scholarly journals Binding of Ferric Enterobactin by theEscherichia coli Periplasmic Protein FepB

2000 ◽  
Vol 182 (19) ◽  
pp. 5359-5364 ◽  
Author(s):  
Cathy Sprencel ◽  
Zhenghua Cao ◽  
Zengbiao Qi ◽  
Daniel C. Scott ◽  
Marjorie A. Montague ◽  
...  
Keyword(s):  
Specific Activity ◽  
Solid Phase ◽  
Cell Envelope ◽  
Binding Protein ◽  
Gel Filtration ◽  
Periplasmic Protein ◽  
Left Handed ◽  
Fluorescence Measurements ◽  
Rate Limiting Step

ABSTRACT The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the Kd of the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (Kd = 30 nM) and ferric enantioenterobactin (Kd = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (Kd = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

Purification and characterization of rat epididymal neutral β-galactosidase and its changes during in vivo development

1993 ◽  
Vol 71 (1-2) ◽  
pp. 22-26 ◽  
Author(s):  
Pratima Dutta ◽  
Gopal C. Majumder
Keyword(s):  
Concanavalin A ◽  
Sexual Maturity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Tissue Homogenate ◽  
Affinity Column ◽  
Ph Optimum

A neutral β-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A - agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with α-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral β-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral β-galactosidase during sexual maturity of epididymis in vivo.Key words: neutral β-galactosidase, rat epididymal, cytosolic, developmental, sexual maturity.

scholarly journals Characterization of bacteriocin ABC transporter ATP-binding protein produced by a newly isolated Enterococcus casseliflavus MI001 strain

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Indira Mikkili ◽  
Venkateswarulu TC ◽  
Abraham Peele Karlapudi ◽  
Vidya Prabhakar Kodali ◽  
Krupanidhi Srirama
Keyword(s):  
Abc Transporter ◽  
Specific Activity ◽  
Binding Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Food Preservation ◽  
Atp Binding ◽  
Transporter Protein ◽  
Enterococcus Casseliflavus ◽  
Atp Binding Protein

Abstract Background ATP-binding cassette (ABC) transporters constitute one of the largest transporter protein families and play a role in diverse biological processes. Results In the present study, bacteriocin isolated from the Enterococcus casseliflavus MI001 strain was identified as an ABC transporter ATP-binding protein. The optimal conditions for the production of bacteriocin were found to be at 35 °C, a pH 5.5, and an incubation time of 24 h. Purification was performed using ammonium sulphate precipitation, gel filtration, and DEAE ion exchange chromatography. The bacteriocin was purified with an eightfold purification scheme resulting with a specific activity of 15,000 AU/mg. The NMR spectrum of purified bacteriocin revealed the presence of amino acids, namely lysine, methionine, cysteine, proline, threonine, tryptophan, and histidine. Further, the bacteriocin ABC transporter showed antimicrobial activity against food spoilage microorganisms. Conclusions The ABC transporter ATP-binding protein could be used as a potential alternative for food preservation, and it may be considered as a bio-preservative agent in food processing industries.

IMMUNOAFFINITY PURIFICATION OF PLASMA INHIBITOR FOR ACTIVATED PROTEIN C

1987 ◽  
Author(s):  
M Laurell ◽  
T Carlsson ◽  
J Stenflo
Keyword(s):  
Monoclonal Antibodies ◽  
Protein C ◽  
Solid Phase ◽  
Gel Filtration ◽  
Activated Protein C ◽  
Specific Inhibitor ◽  
Sds Page ◽  
Fresh Plasma ◽  
Barbital Buffer

