scholarly journals Purification and kinetic analysis of a baculovirus ecdysteroid UDP-glucosyltransferase

1998 ◽  
Vol 330 (3) ◽  
pp. 1265-1270 ◽  
Author(s):  
P. Owain EVANS ◽  
David R. O'REILLY
Keyword(s):  
Kinetic Analysis ◽  
Biochemical Characterization ◽  
Active Form ◽  
Gel Filtration ◽  
Enzyme Mechanism ◽  
The Other ◽  
Kinetic Properties ◽  
Autographa Californica Nucleopolyhedrovirus ◽  
Moulting Hormones

The baculovirus ecdysteroid UDP-glucosyltransferase (EGT) disrupts the hormonal balance of the insect host by catalysing the conjugation of ecdysteroids, the moulting hormones, with the sugar moiety from UDP-glucose or UDP-galactose. In this study, Autographa californica nucleopolyhedrovirus EGT has been overproduced and purified, and its kinetic properties determined. The enzyme was purified 1100-fold to near-homogeneity using only two major steps, ion-exchange and gel-filtration chromatography. EGT activity was eluted from the gel-filtration column as a single peak corresponding to a 260±50 kDa protein, suggesting that the enzyme is an oligomer of three to five subunits, as the subunit molecular mass is approximately 56 kDa. Kinetic analysis showed that EGT has broadly similar specificities for UDP-galactose and UDP-glucose (kcat/Km = 1790.8 and 902.1 respectively) when ecdysone is used as the other substrate. On the other hand, it shows marked differences in specificity for the various ecdysteroids tested. Ecdysone seems to be the optimal substrate (kcat/Km = 7101.1), whereas 3-dehydroecdysone, an ecdysone precursor in Lepidoptera, is seven times less favourable (kcat/Km = 1085.7). Notably, 20-hydroxyecdysone, the active form of the hormone, is conjugated very poorly (kcat/Km = 31.6). Analysis of the data revealed that the enzyme mechanism involves the formation of an ecdysteroid-UDP-sugar-enzyme ternary complex. This work represents the most detailed biochemical characterization of an EGT to date.

scholarly journals Characterization of two β-galactosidases LacZ and WspA1 from Nostoc flagelliforme with focus on the latter’s central active region

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiang Gao ◽  
Litao Liu ◽  
Lijuan Cui ◽  
Tao Zheng ◽  
Boyang Ji ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Active Form ◽  
Gel Filtration ◽  
Special Role ◽  
Nostoc Flagelliforme ◽  
Reporter Protein ◽  
Gfp Reporter ◽  
Potential Applications ◽  
Ic50 Values

AbstractThe identification and characterization of new β-galactosidases will provide diverse candidate enzymes for use in food processing industry. In this study, two β-galactosidases, Nf-LacZ and WspA1, from the terrestrial cyanobacterium Nostoc flagelliforme were heterologously expressed in Escherichia coli, followed by purification and biochemical characterization. Nf-LacZ was characterized to have an optimum activity at 40 °C and pH 6.5, different from that (45 °C and pH 8.0) of WspA1. Two enzymes had a similar Michaelis constant (Km = 0.5 mmol/liter) against the substrate o-nitrophenyl-β-D-galactopyranoside. Their activities could be inhibited by galactostatin bisulfite, with IC50 values of 0.59 µM for Nf-LacZ and 1.18 µM for WspA1, respectively. Gel filtration analysis suggested that the active form of WspA1 was a dimer, while Nf-LacZ was functional as a larger multimer. WspA1 was further characterized by the truncation test, and its minimum central region was found to be from residues 188 to 301, having both the glycosyl hydrolytic and transgalactosylation activities. Finally, transgenic analysis with the GFP reporter protein found that the N-terminus of WspA1 (35 aa) might play a special role in the export of WspA1 from cells. In summary, this study characterized two cyanobacterial β-galactosidases for potential applications in food industry.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization

