2004 Oct 01.
Article in English | MEDLINE | ID: mdl-15450856

ABSTRACT

Strictosidine synthase is a central enzyme involved in the biosynthesis of almost all plant monoterpenoid indole alkaloids. Strictosidine synthase from Rauvolfia serpentina was heterologously expressed in Escherichia coli. Crystals of the purified recombinant enzyme have been obtained by the hanging-drop technique at 303 K with potassium sodium tartrate tetrahydrate as precipitant. The crystals belong to the space group R3 with cell dimensions of a=b=150.3 A and c=122.4 A. Under cryoconditions (120 K), the crystals diffract to about 2.95 A.


Subject(s)
Carbon-Nitrogen Lyases/chemistry , Rauwolfia/enzymology , Base Sequence , Carbon-Nitrogen Lyases/genetics , Carbon-Nitrogen Lyases/isolation & purification , Carbon-Nitrogen Lyases/metabolism , Crystallography, X-Ray , DNA, Plant/genetics , Escherichia coli/genetics , Rauwolfia/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
11.
Anal Bioanal Chem ; 377(6): 1071-8, 2003 Nov.
Article in English | MEDLINE | ID: mdl-13680069

ABSTRACT

A new flow injection (FI) method for photometric monitoring of cyanate in bioremediation processes using immobilised native cyanase is described. The method is based on the catalytic reaction between cyanate and bicarbonate to produce ammonia and carbon dioxide in the presence of an inducible native cyanase, immobilised in a reactor packed with glass beads. Two degrees of purification of the biocatalyst were used-heated cell-free extract and purified extract of cyanase from Pseudomonas pseudoalcaligenes CECT 5344. The ammonia produced by the enzymatic reaction is finally monitored photometrically at 700 nm using a modification of the conventional Berthelot method. The method furnishes different calibration curves depending on the degree of purification of the cyanase, with linear ranges between 1.23 and 616.50 micromol L(-1) ( r(2)=0.9979, n=7) and between 1.07 and 308.25 micro mol L(-1) ( r(2)= 0.9992, n=7) for the heated cell-free extract and the purified cyanase extract, respectively. No statistically significant differences between the samples were found in the precision study evaluated at two cyanate concentration levels using one-way analysis of variance. A sampling frequency of 15 h(-1) was achieved. The method was used to monitor cyanate consumption in a cyanate bioremediation tank inoculated with Pseudomonas pseudoalcaligenes CECT 5344 strain. The correlation between cyanate degradation and ammonia production was tested using a conventional method. Finally, the method was applied to different samples collected from the bioremediation tank using the standard addition method; recoveries between 85.9 and 97.4% were obtained.


Subject(s)
Carbon-Nitrogen Lyases/metabolism , Cyanates/analysis , Enzymes, Immobilized/metabolism , Flow Injection Analysis/methods , Spectrophotometry/methods , Biodegradation, Environmental , Carbon-Nitrogen Lyases/isolation & purification , Environmental Monitoring/methods , Enzyme Stability , Enzymes, Immobilized/isolation & purification , Pseudomonas pseudoalcaligenes/enzymology
12.
Biochemistry ; 41(46): 13755-66, 2002 Nov 19.
Article in English | MEDLINE | ID: mdl-12427038

ABSTRACT

Two recombinant human isopeptidase T isoforms, ISOT-S and ISOT-L, differing by an insertion of 23 amino acids in ISOT-L, were previously classified as thiol proteases. Both contain one Zn2+-binding site of high-affinity, which is part of a cryptic nitrilo-triacetate-resistant pocket (site 1). A second Zn2+ site (site 2) was disclosed when both isoforms of the holoenzyme were incubated with an excess of Zn2+. The firmly bound Zn2+ of site 1 could be removed either slowly by dialysis against 1,10-phenanthroline at pH 5.5 or rapidly by treatment at pH 3.0 in the presence of 6 M urea followed by gel filtration at neutral pH. Zn2+ in site 1, but not in site 2, is essential for proteolytic activity because apoproteins were inactive. Inhibition of the catalytic activity was not due to a loss of ubiquitin binding capacity. CD spectra of both isoforms disclosed no major structural differences between the apo- and holoenzymes. The reconstitution of apoenzyme with Zn2+ under nondenaturing conditions at pH 5.5 completely restored enzymatic activity, which was indistinguishable from the reconstitution carried out in urea at pH 3.0. Thus, both human ISOTs are either thiol proteases with a local structural Zn2+ or monozinc metalloproteases that might use in catalysis a Zn2+-activated hydroxide ion polarized by Cys335.


