scholarly journals The biosynthesis of GDP-d-arabinopyranose in Crithidia fasciculata: characterization of a d-arabino-1-kinase activity and its use in the synthesis of GDP-[5-3H]d-arabinopyranose

1995 ◽  
Vol 311 (1) ◽  
pp. 307-315 ◽  
Author(s):  
P Schneider ◽  
A Nikolaev ◽  
M A Ferguson
Keyword(s):  
Molecular Mass ◽  
Kinase Activity ◽  
Substrate Inhibition ◽  
Size Exclusion ◽  
Crithidia Fasciculata ◽  
Optimum Ph ◽  
Exclusion Chromatography ◽  
Trypanosomatid Parasites

GDP-D-arabinopyranose (GDP-D-Ara) is the precursor of the uncommon D-arabinopyranose residues present in the glycoconjugates of a few trypanosomatid parasites. Biosynthetic labelling experiments with Crithidia fasciculata showed that GDP-D-Ara could be labelled with [3H]D-Ara, [2-3H]D-Glc and [6-3H]D-Glc, but not with [1-3H]D-Glc, suggesting that D-Ara can be either taken up directly by the parasite or derived from D-Glc through a pathway involving the loss of carbon C-1. In vivo pulse-chase experiments indicated that D-Ara was sequentially incorporated into D-Ara-1-PO4 and GDP-D-Ara prior to transfer to the acceptor glycoconjugate, lipoarabinogalactan. An MgATP-dependent D-arabino-1-kinase activity present in soluble extracts of C. fasciculata was purified away from phosphatase activities by size-exclusion chromatography. The D-arabino-1-kinase had an apparent molecular mass of 600 kDa, a neutral optimum pH, and displayed substrate inhibition at D-Ara concentrations above 100 microM. It had a KmATP of 1.7 mM and a KmAra of 24 microM. Competition studies indicated that the orientation of every single hydroxyl residue was important for D-Ara recognition by the enzyme, but that methyl or hydroxymethyl groups could be tolerated as equatorial substituents on C-5 of D-Ara. The partially purified D-arabino-1-kinase activity was used in the chemico-enzymic synthesis of GDP-[5-3H]D-Ara from [6-3H]D-GlcN.

Purification and characterization of an enzyme from a strain ofOchrobactrum anthropithat degrades condensation products of urea and formaldehyde (ureaform)

1997 ◽  
Vol 43 (12) ◽  
pp. 1111-1117 ◽  
Author(s):  
Thomas Jahns ◽  
Roswitha Schepp ◽  
Heinrich Kaltwasser
Keyword(s):  
Molecular Mass ◽  
Enzyme Synthesis ◽  
Size Exclusion ◽  
Ochrobactrum Anthropi ◽  
Purification And Characterization ◽  
Molar Ratios ◽  
Condensation Products ◽  
Tetrameric Structure ◽  
Exclusion Chromatography

An enzyme hydrolyzing the condensation products of urea and formaldehyde (ureaform) was purified and characterized from a bacterium isolated from soil and described as Ochrobactrum anthropi UF4. The enzyme designated as methylenediurea amidinohydrolase (methylenediurea deiminase) hydrolyzed ureaform condensation products of different length (methylenediurea, dimethylenetriurea, trimethylenetetraurea) to ammonium, formaldehyde, and urea at molar ratios of 2:1:1 (methylenediurea), 4:2:1 (dimethylenetriurea), and 6:3:1 (trimethylenetetraurea). Two other substrates, ureidoglycolate and allantoate, were also hydrolyzed, yielding glyoxylate and urea (ureidoglycolate) and glyoxylate, urea, and ammonium (allantoate), respectively. The molecular mass of the enzyme was determined by size exclusion chromatography to be 140 ± 25 kDa; the enzyme was composed of identical subunits of 38 ± 5 kDa, indicating that the native enzyme has a tetrameric structure. Growth of the bacterium in the presence of ureaform specifically induced the methylenediurea deiminase and no complete repression of enzyme synthesis by ammonium was observed.Key words: ureaformaldehyde, methylenediurea deiminase, fertilizer, Ochrobactrum anthropi.

scholarly journals Isolation and characterization of BanLec-I, a mannoside-binding lectin from Musa paradisiac (banana)

1990 ◽  
Vol 272 (3) ◽  
pp. 721-726 ◽  
Author(s):  
V L Koshte ◽  
W van Dijk ◽  
M E van der Stelt ◽  
R C Aalberse
Keyword(s):  
Cell Proliferation ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Size Exclusion ◽  
T Cell Proliferation ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Binding Lectin

