scholarly journals Insolubilized enzymes. Kinetic behaviour of glucose oxidase bound to porous glass particles

1971 ◽  
Vol 124 (4) ◽  
pp. 801-807 ◽  
Author(s):  
Michael K. Weibel ◽  
Harold J. Bright
Keyword(s):  
Glucose Oxidase ◽  
Porous Glass ◽  
Fold Increase ◽  
Kinetic Mechanism ◽  
Diffusion Control ◽  
Kinetic Properties ◽  
Substrate Diffusion ◽  
Catalytically Active ◽  
Absolute Magnitudes ◽  
Covalently Linked

1. The spectrophotometric and steady-state kinetic properties of glucose oxidase (EC 1.1.3.4, from Aspergillus niger) that is covalently linked to porous glass beads have been examined. These properties have been compared with those of soluble glucose oxidase, for which the kinetic mechanism at pH5.5 and 25°C has been established previously by a combination of conventional and rapid-reaction techniques to be the following: [Formula: see text] where Eo and Er represent oxidized and reduced forms of the enzyme, respectively. 2. The ratio k+4/k+2 is unchanged after insolubilization, and evidence is presented which suggests that the absolute magnitudes of k+4 and k+2 are unchanged. 3. The kinetic efficiency of the insolubilized enzyme is greatly enhanced because of a 14-fold increase in the apparent affinity of glucose for Eo. This effect is attributed either to the binding of glucose to the glass surface or to a change in enzyme structure imposed by the insolubilization process. 4. Only 6% of the insolubilized enzyme which can be reduced by glucose is catalytically active. It is shown by calculation and direct experimental evidence that this fraction of catalytically active enzyme is bound to the exterior bead surface. The remaining 94% of the enzyme is bound within the pore network and may be subject to severe substrate diffusion control.

scholarly journals The roles of the conserved pyrimidine bases in hammerhead ribozyme catalysis: evidence for a magnesium ion-binding site

1995 ◽  
Vol 311 (2) ◽  
pp. 487-494 ◽  
Author(s):  
J B Murray ◽  
C J Adams ◽  
J R P Arnold ◽  
P G Stockley
Keyword(s):  
Binding Site ◽  
Hammerhead Ribozyme ◽  
Fold Increase ◽  
Magnesium Ion ◽  
Ion Binding ◽  
Kinetic Properties ◽  
Direct Role ◽  
Single Turnover ◽  
Catalytically Active ◽  
Pyrimidine Bases

We report details of the synthesis and characterization of oligoribonucleotides containing 4-thiouridine or 2-pyrimidinone ribonucleoside (4HC). We have used these probes to examine the roles of the conserved pyrimidines in the central core of the hammerhead ribozyme. The effects on catalysis of singly-substituted hammerhead ribozyme and substrate strands were quantified in multiple-turnover reactions. Various effects were observed on kcat. and Km, with up to a 7-fold decrease and a 3-fold increase respectively. For substitutions with 4HC at positions 3 or 17, catalytic activity in single turnover reactions can be increased up to 8-fold equivalent to 40% of wild-type activity, by increasing the concentration of the Mg2+ cofactor, implying that these substitutions had a deleterious effect on Mg2+ binding. Calculations of the change in the apparent free energy of binding for variants at positions 3, 4 or 17 are each consistent with deletion of a single hydrogen-bond to an uncharged group in the ribozyme. The cytidine 5′ to the scissile phosphate had not previously been thought to play a direct role in catalysis, however, removal of the exocyclic amino group decreased kcat. 4-fold. Recently, the crystal structures of a hammerhead ribozyme bound to either a non-cleavable 2′-deoxy substrate strand or a ribo-substrate strand have been reported. The kinetic properties of the variants described here are consistent with several key interactions seen in the crystals, in particular they provide experimental support for the assignment of the proposed catalytically active magnesium ion-binding site.

scholarly journals A validated collection of mouse monoclonal antibodies to human glycosyltransferases functioning in mucin-type O-glycosylation

Glycobiology ◽  
2019 ◽  
Vol 29 (9) ◽  
pp. 645-656 ◽  
Author(s):  
Catharina Steentoft ◽  
Zhang Yang ◽  
Shengjun Wang ◽  
Tongzhong Ju ◽  
Malene B Vester-Christensen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Secretory Pathway ◽  
Expression Patterns ◽  
In Situ Monitoring ◽  
Kinetic Properties ◽  
Detailed Knowledge ◽  
Cellular Expression ◽  
Wide Range ◽  
Catalytically Active ◽  
In Cells

