scholarly journals Extension of resolution and oligomerization-state studies of 2,4′-dihydroxyacetophenone dioxygenase fromAlcaligenessp. 4HAP

2015 ◽  
Vol 71 (10) ◽  
pp. 1258-1263 ◽  
Author(s):  
J. Guo ◽  
P. Erskine ◽  
A. R. Coker ◽  
J. Gor ◽  
S. J. Perkins ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Weight ◽  
Formic Acid ◽  
Analytical Ultracentrifugation ◽  
Ralstonia Eutropha ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Structural Basis ◽  
Hydroxybenzoic Acid ◽  
Tetrameric Structure

The enzyme 2,4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid. This enzyme is a very unusual dioxygenase in that it cleaves a C—C bond in a substituent of the aromatic ring rather than within the ring itself. Whilst it has been shown that DAD is a tetramer in solution, the recently solved crystal structure of theAlcaligenessp. 4HAP enzyme was in fact dimeric rather than tetrameric. Since the use of limited chymotrypsinolysis, which apparently results in removal of the first 20 or so N-terminal residues of DAD, was necessary for crystallization of the protein, it was investigated whether this was responsible for the change in its oligomerization state. Gel-filtration and analytical ultracentrifugation studies were conducted, which confirmed that chymotrypsinolysed DAD has an apparent molecular weight of around 40 kDa, corresponding to a dimer. In contrast, the native enzyme has a molecular weight in the 70–80 kDa region, as expected for the tetramer. The structural basis for tetramerization has been investigated by the use of several docking servers, and the results are remarkably consistent with the tetrameric structure of a homologous cupin protein fromRalstonia eutropha(PDB entry 3ebr).

Physico-Chemical Properties of Recombinant Desulphatohirudin

1990 ◽  
Vol 63 (03) ◽  
pp. 499-504 ◽  
Author(s):  
A Electricwala ◽  
L Irons ◽  
R Wait ◽  
R J G Carr ◽  
R J Ling ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Alpha Helix ◽  
Apparent Molecular Weight ◽  
Correlation Spectroscopy ◽  
Nmr Studies ◽  
Recombinant Hirudin ◽  
Physico Chemical ◽  
Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose

1976 ◽  
Vol 54 (2) ◽  
pp. 120-129 ◽  
Author(s):  
W. S. Rickert ◽  
P. A. McBride-Warren
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Affinity Column ◽  
Mucor Miehei ◽  
Highly Active ◽  
Elution Buffer ◽  
Turbidimetric Assay ◽  
Ph Range

The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5–8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65 000 and 52 000 were isolated, the 65 000 molecular weight species apparently representing a protomer of concanavalin A (24 000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity.At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including α-methyl D-glucoside and NaCl in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 °C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor rennin did not react with concanavalin A and were not bound to the affinity column.

scholarly journals Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain

1976 ◽  
Vol 156 (1) ◽  
pp. 143-150 ◽  
Author(s):  
R H Quarles
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Weight Class ◽  
Developmental Difference ◽  
Adult Rats ◽  
Neuraminidase Treatment

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.

Mannose containing glycopeptides of cells derived from human teratocarcinoma (line PA 1)

1980 ◽  
Vol 58 (5) ◽  
pp. 384-393 ◽  
Author(s):  
Maija-Liisa Rasilo ◽  
Jorma Wartiovaara ◽  
Ossi Renkonen
Keyword(s):  
Molecular Weight ◽  
Paper Chromatography ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Con A ◽  
Whole Cell ◽  
Undifferentiated State

Human teratocarcinoma derived cells, line PA 1, maintained in the undifferentiated state, yielded upon exhaustive pronase digestion unusually large glycopeptides (fraction A), which showed on gel filtration an apparent molecular weight larger than 7400. These glycopeptides derived from whole cell proteins carried large-sized oligosaccharides as evidenced by repeated pronase treatments, hydrazinolysis, and β-elimination experiments. The oligosaccharides consisted of mannose, fucose, galactose, and N-acetylglucosamine.The PA 1 cells contained also oligomannosyl type glycans, presumably linked to asparagine (fraction C glycopeptides). These glycopeptides were strongly bound to Con A – Sepharose and their oligosaccharides were released by endo-β-N-acetylglucosaminidase H. The liberated glycans ranged from Man5GlcNAc to Man9GlcNAc as analyzed by paper chromatography."Pulse–chase" experiments suggest that there is a precursor–product relationship between the mannose label in the fraction C (oligomannosyl type) glycopeptides and the fraction A glycopeptides.