A lectin (BanLec-I) from banana (Musa paradisiac) with a binding specificity for oligomannosidic glycans of size classes higher than (Man)6GlcNAc was isolated and purified by affinity chromatography on a Sephadex G-75 column. It did not agglutinate untreated human or sheep erythrocytes, but it did agglutinate rabbit erythrocytes. BanLec-I stimulated T-cell proliferation. On size-exclusion chromatography, BanLec-I has a molecular mass of approx. 27 kDa, and on SDS/PAGE the molecular mass is approx. 13 kDa. The isoelectric point is 7.2-7.5. BanLec-I was found to be very effective as a probe in detecting glycoproteins, e.g. on nitrocellulose blots.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

scholarly journals Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

2021 ◽  
Vol 22 (9) ◽  
pp. 4512
Author(s):  
Michał Marcinkowski ◽  
Tomaš Pilžys ◽  
Damian Garbicz ◽  
Jan Piwowarski ◽  
Damian Mielecki ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Size Exclusion Chromatography ◽  
Posttranslational Modifications ◽  
Size Exclusion ◽  
Saxs Data ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Demethylase Activity ◽  
Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

scholarly journals Fibrillar pharmacology of functionalized nanocellulose

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sam Wong ◽  
Simone Alidori ◽  
Barbara P. Mello ◽  
Bryan Aristega Almeida ◽  
David Ulmert ◽  
...  
Keyword(s):  
Aspect Ratio ◽  
Fluorescent Dyes ◽  
Pharmacokinetic Profile ◽  
Water Soluble ◽  
Size Exclusion ◽  
Hydroxyl Groups ◽  
Target Tissue ◽  
Exclusion Chromatography ◽  
Specific Accumulation

AbstractCellulose nanocrystals (CNC) are linear organic nanomaterials derived from an abundant naturally occurring biopolymer resource. Strategic modification of the primary and secondary hydroxyl groups on the CNC introduces amine and iodine group substitution, respectively. The amine groups (0.285 mmol of amine per gram of functionalized CNC (fCNC)) are further reacted with radiometal loaded-chelates or fluorescent dyes as tracers to evaluate the pharmacokinetic profile of the fCNC in vivo. In this way, these nanoscale macromolecules can be covalently functionalized and yield water-soluble and biocompatible fibrillar nanoplatforms for gene, drug and radionuclide delivery in vivo. Transmission electron microscopy of fCNC reveals a length of 162.4 ± 16.3 nm, diameter of 11.2 ± 1.52 nm and aspect ratio of 16.4 ± 1.94 per particle (mean ± SEM) and is confirmed using atomic force microscopy. Size exclusion chromatography of macromolecular fCNC describes a fibrillar molecular behavior as evidenced by retention times typical of late eluting small molecules and functionalized carbon nanotubes. In vivo, greater than 50% of intravenously injected radiolabeled fCNC is excreted in the urine within 1 h post administration and is consistent with the pharmacological profile observed for other rigid, high aspect ratio macromolecules. Tissue distribution of fCNC shows accumulation in kidneys, liver, and spleen (14.6 ± 6.0; 6.1 ± 2.6; and 7.7 ± 1.4% of the injected activity per gram of tissue, respectively) at 72 h post-administration. Confocal fluorescence microscopy reveals cell-specific accumulation in these target tissue sinks. In summary, our findings suggest that functionalized nanocellulose can be used as a potential drug delivery platform for the kidneys.

scholarly journals Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nicole L. McIntosh ◽  
Geoffrey Y. Berguig ◽  
Omair A. Karim ◽  
Christa L. Cortesio ◽  
Rolando De Angelis ◽  
...  
Keyword(s):  
Light Scattering ◽  
Size Exclusion Chromatography ◽  
Molar Mass ◽  
Size Exclusion ◽  
Minimal Sample ◽  
Accuracy And Precision ◽  
Vector Genome ◽  
Exclusion Chromatography ◽  
Novel Applications

AbstractAdeno associated virus (AAV) capsids are a leading modality for in vivo gene delivery. Complete and precise characterization of capsid particles, including capsid and vector genome concentration, is necessary to safely and efficaciously dose patients. Size exclusion chromatography (SEC) coupled to multiangle light scattering (MALS) offers a straightforward approach to comprehensively characterize AAV capsids. The current study demonstrates that this method provides detailed AAV characterization information, including but not limited to aggregation profile, size-distribution, capsid content, capsid molar mass, encapsidated DNA molar mass, and total capsid and vector genome titer. Currently, multiple techniques are required to generate this information, with varying accuracy and precision. In the current study, a new series of equations for SEC-MALS are used in tandem with intrinsic properties of the capsids and encapsidated DNA to quantify multiple physical AAV attributes in one 20-min run with minimal sample manipulation, high accuracy, and high precision. These novel applications designate this well-established method as a powerful tool for product development and process analytics in future gene therapy programs.

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