Subject(s)
Brain/enzymology , Carbon-Nitrogen Lyases/drug effects , Catalysis/drug effects , Chelating Agents/pharmacology , Zinc/pharmacology , Apoenzymes/chemistry , Apoenzymes/metabolism , Carbon-Nitrogen Lyases/genetics , Carbon-Nitrogen Lyases/isolation & purification , Carbon-Nitrogen Lyases/metabolism , Circular Dichroism , Dose-Response Relationship, Drug , Holoenzymes/chemistry , Holoenzymes/metabolism , Humans , Mass Spectrometry , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Conformation , Protein Isoforms , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Substrate Specificity , Ubiquitins/metabolism
13.
Phytochemistry ; 55(2): 177-81, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11065292

ABSTRACT

Deacetylipecoside synthase (DIS), the enzyme catalyzing the condensation of dopamine and secologanin to form the (R)-epimer of deacetylipecoside, has been purified 570-fold from the leaves of Alangium lamarckii and partially characterized. The isolated enzyme is a single polypeptide with Mr 30,000, and has a pH optimum at 7.5 and a temperature optimum at 45 degrees C. The apparent Km values for dopamine and secologanin are 0.7 and 0.9 mM, respectively. DIS exhibits high substrate specificity toward dopamine, whereas neither tyramine nor tryptamine are utilized. The enzyme activity is not inhibited by its substrate dopamine, but is inhibited by alangimakine and dehydroalangimakine with similar I50 values of 10 microM. DIS presumably provides (R)-deacetylipecoside for the formation of tetrahydroisoquinoline monoterpene glucosides that also possess an (R)-configuration at the same chiral center.


Subject(s)
Carbon-Nitrogen Lyases/isolation & purification , Magnoliopsida/enzymology , Carbon-Nitrogen Lyases/antagonists & inhibitors , Carbon-Nitrogen Lyases/metabolism , Catalysis , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Hydrogen-Ion Concentration , Plant Leaves/enzymology , Substrate Specificity
14.
Immunology ; 94(2): 142-8, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9741334

ABSTRACT

Although existence of suppressor T cells is a controversial issue in cellular immunology, several lines of evidence indicate that T-cell-receptor alpha-chain (TCR-alpha) is a critical component of suppressor factors produced by these cells. Monoclonal non-specific suppressor factor (MNSF), a lymphokine produced by murine T-cell hybridoma, possesses pleiotrophic antigen-non-specific suppressive functions. Recently, we have shown that the 70,000-MW MNSF comprises an 8000-MW ubiquitin-like polypeptide and other subunit(s). Here we report that the 8000-MW ubiquitin homologue is associated with an intracellular TCR-alpha (but not TCR-beta)-like molecule and released from the cells. The affinity eluates obtained from the culture supernatants of E17 cells and concanavalin A (Con A)-activated splenocytes with anti-TCR-alpha monoclonal antibody (mAb) showed an antigen-non-specific, major histocompatibility complex (MHC)-non-restricted suppression. Immunoblot analysis demonstrated that anti-TCR-alpha, but not anti-TCR-beta, mAb recognizes native 70,000-MW MNSF. In addition, we found the dissociation of the 8000-MW polypeptide from the 62,000-MW TCR-alpha cross-reactive protein by hydrolase which cleaves isopeptide bonds. Thus the covalent attachment of ubiquitin-like protein(s) may be involved in the underlying mechanism of suppressor T-cells and TCR-alpha-like molecule(s) might be a main link between antigen-specific and non-specific suppression.


Subject(s)
Immune Tolerance , Receptors, Antigen, T-Cell, alpha-beta/immunology , Ubiquitins/immunology , Animals , Carbon-Nitrogen Lyases/isolation & purification , Carbon-Nitrogen Lyases/metabolism , Carbon-Nitrogen Lyases/pharmacology , Cell Culture Techniques , Concanavalin A/immunology , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Molecular Weight , Spleen/enzymology , Spleen/immunology , Suppressor Factors, Immunologic/chemistry , Suppressor Factors, Immunologic/immunology
15.
EMBO J ; 17(12): 3241-50, 1998 Jun 15.
Article in English | MEDLINE | ID: mdl-9628861

ABSTRACT

The ubiquitin pathway has been implicated in the regulation of the abundance of proteins that control cell growth and proliferation. We have identified and characterized a novel human ubiquitin isopeptidase, UBPY, which both as a recombinant protein and upon immunoprecipitation from cell extracts is able to cleave linear or isopeptide-linked ubiquitin chains. UBPY accumulates upon growth stimulation of starved human fibroblasts, and its levels decrease in response to growth arrest induced by cell-cell contact. Inhibition of UBPY accumulation by antisense plasmid microinjection prevents fibroblasts from entering S-phase in response to serum stimulation. By increasing or decreasing the cellular abundance of UBPY or by overexpressing a catalytic site mutant, we detect substantial changes in the total pattern of protein ubiquitination, which correlate stringently with cell proliferation. Our results suggest that UBPY plays a role in regulating the overall function of the ubiquitin-proteasome pathway. Affecting the function of a specific UBP in vivo could provide novel tools for controlling mammalian cell proliferation.


Subject(s)
Carbon-Nitrogen Lyases/genetics , Endopeptidases/genetics , Ubiquitins/metabolism , Animals , Carbon-Nitrogen Lyases/isolation & purification , Carbon-Nitrogen Lyases/metabolism , Cell Culture Techniques , Cell Division/drug effects , Endopeptidases/isolation & purification , Endopeptidases/metabolism , Endosomal Sorting Complexes Required for Transport , Humans , Mice , Mutagenesis, Site-Directed , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Transfection , Ubiquitin Thiolesterase
SELECTION OF CITATIONS
SEARCH DETAIL
...