scholarly journals Identification and characterization of Thermoplasma acidophilum glyceraldehyde dehydrogenase: a new class of NADP+-specific aldehyde dehydrogenase

2006 ◽  
Vol 397 (1) ◽  
pp. 131-138 ◽  
Author(s):  
Jin Hwa Jung ◽  
Sun Bok Lee
Keyword(s):  
Molecular Mass ◽  
Aldehyde Dehydrogenase ◽  
Aromatic Aldehydes ◽  
Thermoplasma Acidophilum ◽  
Genome Database ◽  
New Class ◽  
Cell Extracts ◽  
Sds Page ◽  
Aldehyde Dehydrogenase Superfamily ◽  
Inhibition Studies

Thermoacidophilic archaea such as Thermoplasma acidophilum and Sulfolobus solfataricus are known to metabolize D-glucose via the nED (non-phosphorylated Entner–Doudoroff) pathway. In the present study, we identified and characterized a glyceraldehyde dehydrogenase involved in the downstream portion of the nED pathway. This glyceraldehyde dehydrogenase was purified from T. acidophilum cell extracts by sequential chromatography on DEAE-Sepharose, Q-Sepharose, Phenyl-Sepharose and Affi-Gel Blue columns. SDS/PAGE of the purified enzyme showed a molecular mass of approx. 53 kDa, whereas the molecular mass of the native protein was 215 kDa, indicating that glyceraldehyde dehydrogenase is a tetrameric protein. By MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight MS) peptide fingerprinting of the purified protein, it was found that the gene product of Ta0809 in the T. acidophilum genome database corresponds to the purified glyceraldehyde dehydrogenase. The native enzyme showed the highest activity towards glyceraldehyde, but no activity towards aliphatic or aromatic aldehydes, and no activity when NAD+ was substituted for NADP+. Analysis of the amino acid sequence and enzyme inhibition studies indicated that this glyceraldehyde dehydrogenase belongs to the ALDH (aldehyde dehydrogenase) superfamily. BLAST searches showed that homologues of the Ta0809 protein are not present in the Sulfolobus genome. Possible differences between T. acidophilum (Euryarchaeota) and S. solfataricus (Crenarchaeaota) in terms of the glycolytic pathway are thus expected.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

scholarly journals Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

2000 ◽  
Vol 182 (9) ◽  
pp. 2559-2566 ◽  
Author(s):  
Donald E. Ward ◽  
Servé W. M. Kengen ◽  
John van der Oost ◽  
Willem M. de Vos
Keyword(s):  
Molecular Mass ◽  
Alanine Aminotransferase ◽  
Pyrococcus Furiosus ◽  
Sodium Dodecyl ◽  
Northern Analysis ◽  
Transcriptional Level ◽  
Significant Activity ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts

ABSTRACT Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosusby multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in theP. furiosus genome database. The gene was expressed inEscherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. Thek cat/Km values for alanine and pyruvate formation were 41 and 33 s−1mM−1, respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aatgene. In addition, both the aat and gdh(encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdhgenes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.

Detection of both Type 1 and Type 2 Plasminogen Activator Inhibitors in Human Monocytes

1990 ◽  
Vol 63 (01) ◽  
pp. 067-071 ◽  
Author(s):  
Joan C Castellote ◽  
Enric Grau ◽  
Maria A Linde ◽  
Nuria Pujol-Moix ◽  
Miquel LI Rutllant
Keyword(s):  
Molecular Mass ◽  
Plasminogen Activator ◽  
Human Peripheral Blood ◽  
Direct Interaction ◽  
Apparent Molecular Weight ◽  
Fibrinolytic System ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Single Chain ◽  
Sds Page

SummaryIncreasing evidence suggests the involvement of leukocytes in the fibrinolytic system. Monocytes secrete pro-urokinase (Grau, Thromb Res 1989; 53: 145) and it has been shown that these cells have specific receptors for urokinase and plasminogen (Miles, Thromb Haemostas 1987; 58: 936). The aim of this study was to analyse the presence of plasminogen activator inhibitor(s) in platelet-free suspensions of human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). SDS-PAGE and reverse fibrin autography showed an inhibitory band of 50 kDa in the monocyte extracts (Triton X-100) but not in the PMN extracts. Urokinase (u-PA) was mixed with increasing amounts of monocyte extract for 10 min and the mixtures were added to 125Ifibrin coated wells containing plasminogen. A dose-dependent decrease in the u-PA fibrinolytic activity was observed. The amount of inhibition increased when the monocyte releasates were preincubated with u-PA (40% inhibition after 5 min preincubation and 80% after 15 min), indicating a direct interaction between this activator and an inhibitor(s). After SDS-PAGE of monocyte extracts, immunoblotting and peroxidase staining identified both PAI1 and PAI2, with an apparent molecular weight of 47-50 kDa. Monocyte-associated PAI1 formed complexes with single chain t-PA with a molecular mass 50 kDa higher than the molecular mass of the free PAI1. However, a significant amount of PAI remained unbound to t-PA. This inactive PAI1 could have come from a rapid inactivation of the primary active PAI1. These PAI1 and PAI2 detected in human monocytes may be transcendent in the regulation of the fibrinolytic system.

scholarly journals Properties of subunits of the multicatalytic proteinase complex revealed by the use of subunit-specific antibodies

1991 ◽  
Vol 278 (1) ◽  
pp. 171-177 ◽  
Author(s):  
A J Rivett ◽  
S T Sweeney
Keyword(s):  
Molecular Mass ◽  
Rat Liver ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Rat Tissues ◽  
Specific Antibodies ◽  
Multicatalytic Proteinase ◽  
Cell Extracts ◽  
Liver And Kidney ◽  
Lysosomal Proteinase

