Control of glycoprotein synthesis. The use of oligosaccharide substrates and HPLC to study the sequential pathway for N-acetylglucosaminyltransferases I, II, III, IV, V, and VI in the biosynthesis of highly branched N-glycans by hen oviduct membranes

1988 ◽  
Vol 66 (10) ◽  
pp. 1134-1151 ◽  
Author(s):  
Inka Brockhausen ◽  
Jeremy P. Carver ◽  
Harry Schachter
Keyword(s):  
Enzyme Assay ◽  
Glycoprotein Synthesis ◽  
Substrate Specificities ◽  
Multiple Products ◽  
Glcnac Termini

Glycoproteins isolated from hen oviduct contain highly branched asparagine-linked oligosaccharides (N-glycans). Six N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V, and VI) are involved in initiating the synthesis of these branches, as indicated below:[Formula: see text]where R is GlcNAcβ1—4(+/−Fucα1—6)GlcNAcAsn-X. HPLC has been used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct. Oligosaccharides with free reducing GlcNAc termini were prepared from various glycoproteins by hydrazinolysis–re-N-acetylation and used as GlcNAc-T substrates and HPLC standards. Enzyme assay components were separated on AG1 × 8, followed by HPLC on amine-bonded silica columns eluted with acetonitrile–water mixtures. Absorbance at 195 nm and radioactivity of eluted compounds were monitored. Substrates and products were identified by comparison of their retention times with those of oligosaccharides with known structures. Enzyme assay by HPLC is more rapid and convenient than previous GlcNAc-T assays using lectin columns or electrophoresis. Since some substrates yielded multiple products, these could be used to assay more than one GlcNAc-T in the same incubation. GlcNAc-T VI was shown to act on both bisected and nonbisected GlcNAc-terminating tetraantennary oligosaccharide substrates; GlcNAc-T II, IV, and V acted poorly or not at all on bisected substrates. GlcNAc-T V was the only enzyme among the six transferases studied that could be assayed in the absence of Mn2+.

Isoperoxidases of (IAA oxidase) oxidase in oat coleoptiles

1973 ◽  
Vol 51 (11) ◽  
pp. 2047-2052 ◽  
Author(s):  
William R. Gordon ◽  
James H. M. Henderson
Keyword(s):  
Acetic Acid ◽  
Avena Sativa ◽  
Gel Electrophoresis ◽  
Enzyme Assay ◽  
Polyacrylamide Gel Electrophoresis ◽  
Acid Oxidase ◽  
Iaa Oxidase ◽  
Substrate Specificities ◽  
Electrophoretic Mobilities ◽  
Indole 3 Acetic Acid

Eight constitutive isoperoxidases were separated by the disc method of polyacrylamide gel electrophoresis from a lyophilized extract of 8-day-old oat (Avena sativa L., cv. Victory) coleoptiles. Both anodic and cathodic isoperoxidases were studied and differences in electrophoretic mobilities and hydrogen donor substrate specificities were revealed. In addition, by enzyme assay, cathodic and anodic isoenzymes were shown to possess differences in peroxidase and IAA (indole-3-acetic acid) oxidase activities.Treatment of coleoptiles with 0.07 mM IAA for 24 h resulted in the repression of two slow-migrating anodic isoperoxidases; however, the same treatment also resulted in the induction of two slow-migrating cathodic isoenzymes which were shown to exhibit peroxidase and IAA oxidase activities.

Enzyme assay to study the influence of citalopram on the concentration of psychotropic comedication

2011 ◽  
Vol 44 (06) ◽  
Author(s):  
R Brandl ◽  
R Köber ◽  
T Jahner ◽  
A Dörfelt ◽  
E Haen
Keyword(s):  
Enzyme Assay

Synthesis and Photochemical Properties of Re(I) Tricarbonyl Complexes Bound to Thione and Thiazole-2-ylidene Ligands

2020 ◽  
Author(s):  
Matthew Stout ◽  
Brian Skelton ◽  
Alexandre N. Sobolev ◽  
Paolo Raiteri ◽  
Massimiliano Massi ◽  
...  
Keyword(s):  
Charge Transfer ◽  
Excited States ◽  
Ligand Exchange ◽  
Ligand Substitution ◽  
Bidentate Ligand ◽  
The Other ◽  
Ligand Exchange Reaction ◽  
Multiple Products ◽  
Ligand Charge Transfer ◽  
Photochemical Properties

<p>Three Re(I) tricarbonyl complexes, with general formulation Re(N^L)(CO)<sub>3</sub>X (where N^L is a bidentate ligand containing a pyridine functionalized in the position 2 with a thione or a thiazol-2-ylidene group and X is either chloro or bromo) were synthesized and their reactivity explored in terms of solvent-dependent ligand substitution, both in the ground and excited states. When dissolved in acetonitrile, the complexes bound to the thione ligand underwent ligand exchange with the solvent resulting in the formation of Re(NCMe)<sub>2</sub>(CO)<sub>3</sub>X. The exchange was found to be reversible, and the starting complex was reformed upon removal of the solvent. On the other hand, the complexes appeared inert in dichloromethane or acetone. Conversely, the complex bound to the thiazole-2-ylidene ligand did not display any ligand exchange reaction in the dark, but underwent photoactivated ligand substitution when excited to its lowest metal-to-ligand charge transfer manifold. Photolysis of this complex in acetonitrile generated multiple products, including Re(I) tricarbonyl and dicarbonyl solvato-complexes as well as free thiazole-2-ylidene ligand.</p>

A kinetic analysis of coupled sequential enzyme reactions

2018 ◽  
Author(s):  
Justin Eilertsen ◽  
Santiago Schnell
Keyword(s):  
Enzyme Assay ◽  
Sequential Reaction ◽  
Enzyme Reactions ◽  
Indicator Reaction ◽  
Single Substrate ◽  
Complex Sequences ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Single Enzyme

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis--Menten reaction mechanism. The sequential reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis--Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis--Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

A kinetic analysis of coupled sequential enzyme reactions

2018 ◽  
Author(s):  
Justin Eilertsen ◽  
Santiago Schnell
Keyword(s):  
Enzyme Assay ◽  
Sequential Reaction ◽  
Enzyme Reactions ◽  
Indicator Reaction ◽  
Single Substrate ◽  
Complex Sequences ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Single Enzyme

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis-Menten reaction mechanism. The sequential reaction consists of a single-substrate, single enzyme non-observable reaction followed by another single-substrate, single enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis-Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis-Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

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