Activated protein C (APC) is an important regulator of blood coagulation in vivo. In plasma this serine protease is slowly inhibited by a specific inhibitor, activated protein C inhibitor (PCI), (Suzuki et al. (1983) J.Biol.Chem. 258 , 163-168). We have now made monoclonal antibodies against PCI by immunizing with the APC-PCI complex. Positive clones were identified by solid phase immunoassay with 125I labelled partially purified inhibitor. After subcloning and expansion in mice, one of the monoclonal antibodies was immobilized on Sepharose 4B and used in the purification of the inhibitor. A two step purification procedure was deviced starting with passage of fresh human plasma over the column. Following extensive washing the inhibitor was eluted with 50 mM triethylamine- HCI,0.5 M NaCl, pH 11.0, from the column together with a small amount of high molecular weight material. After gel filtration on a column packed with AcA 44 the inhibitor appeared homogenous on SDS - PAGE. Approximatly 0.5 mg inhibitor was obtained from 200 ml of fresh plasma. The apparent Mr of the inhibitor was 57000 kDa on SDS -PAGE. The purified protein formed a complex (Mr =110000 kDa) with human APC. At the same time a band (Mr = 54000 kDa ) appeared that represented the modified inhibitor. When analyzed on agarose gel electrophoresis (75mM barbital buffer, 2mM EDTA at pH 8.7 ) the PCI migrated to the β2- region, whereas the modified inhibitor had a slightly more anodal mobility. The APC-PCI komplex migrated to the α2- region.Two immunoradiometric assays were constructed with the monoclonal antibodies. One measured the complexes between APC and PCI while the other one measured the total amount of PCI present. These assays were used to study complex formation in buffer and plasma.

Studies on uroporphyrinogen decarboxylase from Chlorella kessleri (Trebouxiophyceae, Chlorophyta)

2007 ◽  
Vol 53 (2) ◽  
pp. 303-312 ◽  
Author(s):  
Angela B. Juárez ◽  
Carmen Aldonatti ◽  
María S. Vigna ◽  
María del C. Ríos de Molina
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Exponential Growth Phase ◽  
Uroporphyrinogen Decarboxylase ◽  
Chlorella Kessleri ◽  
Rate Limiting Step ◽  
Lower Molecular Mass ◽  
Green Alga Chlorella ◽  
Two Stages ◽  
First Time

Uroporphyrinogen decarboxylase (UroD) (EC 4.1.1.37) is an enzyme from the tetrapyrrole biosynthetic pathway, in which chlorophyll is the main final product in algae. This is the first time that a study on UroD activity has been performed in a green alga ( Chlorella ). We isolated and partially purified the enzyme from a Chlorella kessleri (Trebouxiophyceae, Chlorophyta) strain (Copahue, Neuquén, Argentina), and describe for the first time some of its properties. In C. kessleri, the decarboxylation of uroporphyrinogen III occurs in two stages, via 7 COOH and then 6 and 5 COOH intermediates, with the decarboxylation of the 7 COOH compound being the rate-limiting step for the reaction. Cultures in the exponential growth phase showed the highest specific activity values. The most suitable conditions to measure UroD activity in C. kessleri were as follows: 0.23–0.3 mg protein/mL, ?6–8 μmol/L uroporphyrinogen III, and 20 min incubation time. Gel filtration chromatography and Western blot assays indicated that UroD from C. kessleri is a dimer of approximately 90 kDa formed by species of lower molecular mass, which conserves enzymatic activity.

scholarly journals A high-affinity oestrogen-binding protein in cockerel liver cytosol

1979 ◽  
Vol 180 (2) ◽  
pp. 347-353 ◽  
Author(s):  
C B Lazier ◽  
A J Haggarty
Keyword(s):  
Binding Sites ◽  
Proteinase Inhibitors ◽  
Specific Binding ◽  
Binding Protein ◽  
Gel Filtration ◽  
Binding Activity ◽  
Single Class ◽  
High Affinity ◽  
Significant Concentration

In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1–4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.

scholarly journals Development of a radiolabeled β-human chorionic gonadotropin

2009 ◽  
Vol 59 (4) ◽  
pp. 421-429 ◽  
Author(s):  
Amir Jalilian ◽  
Mohammad Khoshdel ◽  
Javad Garousi ◽  
Hassan Yousefnia ◽  
Mohammad Hosseini ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
High Performance ◽  
Radiochemical Purity ◽  
Specific Activity ◽  
Solid Phase ◽  
Biodistribution Studies ◽  
Β Hcg ◽  
Optimized Conditions