The multicatalytic proteinase (MCP) is a high-molecular-mass non-lysosomal proteinase that gives rise to a characteristic pattern of bands of molecular mass 22-34 kDa on SDS/PAGE gels. Isoelectric-focusing gels of the enzyme purified from rat liver show 16 bands with isoelectric points in the range of pH 5-8.5. Two-dimensional PAGE gels reveal that there are more than the previously reported 13 polypeptides associated with the MCP from rat liver and show a pattern of 15-20 major spots and several minor ones, similar to that of MCP isolated from some other sources. Possible relationships between the different polypeptides were investigated by immunoblot analysis of electrophoretically purified proteinase subunits with affinity-purified subunit-specific antibodies as well as antibodies raised against individual denatured subunits of the complex. The results demonstrate that many of the major polypeptide components of the MCP complex are antigenically distinct. Moreover comparison of immunoreactive material in crude cell extracts with that in purified MCP preparations has shown that the polypeptides are not derived from a smaller number of higher-molecular-mass subunits. Also, individual subunits have the same apparent molecular mass in a variety of rat tissues, suggesting close similarity between MCPs of different tissues. The highest concentrations of MCP subunits occur in liver and kidney. Gel-filtration analysis of crude extracts has demonstrated that MCP polypeptides are also associated with a higher-molecular-mass complex, which may be the 26 S proteinase that has been implicated in the degradation of ubiquitin-protein conjugates.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins

1997 ◽  
Vol 110 (5) ◽  
pp. 611-621 ◽  
Author(s):  
M. Gimona ◽  
Z. Lando ◽  
Y. Dolginov ◽  
J. Vandekerckhove ◽  
R. Kobayashi ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Epithelial Cell Line ◽  
Chemical Crosslinking ◽  
Related Function ◽  
Differentiated Cells ◽  
Cell Extracts ◽  
Sds Page ◽  
Functional Implications ◽  
Dependent Interaction

Zero-length chemical crosslinking with 1-ethyl-3-[3-(dimethyl amino)propyl]carbodiimide (EDC) indicated an association of the Ca2+-binding protein S100A2 with tropomyosin (TM) in vitro. The mobility of the crosslinked product on SDS-PAGE gels indicated the formation of a 1:1 complex between S100A2 and TM and the interaction was Ca2+ dependent. Monoclonal antibodies were raised against S100A2 and used to determine its cellular localization in the porcine epithelial cell line LLC PK1. It was found that the localization of S100A2 depended on the differentiation state of the cells, being absent from actin stress fibers in sparsely seeded cultures, but present in the actin-containing microvilli characteristic of differentiated cells. Immunoprecipitations of [35S]methionine-labeled extracts using S100A2 as well as TM-specific antibodies failed to co-precipitate TM and S100A2, indicating a transient association between these two molecules in solution. Affinity chromatography of cell extracts on immobilized recombinant TMs, however, confirmed the Ca2+-dependent interaction between S100A2 and both muscle TMs as well as with high and low molecular mass nonmuscle TMs, suggesting that the binding site resides in one of the conserved regions of TM. Our data demonstrate the possible interaction of S100A2 with TM that is not bound to the microfilaments and indicate a differentiation-related function for S100A2 in LLC PK1 cells. The possible functional implications of this interaction are discussed.

scholarly journals Synthesis of new Cα-tetrasubstituted α-amino acids

2009 ◽  
Vol 5 ◽  
Author(s):  
Andreas A Grauer ◽  
Burkhard König
Keyword(s):  
Amino Acids ◽  
Secondary Structure ◽  
Aromatic Aldehydes ◽  
Building Blocks ◽  
Reaction Conditions ◽  
Peptide Backbone ◽  
Aliphatic Aldehydes ◽  
New Class ◽  
Cyclic Amino Acids

Cα-Tetrasubstituted α-amino acids are important building blocks for the synthesis of peptidemimetics with stabilized secondary structure, because of their ability to rigidify the peptide backbone. Recently our group reported a new class of cyclic Cα-tetrasubstituted tetrahydrofuran α-amino acids prepared from methionine and aromatic aldehydes. We now report the extension of this methodology to aliphatic aldehydes. Although such aldehydes are prone to give aldol products under the reaction conditions used, we were able to obtain the target cyclic amino acids in low to moderate yields and in some cases with good diastereoselectivity.

scholarly journals Heparin- and Zn2+-binding proteins from boar seminal plasma.

1999 ◽  
Vol 46 (4) ◽  
pp. 935-939 ◽  
Author(s):  
D Hołody ◽  
J Strzezek
Keyword(s):  
Amino Acid ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Seminal Plasma ◽  
Binding Proteins ◽  
Reproductive Tract ◽  
Female Reproductive Tract ◽  
Heparin Binding ◽  
Sds Page ◽  
Protein Components

Low molecular mass, heparin-binding proteins from seminal plasma play an important role in gametes interaction whereas plasmatic Zn2+-binding proteins stabilize chromatin and plasmalemma structures and protect spermatozoa in the female reproductive tract. By means of affinity chromatography the heparin- and Zn2+-binding proteins were isolated from boar seminal plasma and both preparations were analyzed by reverse HPLC. Most of the proteins bound to heparine and Zn2+-ions were classified as spermadhesins. Three fractions binding exclusively Zn2+ were isolated. They differ in amino-acid composition, content of glucosamine and content of protein components revealed by SDS/PAGE.

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