scholarly journals Homocitrate synthase from Penicillium chrysogenum. Localization, purification of the cytosolic isoenzyme, and sensitivity to lysine

1990 ◽  
Vol 269 (1) ◽  
pp. 247-253 ◽  
Author(s):  
W M Jaklitsch ◽  
C P Kubicek
Keyword(s):  
Molecular Mass ◽  
Penicillium Chrysogenum ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Protamine Sulphate ◽  
Kinetic Behaviour ◽  
Saturation Kinetics ◽  
Homocitrate Synthase ◽  
Ph Dependent ◽  
Inhibition Pattern

Subcellular fractionation of cell-free extracts obtained by nitrogen cavitation showed that Penicillium chrysogenum Q176 contains a cytosolic as well as a mitochondrial homocitrate synthase activity. The cytosolic isoenzyme was purified about 500-fold, and its kinetic and molecular properties were investigated. Native homocitrate synthase shows a molecular mass of 155 +/- 10 kDa as determined by gel filtration and a pH of 4.9 +/- 0.1 as determined by chromatofocusing. The kinetic behaviour towards 2-oxoglutarate is hyperbolic, with Km = 2.2 mM; with respect to acetyl-CoA the enzyme shows sigmoidal saturation kinetics, with [S]0.5 = 41 microM and h = 2.6. The enzyme was inhibited strongly by L-lysine (Ki = 8 +/- 2 microM; 50% inhibition by 53 microM at 6 mM-2-oxoglutarate), competitively with 2-oxoglutarate, in protamine sulphate-treated and desalted cell-free extracts and in partially purified preparations. The extent of this inhibition was strongly pH-dependent. Both isoenzymes are equally susceptible to inhibition by lysine. The same inhibition pattern is shown by the enzyme from strain D6/1014A, which is a better producer of penicillin than strain Q176.

scholarly journals Purification and characterization of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase from Penicillium chrysogenum

1997 ◽  
Vol 327 (1) ◽  
pp. 185-191 ◽  
Author(s):  
Hanne B.Aa. THEILGAARD ◽  
Klaus N. KRISTIANSEN ◽  
Claus M. HENRIKSEN ◽  
Jens NIELSEN
Keyword(s):  
Molecular Mass ◽  
Penicillium Chrysogenum ◽  
Feedback Inhibition ◽  
Glucose Repression ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Exchange Chromatography ◽  
Protamine Sulphate ◽  
Gradient Gel Electrophoresis ◽  
Single Polymer Chain

δ-(L-α-Aminoadipyl)-L-cysteinyl-d-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)2SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE. The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa. The denatured enzyme had an estimated molecular mass of 440 kDa. The influence of different reaction parameters such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The Km values for the three precursor substrates L-α-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 μM respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM MgCl2). The dimer of the reaction product bis-δ-(L-α-aminoadipyl)-l-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter KbisACV was determined as 1.4 mM. Furthermore dithiothreitol was shown to inhibit the purified ACVS. From the addition of a glucose pulse to a steady-state glucose-limited continuous culture of P. chrysogenum it was found that there is glucose repression of the synthesis of ACVS and that there must be a constant turnover of ACVS owing to synthesis and degradation.

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 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.

scholarly journals Characterization of [3H]palmitate- and [3H]ethanolamine-labelled proteins in the multicellular parasitic trematode Schistosoma mansoni

1988 ◽  
Vol 254 (2) ◽  
pp. 419-426 ◽  
Author(s):  
P M Wiest ◽  
E J Tisdale ◽  
W L Roberts ◽  
T L Rosenberry ◽  
A A F Mahmoud ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Free Amino ◽  
Protein Modification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Reductive Methylation ◽  
Amide Linkage

Biosynthetic labelling experiments with cercariae and schistosomula of the multicellular parasitic trematode Schistosoma mansoni were performed to determine whether [3H]palmitate or [3H]ethanolamine was incorporated into proteins. Parasites incorporated [3H]palmitate into numerous proteins, as judged by SDS/polyacrylamide-gel electrophoresis and fluorography. The radiolabel was resistant to extraction with chloroform, but sensitive to alkaline hydrolysis, indicating the presence of an ester bond. Further investigation of the major 22 kDa [3H]palmitate-labelled species showed that the label could be recovered in a Pronase fragment which bound detergent and had an apparent molecular mass of 1200 Da as determined by gel filtration on Sephadex LH-20. Schistosomula incubated with [3H]ethanolamine for up to 24 h incorporated this precursor into several proteins; labelled Pronase fragments recovered from the three most intensely labelled proteins were hydrophilic and had a molecular mass of approx. 200 Da. Furthermore, reductive methylation of such fragments showed that the [3H]ethanolamine bears a free amino group, indicating the lack of an amide linkage. We also evaluated the effect of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: [3H]palmitate-labelled proteins of schistosomula and surface-iodinated proteins were resistant to hydrolysis with this enzyme. In conclusion, [3H]palmitate and [3H]ethanolamine are incorporated into distinct proteins of cercariae and schistosomula which do not bear glycophospholipid anchors. The [3H]ethanolamine-labelled proteins represent a novel variety of protein modification.

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.

Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays

1995 ◽  
Vol 41 (9) ◽  
pp. 1273-1282 ◽  
Author(s):  
Z Chen ◽  
A Prestigiacomo ◽  
T A Stamey
Keyword(s):  
Molecular Mass ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Free Psa ◽  
Apparent Homogeneity ◽  
International Standardization

Abstract We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.

scholarly journals Iron release from haemosiderin and ferritin by therapeutic and physiological chelators

1989 ◽  
Vol 260 (3) ◽  
pp. 903-907 ◽  
Author(s):  
M J O'Connell ◽  
R J Ward ◽  
H Baum ◽  
T J Peters
Keyword(s):  
Lipid Peroxidation ◽  
Molecular Mass ◽  
Absorption Spectra ◽  
Gel Filtration ◽  
Phosphate Content ◽  
Core Size ◽  
Iron Release ◽  
Release Rates ◽  
Stimulate Lipid Peroxidation

Iron release from both human and horse spleen haemosiderin to desferrioxamine was substantially less than that released from ferritin samples. This finding contradicts a previous report [Kontoghiorges, Chambers & Hoffbrand (1987) Biochem. J. 241, 87-92]. Differences in phosphate content of cores and in core size between haemosiderin and ferritin did not account for the different iron-release rates. Iron released to acetate was found to stimulate lipid peroxidation in liposomes, whereas that released to stronger chelators such as citrate and desferal did not. Absorption spectra and gel-filtration studies suggest that the acetate-solubilized iron was in the form of low-molecular-mass (less than 5 kDa) ferrihydrite fragments.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

scholarly journals Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24

1996 ◽  
Vol 319 (3) ◽  
pp. 977-983 ◽  
Author(s):  
Jeong Heon KO ◽  
Cheorl Ho KIM ◽  
Dae-Sil LEE ◽  
Yu Sam KIM
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Gel Filtration ◽  
Higher Plant ◽  
Sds Page ◽  
Thermus Caldophilus ◽  
Internal Peptide ◽  
Adp Glucose Pyrophosphorylase ◽  
Fructose 1,6 Bisphosphate

An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases. The enzyme was most active at pH 6.0. The activity was maximal at 73–78 °C and its half-life was 30 min at 95 °C. Kinetic and regulatory properties were characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala+Gly residues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related.

Sign in / Sign up

Export Citation Format

Share Document