Abstract Complex carbohydrates serve a wide range of biological functions in cells and tissues, and their biosynthesis involves more than 200 distinct glycosyltransferases (GTfs) in human cells. The kinetic properties, cellular expression patterns and subcellular topology of the GTfs direct the glycosylation capacity of a cell. Most GTfs are ER or Golgi resident enzymes, and their specific subcellular localization is believed to be distributed in the secretory pathway according to their sequential role in the glycosylation process, although detailed knowledge for individual enzymes is still highly fragmented. Progress in quantitative transcriptome and proteome analyses has greatly advanced our understanding of the cellular expression of this class of enzymes, but availability of appropriate antibodies for in situ monitoring of expression and subcellular topology have generally been limited. We have previously used catalytically active GTfs produced as recombinant truncated secreted proteins in insect cells for generation of mouse monoclonal antibodies (mAbs) to human enzymes primarily involved in mucin-type O-glycosylation. These mAbs can be used to probe subcellular topology of active GTfs in cells and tissues as well as their presence in body fluids. Here, we present several new mAbs to human GTfs and provide a summary of our entire collection of mAbs, available to the community. Moreover, we present validation of specificity for many of our mAbs using human cell lines with CRISPR/Cas9 or zinc finger nuclease (ZFN) knockout and knockin of relevant GTfs.

Nitroalkanes as Reductive Substrates for Flavoprotein Oxidases

1972 ◽  
Vol 27 (9) ◽  
pp. 1052-1053 ◽  
Author(s):  
David J. T. Porter ◽  
Judith G. Voet ◽  
Harold J. Bright
Keyword(s):  
Hydrogen Peroxide ◽  
Amino Acid ◽  
Glucose Oxidase ◽  
Ph Dependence ◽  
Kinetic Mechanism ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Kinetics Of ◽  
Oxidase Reaction ◽  
Detailed Kinetic Mechanism

Nitroalkanes have been found to be general reductive substrates for D-amino acid oxidase, glucose oxidase and L-amino acid oxidase. These enzymes show different specificities for the structure of the nitroalkane substrate.The stoichiometry of the D-amino acid oxidase reaction is straightforward, consisting of the production of one mole each of aldehyde, nitrite and hydrogen peroxide for each mole of nitroalkane and oxygen consumed. The stoichiometry of the glucose oxidase reaction is more complex in that less than one mole of hydrogen peroxide and nitrite is produced and nitrate and traces of 1-dinitroalkane are formed.The kinetics of nitroalkane oxidation show that the nitroalkane anion is much more reactive in reducing the flavin than is the neutral substrate. The pH dependence of flavin reduction strongly suggests that proton abstraction is a necessary event in catalysis. A detailed kinetic mechanism is presented for the oxidation of nitroethane by glucose.It has been possible to trap a form of modified flavin in the reaction of D-amino acid oxidase with nitromethane from which oxidized FAD can be regenerated in aqueous solution in the presence of oxygen.

Regulation of Fructose 1,6-Bisphosphatase Activity of Chlorella by Mole Mass Change

1996 ◽  
Vol 51 (9-10) ◽  
pp. 639-645 ◽  
Author(s):  
N. Grotjohann
Keyword(s):  
Enzyme Regulation ◽  
Fold Increase ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Enzyme Form ◽  
Phosphorylated Compounds ◽  
Fructose 1,6 Bisphosphatase ◽  
Chloroplast Stroma ◽  
Fructose 1,6 Bisphosphate

Fast protein liquid chromatography on Superose 6 of partially purified FBPase II from Chlorella reveals a 1350 kDa-form at pH 6.0 and a 67 kDa-form at pH 8.5. Treatment of the large enzyme form with 5mᴍ concentrations of Mg2+, F1,6P2, DTT or ATP leads to dissociation into smaller ones of 215 -470 kDa. Aggregation/dissoziation is a reversible process, as has been shown for the effect of F1,6P2 and of pH, by rechromatography. The change in mole mass results in alterations of the activitiy and of the kinetic properties of the enzyme forms, obtained. Dissociation results in a 4 - 6 fold increase in activity, as can be shown for F1,6P2-treated samples. Halfsaturation constants, as well as the degree of cooperativity of the 67- and the 1350- kDa form, are different for substrate affinity, activation by Mg2+ and DTT, and for inhibition by ATP. Both enzyme forms hydrolyse fructose 1,6 bisphosphate and seduheptulose 1,7 bisphosphate better than other phosphorylated compounds. The ratio of F1,6P2- to SDP-cleavage is 100:58 for the small enzyme form and 100: 84 for the large one. Activation of FBPase II in the light and inactivation in the dark is discussed on the basis of different oligomeric forms of the enzyme, generated by changes in the concentration of intermediates and effectors in the chloroplast stroma, leading to dissociation or aggregation. The conclusion is drawn that oligomerization of key enzymes, resulting in enzyme forms with different activities and different kinetic properties, might provide an effective mechanism for enzyme regulation in vivo