Development of a radiolabeled β-human chorionic gonadotropinβ-Human chorionic gonadotropin (β-hCG) was successively labeled with [67Ga]-gallium chloride after conjugation with freshly prepared diethylenetriaminepentaacetic acid dianhydride (ccDTPA). After solid phase purification of the radiolabeled hormone, high performance liquid chromatography showed radiochemical purity higher than 95 % under optimized conditions (specific activity = 22-23 TBq mM-1, labeling efficiency 80 %). Preliminaryin vivostudies (ID g-1, %) in male wild-type rats showed marked gonadal uptake of the tracer after 240 minutes in agreement with the biodistribution studies and reported β-hCG receptors. Target to blood ratios were 5.1 and 15.2 after 3 and 24 hours, respectively, while target to muscle ratios were 35 and 40 after 3 and 24 hours, respectively.

scholarly journals Direct Interaction of the EpsL and EpsM Proteins of the General Secretion Apparatus in Vibrio cholerae

1999 ◽  
Vol 181 (10) ◽  
pp. 3129-3135 ◽  
Author(s):  
Maria Sandkvist ◽  
Lloyd P. Hough ◽  
Mira M. Bagdasarian ◽  
Michael Bagdasarian
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Cytoplasmic Membrane ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Direct Interaction ◽  
Stable Complex ◽  
Extracellular Secretion ◽  
Triton X

ABSTRACT The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM ofVibrio cholerae, have been purified and characterized. Based on gel filtration analysis, both purified EpsM(His)6 and wild-type EpsL present in anEscherichia coli Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in E. coli, they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.

Synthesis of peptides by the solid-phase method. V. Substance P and analogs

1980 ◽  
Vol 58 (4) ◽  
pp. 272-280 ◽  
Author(s):  
A. Fournier ◽  
R. Couture ◽  
J. Magnan ◽  
M. Gendreau ◽  
D. Regoli ◽  
...  
Keyword(s):  
Substance P ◽  
Solid Phase ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Exchange Chromatography ◽  
Phase Method ◽  
Elemental Analyses ◽  
Solid Phase Method

We have synthesized a series of 12 analogs of the undecapeptide substance P in order to perform a structure–activity study of this peptide. In the present work, each residue was substituted by L-alanine, and the C-terminal amide was replaced by the free carboxyl in order to pinpoint biologically important side chains and functional groups. The synthesis of the analogs was carried out by the automatic solid-phase method. Couplings were performed by the symmetrical anhydride procedure. After cleavage with liquid HF, the peptides were purified by gel filtration and ion-exchange chromatography. Their purity was assessed by thin-layer chromatography, paper electrophoresis, amino acid and elemental analyses, and high pressure liquid chromatography. They were tested for biological activity in vitro on the ileum of the guinea pig, the mesenteric vein of the rabbit, and the vas deferens of the rat, and in vivo by measuring their effect on the blood pressure of the rat.

Rat intestinal angiotensin-converting enzyme: purification, properties, expression, and function

1992 ◽  
Vol 263 (4) ◽  
pp. G466-G473 ◽  
Author(s):  
R. H. Erickson ◽  
Y. Suzuki ◽  
A. Sedlmayer ◽  
I. S. Song ◽  
Y. S. Kim
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Maximal Velocity ◽  
Converting Enzyme ◽  
And Function

Angiotensin-converting enzyme [ACE (peptidyl-dipeptidase A, EC 3.4.15.1)] was purified from a total cell membrane fraction of rat intestinal mucosa. A 4,500-fold purification was achieved after affinity chromatography with lisinopril-Sepharose and gel filtration. The final preparation was judged to be homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 160,000. The purified protein is a glycoenzyme containing 12% N-linked carbohydrate. Purified ACE had a specific activity of 65 U/mg protein with benzoyl-Gly-His-Leu as substrate. A kinetic analysis showed that the enzyme had the maximal velocity with substrates containing proline at the COOH-terminal end. Inhibitor studies indicated that the enzyme is a metalloprotein. Along the proximal-distal axis of the small intestine, ACE activity is most predominant in the proximal to middle portions, decreasing toward the distal end. This pattern was also observed for ACE mRNA and protein, suggesting that ACE expression is controlled at the level of mRNA. Perfusion of benzoyl-Gly-His-Leu in vivo through a segment of intestinal jejunum demonstrated that ACE is an important intestinal dipeptidyl carboxypeptidase, participating in the digestion and assimilation of dietary peptides.

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