2006 ◽  
Vol 395 (2) ◽  
pp. 295-301 ◽  
Author(s):  
Chiara Ciaccio ◽  
Alessandra Gambacurta ◽  
Giampiero DE Sanctis ◽  
Domenico Spagnolo ◽  
Christina Sakarikou ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Biochemical Characterization ◽  
Expression System ◽  
Gel Filtration ◽  
Proteolytic Processing ◽  
Kinetic Properties ◽  
Specific Substrate ◽  
Human Eosinophil ◽  
Eosinophil Peroxidase ◽  
Pichia Pastoris Expression System

A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast α-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN−, Br− and Cl−. On the basis of the estimated Km and kcat values it is evident that the pseudohalide SCN− is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.

scholarly journals Purification and Characterization of the Recombinant Thermus sp. Strain T2 α-Galactosidase Expressed in Escherichia coli

2001 ◽  
Vol 67 (4) ◽  
pp. 1601-1606 ◽  
Author(s):  
Mitsunori Ishiguro ◽  
Satoshi Kaneko ◽  
Atsushi Kuno ◽  
Yoshinori Koyama ◽  
Shigeki Yoshida ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Side Chain ◽  
Catalytic Function ◽  
Native Polyacrylamide Gel Electrophoresis

ABSTRACT The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable α-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M r, 53,514). The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus brockianus was over 70%.Thermus sp. strain T2 α-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75°C forp-nitrophenyl-α-d-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70°C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40°C for 1 h. The enzyme acted on the terminal α-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea α-galactosidase I. The enzyme has only one Cys residue in the molecule.para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.

scholarly journals Ultrastructural and Partial Biochemical Characterization of Platelets from Chediak-Higashi Cattle

1977 ◽  
Author(s):  
K. M. Meyers ◽  
C. I. Seacord ◽  
G. Hopkins ◽  
H. Holmsen
Keyword(s):  
Electron Micrographs ◽  
Biochemical Characterization ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Human Platelets ◽  
Additional Information ◽  
Storage Pool ◽  
Calcium And Magnesium ◽  
Chediak Higashi Syndrome

To provide additional information on the platelet defect which is associated with the Chediak-Higashi syndrome (CHS), platelet rich plasma from normal and CHS cattle was incubated with 14C-adenine. Platelets were then isolated by gel filtration and treated with thrombin. Both the resting amount and extent of secretion of ATP, ADP, several acid hydrolasis, serotonin, calcium and magnesium was determined. Nucleotide profiles and electron micrographs of resting and thrombin treated platelets were also obtained. The markedly reduced secretion of nucleotides, serotonin, and metals demonstrate that CHS cattle have a storage pool defect. Furthermore, there appears to be significant differences in both the resting amount and extent of secretion of several of these measured substances between normal cattle and human platelets.

scholarly journals Biochemical characterization of guanidinium chloride-soluble dentine collagen from lathyritic-rat incisors

1979 ◽  
Vol 181 (3) ◽  
pp. 667-676 ◽  
Author(s):  
M Wohllebe ◽  
D J Carmichael
Keyword(s):  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Neutral Salt ◽  
Guanidinium Chloride ◽  
Lysine Residues ◽  
Cross Links ◽  
Lysine Hydroxylation

alpha- and beta-Chains were isolated by sequential ion-exchange and gel-filtration chromatography of guanidinium chloride-soluble dentine collagen obtained from Tris/NaCl-extracted EDTA-demineralized lathyritic-rat incisors. The alpha-chains were identified as alpha 1 I and alpha 2 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and amino acid analysis of the intact chains and their CNBr peptides. The dentine alpha-chains exhibited higher lysine hydroxylation and phosphate content, but lower hydroxylysine glycosylation, than alpha-chains from skin. Increased lysine hydroxylation was observed in the helical sequences. The alpha 1 I/alpha 2 ratio was approx. 3:1, and was presumably due to the presence of (alpha 1 I)3 molecules along with (alpha 1 I)2 alpha 2 molecules as shown recently for neutral-salt-soluble dentine collagen [Wohllebe & Carmichael (1978) Eur. J. Biochem. 92, 183–188]. In the borohydride-reduced beta 11- and beta 12-chains from guanidinium chloride-soluble dentine collagen, the reduced cross-links hydroxylysinohydroxynorleucine and hydroxylysinonorleucine were present. A higher proportion of hydroxylysinonorleucine in the reduced beta 12-chain probably reflects differences in extent of hydroxylation of specific lysine residues of the alpha 1 I- and alpha 2-chains.