Kinetic Study on Microwave Magnetizing Roast of Fe2O3 Powders

2020 ◽  
Vol 304 ◽  
pp. 91-97
Author(s):  
Lei Wang ◽  
Ling Bing Kong ◽  
Pei Min Guo ◽  
Jie Li
Keyword(s):  
Reaction Rate ◽  
Reaction Kinetic ◽  
Three Dimensional ◽  
Kinetic Mechanism ◽  
Electrical Heating ◽  
Diffusion Control ◽  
Weight Loss Curve ◽  
Dimensional Diffusion ◽  
Boundary Reaction ◽  
Phase Boundary Reaction

In this paper, the reaction kinetic mechanism of Fe2O3 powder containing carbon was studied by microwave magnetizing roast. Based on the temperature-rise curve and weight loss curve of Fe2O3 powder by microwave magnetizing roast, the kinetic parameters of Fe2O3 powder microwave magnetizing roast were calculated by non-isothermal methods. The controlling steps of different temperature-rising periods in microwave magnetizing roast process of Fe2O3 powder were calculated by the Achar-Brindley-Sharp-Wendworth method. The results indicated that the controlling step of microwave magnetizing roast was phase boundary reaction control of contracted cylinder in 250~450°C, and it was three-dimensional diffusion control of spherical symmetry in 450~650°C. The results showed that the starting temperature of reduction roasting of Fe2O3 powder was 250°C, which was lower than that under electrical heating, thereby, it proved in theory that microwave heating can enhance reaction rate.

scholarly journals The Yeast Nα-Acetyltransferase NatA Is Quantitatively Anchored to the Ribosome and Interacts with Nascent Polypeptides

2003 ◽  
Vol 23 (20) ◽  
pp. 7403-7414 ◽  
Author(s):  
Matthias Gautschi ◽  
Sören Just ◽  
Andrej Mun ◽  
Suzanne Ross ◽  
Peter Rücknagel ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mating Type ◽  
Temperature Sensitivity ◽  
Temperature Sensitive ◽  
Wide Range ◽  
Close Proximity ◽  
N Terminus ◽  
Catalytically Active ◽  
Covalently Linked ◽  
Silent Mating Type Loci

ABSTRACT The majority of cytosolic proteins in eukaryotes contain a covalently linked acetyl moiety at their very N terminus. The mechanism by which the acetyl moiety is efficiently transferred to a large variety of nascent polypeptides is currently only poorly understood. Yeast Nα -acetyltransferase NatA, consisting of the known subunits Nat1p and the catalytically active Ard1p, recognizes a wide range of sequences and is thought to act cotranslationally. We found that NatA was quantitatively bound to ribosomes via Nat1p and contained a previously unrecognized third subunit, the Nα -acetyltransferase homologue Nat5p. Nat1p not only anchored Ard1p and Nat5p to the ribosome but also was in close proximity to nascent polypeptides, independent of whether they were substrates for Nα -acetylation or not. Besides Nat1p, NAC (nascent polypeptide-associated complex) and the Hsp70 homologue Ssb1/2p interact with a variety of nascent polypeptides on the yeast ribosome. A direct comparison revealed that Nat1p required longer nascent polypeptides for interaction than NAC and Ssb1/2p. Δnat1 or Δard1 deletion strains were temperature sensitive and showed derepression of silent mating type loci while Δnat5 did not display any obvious phenotype. Temperature sensitivity and derepression of silent mating type loci caused by Δnat1 or Δard1 were partially suppressed by overexpression of SSB1. The combination of data suggests that Nat1p presents the N termini of nascent polypeptides for acetylation and might serve additional roles during protein synthesis.