scholarly journals Studies on the appearance of a hepatic copper-binding protein in normal and zinc-deficient rats

1976 ◽  
Vol 36 (1) ◽  
pp. 101-112 ◽  
Author(s):  
I. Bremner ◽  
N. T. Davies
Keyword(s):  
Molecular Weight ◽  
Metal Binding ◽  
Binding Protein ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Low Molecular Weight ◽  
Copper Binding ◽  
Hepatic Copper ◽  
Copper Binding Protein ◽  
Molecular Weight Form

1. A study has been made by gel-filtration techniques of the soluble copper- and zinc-binding proteins in rat liver after both intraperitoneal injection of Cu and dietary Cu supplementation.2. Liver Cu and Zn concentrations increased after injection of Cu, both metals accumulating in the cytosol, mainly in a fraction with an apparent molecular weight of (about 12 000)3. When Zn-deficient rats were injected with Cu, there was little change in liver Zn concentration and the occurrence of Cu in the low-molecular-weight form (about 12 000) was more transient. At most periods after injection, Cu accumulated mainly in a fraction with a molecular weight greater than 65 000.4. When the rats were Cu-loaded by dietary supplementation, virtually no Cu or Zn was found in the low-molecular-weight form in Zn-deficient rats, although they were found in the Zn-supplemented animals.5. The results suggest that Zn is essential for the accumulation of Cu in this form, but not for Cu to stimulate production of the metal-binding protein by a process requiring active protein synthesis.

scholarly journals Isolation and function of a human endothelial cell C1q receptor

1993 ◽  
Vol 2 (6) ◽  
pp. 447-452 ◽  
Author(s):  
M. R. Daha ◽  
L. Dunn ◽  
R. van den Berg ◽  
Y. Muizert-de Lange ◽  
A. Gerritsen ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Primary Immune Response ◽  
Reduced Form ◽  
Human Umbilical Cord ◽  
Dependent Manner ◽  
Western Blots

It has been shown previously that cultured human venous and arterial endothelial cells (EC) bind C1q in a time- and dose-dependent manner. Cultured human endothelial cells express an average number of 5.2 × 105binding sites/cell. In the present study the putative receptor for C1q (C1qR) was isolated from the membranes of 1–5 × 109human umbilical cord EC by affinity chromatography on C1q–Sepharose. During isolation, C1qR was detected by its capacity to inhibit the lysis of EAC1q in C1q-deficient serum. The eluate from C1q–Sepharose was concentrated, dialysed and subjected to QAE-A50 chromatography and subsequently to gel filtration on HPLC–TSK 3000. C1qR filtered at an apparent molecular weight of 60 kDa. Purified C1qR exhibited an apparent molecular weight of 55–62 kDa in the unreduced state and a molecular weight of 64–68 kDa in reduced form. Two IgM monoclonal antibodies (mAb) D3 and D5 were raised following immunization of mice with purified receptor preparations. Both monoclonal antibodies increased the binding of125I-C1q to endothelial cells but F(ab')2anti-C1qR mAb inhibited the binding of a125I-C1q to EC in a dosedependent manner. The D3 mAb recognized a band of 54–60 kDa in Western blots of membranes of human EC and polymorphonuclear leukocytes. Previously, the authors showed that C1q induces the binding of IgM-containing immune complexes to EC. Therefore, it was hypothesized that during a primary immune response generation of IgM-IC may occur, resulting in binding and activation of C1, dissociation of activated C1 by C1 inhibitor and subsequent interaction of IgM-IC bearing C1q with EC–C1qR.

scholarly journals Macromolecules stimulating human granulocytic colony-forming cells, precursors of these cells, and primitive erythroid progenitors: some apparent nonidentities

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 960-966 ◽  
Author(s):  
T Hoang ◽  
NN Iscove ◽  
N Odartchenko
Keyword(s):  
Molecular Weight ◽  
Conditioned Medium ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
The Other ◽  
Erythroid Progenitors ◽  
Granulocytic Colony ◽  
Ph Range ◽  
Direct Primary ◽  
The Relationship