scholarly journals Molecular Characterization of Saccharomyces cerevisiae TFIID

2002 ◽  
Vol 22 (16) ◽  
pp. 6000-6013 ◽  
Author(s):  
Steven L. Sanders ◽  
Krassimira A. Garbett ◽  
P. Anthony Weil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Molecular Mass ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Calculated Molecular Mass ◽  
General Transcription Factor

ABSTRACT We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

scholarly journals Purification and characterization of fat body lipase from the Greater Wax Moth Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Author(s):  
Rahma R.Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M.S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

AbstractLipid mobilization and transport in insects is under investigation, especially lipases and lipophorin because of their roles in energy production and transport of lipids at flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from last larval instar of Galleria mellonella. Purification methods by combination of ammonium sulfate precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633±0.000551 mg/ml and 1.5754±0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore biochemical characterization of fat body lipase was carried out through testing its activities against several factors such as; different temperatures, pH ranges, metal ions and inhibitors ending by determination of their kinetic parameters with the use of p-Nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35-37°C and 37-40°C and pH ranges of 7-9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+ and Na+ metal ions indicating that fat body lipase is metalloproteinase. Additionally, lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfony fluoride (PMSF), ethylene-diaminetetractic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA) providing an evidence of presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 301.95mM Km and 0.316 Umg−1 Vmax. By considering the purification of fat body lipase from larvae and using some inhibitors especially ion chelating agents, it is suggested to develop this study by using lipase inhibitors to reach a successful control of Galleria mellonella in the near future.

Biochemical Characterization of Chloromethane Emission from the Wood-Rotting Fungus Phellinus pomaceus

1998 ◽  
Vol 64 (8) ◽  
pp. 2831-2835 ◽  
Author(s):  
Deepti Saxena ◽  
Saleh Aouad ◽  
Jihad Attieh ◽  
Hargurdeep S. Saini
Keyword(s):  
Atmospheric Chemistry ◽  
Biochemical Characterization ◽  
Active Form ◽  
Methyl Chloride ◽  
Bound State ◽  
Ph Optimum ◽  
Methyl Donors ◽  
Membrane Bound

ABSTRACT Many wood-rotting fungi, including Phellinus pomaceus, produce chloromethane (CH3Cl). P. pomaceus can be cultured in undisturbed glucose mycological peptone liquid medium to produce high amounts of CH3Cl. The biosynthesis of CH3Cl is catalyzed by a methyl chloride transferase (MCT), which appears to be membrane bound. The enzyme is labile upon removal from its natural location and upon storage at low temperature in its bound state. Various detergents failed to solubilize the enzyme in active form, and hence it was characterized by using a membrane fraction. The enzyme had a sharp pH optimum between 7 and 7.2. Its apparent Km for Cl− (ca. 300 mM) was much higher than that for I− (250 μM) or Br− (11 mM). A comparison of theseKm values to the relative in vivo methylation rates for different halides suggests that the realKm for Cl− may be much lower, but the calculated value is high because the CH3Cl produced is used immediately in a coupled reaction. Among various methyl donors tested, S-adenosyl-l-methionine (SAM) was the only one that supported significant methylation by MCT. The reaction was inhibited by S-adenosyl-l-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings advance our comprehension of a poorly understood metabolic sector at the origin of biogenic emissions of halomethanes, which play an important role in atmospheric chemistry.

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