Properties of Inosinic Acid Dehydrogenase from Bacillus subtilis. I. Purification and Physical Properties

1973 ◽  
Vol 51 (10) ◽  
pp. 1380-1390 ◽  
Author(s):  
Tai-Wing Wu ◽  
K. G. Scrimgeour
Keyword(s):  
Bacillus Subtilis ◽  
Quaternary Structure ◽  
Active Form ◽  
Kinetic Properties ◽  
Purification Method ◽  
Molecular Weights ◽  
Inosinic Acid ◽  
Apparent Homogeneity ◽  
Catalytically Active ◽  
Membrane Bound

IMP (inosinic acid or inosine-5′-phosphate) dehydrogenase has been purified to apparent homogeneity from Bacillus subtilis. The purification method yields an enzyme preparation that retains a constant level of inhibition by guanosine 5′-phosphate. The enzyme is membrane bound, and can be removed from membrane material after treatment either with detergents or with phospholipase A. Both the membrane-bound and solubilized forms of IMP dehydrogenase have similar kinetic properties. The soluble enzyme can occur in a number of oligomeric forms, with molecular weights that are multiples of 100 000 daltons. Although both the tetramer and the dimer appear to be catalytically active, no conclusions can yet be drawn about the quaternary structure of the enzymically active form(s).

scholarly journals Enhanced Performance of Bioelectrodes Made with Amination-Modified Glucose Oxidase Immobilized on Carboxyl-Functionalized Ordered Mesoporous Carbon

Nanomaterials ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3086
Author(s):  
Chuhan Lv ◽  
Xuewei Yang ◽  
Zongkang Wang ◽  
Ming Ying ◽  
Qingguo Han ◽  
...  
Keyword(s):  
Glucose Oxidase ◽  
Mesoporous Carbon ◽  
Fold Increase ◽  
Electrochemical Analysis ◽  
Electron Transfer Rate ◽  
Anodic Reaction ◽  
Ordered Mesoporous ◽  
Electron Transfer Rate Constant ◽  
Improved Performance ◽  
Charge Transfer Coefficient

This research reveals the improved performance of bioelectrodes made with amination-modified glucose oxidase (GOx-NH2) and carboxyl-functionalized mesoporous carbon (OMC-COOH). Results showed that when applied with 10 mM EDC amination, the functional groups of NH2 were successfully added to GOx, according to the analysis of 1H-NMR, elemental composition, and FTIR spectra. Moreover, after the aminated modification, increased enzyme immobilization (124.01 ± 1.49 mg GOx-NH2/g OMC-COOH; 2.77-fold increase) and enzyme activity (1.17-fold increase) were achieved, compared with those of non-modified GOx. Electrochemical analysis showed that aminated modification enhanced the peak current intensity of Nafion/GOx-NH2/OMC-COOH (1.32-fold increase), with increases in the charge transfer coefficient α (0.54), the apparent electron transfer rate constant ks (2.54 s−1), and the surface coverage Γ (2.91 × 10−9 mol·cm−2). Results showed that GOx-NH2/OMC-COOH exhibited impressive electro-activity and a favorable anodic reaction.

Effect of aldosterone on abundance and phosphorylation kinetics of Na-K-ATPase of toad urinary bladder

1984 ◽  
Vol 246 (4) ◽  
pp. F509-F516
Author(s):  
C. S. Park ◽  
I. S. Edelman
Keyword(s):  
Urinary Bladder ◽  
Cell Membranes ◽  
Specific Activity ◽  
Short Circuit ◽  
Fold Increase ◽  
Sodium Deoxycholate ◽  
Kinetic Properties ◽  
Toad Urinary Bladder ◽  
Bladder Cell ◽  
Phosphorylation Reaction

The possibility was explored that aldosterone-dependent modulation of either the abundance or the kinetic properties of the Na+ pump (Na-K-ATPase) is involved in the mechanism of natriferic action on toad urinary bladder. Cell membranes from the epithelium of urinary bladders of toads were prepared by a sucrose-Ficoll step gradient method, which yielded a 10-fold increase in specific activity of plasma membrane marker enzymes and only minor contamination with other subcellular fractions. Phosphorylation of Na-K-ATPase by gamma-PO4 of [gamma-32P]ATP in the presence of Mg2+ was Na+ dependent, whereas dephosphorylation was K+ dependent. Km for phosphorylation by ATP was 3 X 10(-8) M and K1/2 (half-maximal stimulation) for Na+ was 13 +/- 2 mM. Pretreatment of cell membranes with sodium deoxycholate (DOC) increased the maximal extent of phosphorylation (Nmax) about threefold without changing the Km for ATP. Aldosterone (5 X 10(-8) M) stimulated transepithelial Na+ transport two- to threefold in 5 h but had no significant effect on Km for ATP in the phosphorylation reaction (with or without activation by DOC) or on the abundance of the enzyme inferred from Nmax of phosphorylation in the absence of activation by DOC. After pretreatment with DOC, average Nmax was 13% greater in the aldosterone-treated population. Regression analysis of these responses revealed no significant correlation between increments in short-circuit currents and Nmax values of phosphorylation with or without pretreatment with DOC.(ABSTRACT TRUNCATED AT 250 WORDS)

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