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Enzyme assay

About: Enzyme assay is a(n) research topic. Over the lifetime, 25406 publication(s) have been published within this topic receiving 690706 citation(s).


Papers
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Journal ArticleDOI
TL;DR: A photometric method for determining acetylcholinesterase activity of tissue extracts, homogenates, cell suspensions, etc., has been described and Kinetic constants determined by this system for erythrocyte eholinesterases are presented.
Abstract: A photometric method for determining acetylcholinesterase activity of tissue extracts, homogenates, cell suspensions, etc., has been described. The enzyme activity is measured by following the increase of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion. It is based on coupling of these reactions: The latter reaction is rapid and the assay is sensitive (i.e. a 10 μ1 sample of blood is adequate). The use of a recorder has been most helpful, but is not essential. The method has been used to study the enzyme in human erythrocytes and homogenates of rat brain, kidney, lungs, liver and muscle tissue. Kinetic constants determined by this system for erythrocyte eholinesterase are presented. The data obtained with acetylthiocholine as substrate are similar to those with acetylcholine.

20,867 citations

Journal ArticleDOI
TL;DR: A sensitive, fixed-time, spectrophotometric assay for angiotensin-converting enzyme measures the rate of production of hippuric acid from hippuryl-L -histidyl- L -leucine (HHL).
Abstract: A sensitive, fixed-time, spectrophotometric assay for angiotensin-converting enzyme measures the rate of production of hippuric acid from hippuryl- L -histidyl- L -leucine (HHL). The angiotensin-converting enzyme from rabbit lung acetone powder extract, when assayed by this method, is optimally active at pH 8.1 to 8.3 at a chloride ion concentration of 300 mM and an HHL concentration of 5–10 mM; the K m for HHL is 2–6 mM. The enzyme was inhibited by metal-chelating agents, heavy metal salts and certain peptides. The most effective inhibitors were EDTA; CdBr 2 ; angiotensin II; bradykinin; and a pentapeptide, L -pyroglutamyl- L -lysyl- L- tryptophyl- L -alanyl- L -proline, a component of Bothrops jararaca venom. Enzyme inhibited by 0.1 mM EDTA was completely reactivated after removal of EDTA by dialysis but, after prolonged dialysis of the enzyme against 1 mM EDTA, reactivation could only be achieved by addition of metal ions: MnCI 2 (40%), ZnCl 2 (100%) or Co (NO 3 )2 (160%). The angiotensin-converting enzyme of rabbit lung is a stable, chloride ion-activated metalloenzyme, similar to both the angiotensin-converting enzyme and kininase II of plasma.

2,387 citations

Book
01 Jan 1979
TL;DR: Basic Principles of Chemical Kinetics Introduction to Enzyme Kinetics "Alternative" Enzymes Practical Aspects of Kinetics Deriving Steady-state Rate Equations Reversible Inhibition and Activation Tight-binding and Irreversible Inhibitors
Abstract: Basic Principles of Chemical Kinetics Introduction to Enzyme Kinetics "Alternative" Enzymes Practical Aspects of Kinetics Deriving Steady-state Rate Equations Reversible Inhibition and Activation Tight-binding and Irreversible Inhibitors Reactions of More than One Substrate Use of Isotopes for Studying Enzyme Mechanisms Effect of pH on Enzyme Activity Temperature Effects on Enzyme Activity Regulation of Enzyme Activity Multienzyme Systems Fast Reactions Estimation of Kinetic Constants Standards for Reporting Enzymology Data Solutions and Notes to Problems Index

2,219 citations

Journal ArticleDOI
02 Dec 1999-Nature
TL;DR: A membrane-bound enzyme activity that cleaves full-length APP at the β-secretase cleavage site is described and found to be the predominant β-cleavage activity in human brain, and it is found that human brain β- secretase is a new membrane- bound aspartic proteinase.
Abstract: Proteolytic processing of the amyloid precursor protein (APP) generates amyloid β (Aβ) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease Cleavage by β-secretase at the amino terminus of the Aβ peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of β-cleaved soluble APP1, and a corresponding cell-associated carboxy-terminal fragment Cleavage of the C-terminal fragment by γ-secretase(s) leads to the formation of Aβ The pathogenic mutation K670M671 → N670L671 at the β-secretase cleavage site in APP2, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased β-secretase cleavage of the mutant substrate3 Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the β-secretase cleavage site, and find it to be the predominant β-cleavage activity in human brain We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for β-secretase Cloning and expression of the enzyme reveals that human brain β-secretase is a new membrane-bound aspartic proteinase

1,728 citations

Journal ArticleDOI
TL;DR: The spectrophotofluorometric determination of hydroxylated benzo[a]pyrene products is sufficiently sensitive to detect 10-12 mole per ml and has great utility in measuring the hydroxymatic activity of cells grown in culture.
Abstract: The aryl hydroxylase enzyme system is inducible in hamster fetus cell cultures. The enzyme system is localized in the 105,000 x g pellet ("microsomal fraction"), has an absolute requirement for NADPH and molecular O2, a pH optimum of pH 7.5, and a partial requirement for divalent cations. Exposure to carbon monoxide reduces the enzyme activity. Treatment with ethylenediaminetetraacetate or trypsin completely prevents enzymatic activity. The Km for the hydroxylase is approximately 0.6 µm with benzo[a]pyrene as substrate. Benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, dibenz[a,h]anthracene, and dibenz[a,c]anthracene are also substrates for the enzyme system. The spectrophotofluorometric determination of hydroxylated benzo[a]pyrene products is sufficiently sensitive to detect 10-12 mole per ml and, hence, has great utility in measuring the hydroxylase activity of cells grown in culture. This mammalian cell culture system is advantageous for the study of the mechanism of microsomal enzyme induction and the related areas of carcinogenesis and drug and steroid metabolism.

1,660 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202214
2021239
2020330
2019392
2018415
2017430