Abstract The relationship between molecules having granulocyte colony- stimulating activity (G-CSA), erythroid burst-promoting activity (E- BPA), and activity promoting increase in the number of granulocytic progenitors in liquid culture (delta GPA) was explored in conditioned medium from human leukocytes (HLCM) and human placenta (HPCM). As tested on human hemopoietic progenitors in culture, G-CSA eluted from Sephadex G100 as a single peak with apparent molecular weight of 25,000, separating partially from E-BPA and delta GPA, which both had an apparent molecular weight of 45,000. All three activities eluted together from hydroxyapatite at low molarity phosphate. Their charge properties were also similar and all three electrofocused in flat gel beds in the pH range near 5.4. On both hydroxyapatite and isoelectric focusing, delta GPA sometimes separated partially from the other two activities but not consistently. The gel filtration result shows that in conditioned medium of human origin, molecules having G-CSA are not the same as those having delta GPA, suggesting a dual factor requirement in the granulocytic lineage reminiscent of that in the erythroid pathway. The results suggesting that delta GPA might differ from E-BPA, on the other hand, were not consistent enough to establish their nonidentity. Single micromanipulated cells proved capable of forming erythroid or granulocytic colonies in the presence of either crude or partially purified activity. The results establish that human colony-forming cells are direct primary targets of growth factors in HLCM and HPCM.

scholarly journals The Structure and Function of Modular Escherichia coli O157:H7 Bacteriophage FTBEc1 endolysin, LysT84: Defining a New Endolysin Catalytic Subfamily

2021 ◽  
Author(s):  
Michael Love ◽  
David Coombes ◽  
Salim Ismail ◽  
Craig Billington ◽  
Renwick CJ Dobson
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Antibacterial Agents ◽  
Analytical Ultracentrifugation ◽  
Structural Data ◽  
Scattering Data ◽  
Structural Basis ◽  
Escherichia Coli O157 ◽  
Dynamic Simulations ◽  
Enzymatic Function

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level—key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1–4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.

Purification of Ristocetin Willebrand Factor (RWF)

1975 ◽  
Author(s):  
J. D. Olson ◽  
D. N. Fass ◽  
W. J. Brockway ◽  
E. J. W. Bowie ◽  
K. G. Mann
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Amino Sugar ◽  
Procoagulant Activity ◽  
Factor Viii Inhibitor ◽  
Induced Aggregation

A component required for the ristocetin-induced aggregation of platelets was isolated in yields of 20-30% from pooled human and porcine plasma by cryoethanol concentration of the RWF, after removal of the vitamin K dependent coagulation factors on barium citrate. The concentrate was further purified using gel filtration (4% agarose) and ion exchange (DEAE-cellulose) chromatography. Sodium dodecyl sulfate polyacrylamide gels of the isolated factor indicated an apparent molecular weight of greater than 500,000. After reduction of RWF with mercaptoethanol, a single band is resolved with an apparent molecular weight of 230,000. The purified component had no factor VIII procoagulant activity and did not compete for the activity of naturally occurring factor VIII inhibitor (human). Antisera raised in rabbits directed against the purified component inhibited the RWF activity but not the factor VIII procoagulant activity of plasma. Amino acid analysis indicated the presence of all normal amino acids and failed to detect any amino sugar. Analysis of lipid revealed a significant amount of lipid composed of mono, di and triglycerides, cholesterol, cholesterol esters and free fatty acid, with small portions of phospholipids.

Characterisation of a Neutral Protease Produced by Micrococcus luteus

1996 ◽  
Vol 51 (5-6) ◽  
pp. 429-431 ◽  
Author(s):  
M.O. Ilori ◽  
O.O. Amund ◽  
O. Omidiji
Keyword(s):  
Molecular Weight ◽  
Citric Acid ◽  
Proteolytic Enzyme ◽  
Micrococcus Luteus ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Optimum Ph

Abstract A proteolytic enzyme produced by a cassava-ferment­ing strain of Micrococcus luteus was extracted and puri­fied 50-fold by gel filtration and ion exchange chromatography. The optimum pH for the enzyme was 7.0, the optimum temperature 25 °C, the apparent molecular weight 42 kDa and the Km value, 0.45 mg ml-1 with casein as substrate. The enzyme was stimulated by Ca2+ and Mg2+ but inhibited by Zn2+ and Co2+ ions. Other inhibitors were EDTA, KCN, citric acid and L-cysteine indicating the enzyme to be a metalloprotease.

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