scholarly journals MICROTEST FOR THE DETERMINATION OF THE AMYLOLYTIC ACTIVITY OF SALIVA ALPHA-AMYLASE

2019 ◽  
Vol 23 (3-4) ◽  
pp. 115-117
Author(s):  
A. P Godovalov ◽  
M. V Yakovlev ◽  
I. I Zadorina
Keyword(s):  
Enzyme Activity ◽  
Catalytic Activity ◽  
Enzyme Concentration ◽  
Alpha Amylase ◽  
Proportional Increase ◽  
Activity Of Enzymes ◽  
Student’S T ◽  
Large Groups

It is known that the concentration of alpha-amylase in saliva can determine its catalytic activity, the decrease of which occurs during various pathological processes in the oral cavity. The overwhelming number of methods for determining the catalytic activity of enzymes involve the use of a large volume of reagents and samples, which makes it difficult to study saliva in large groups. The purpose of the study is to evaluate the possibility of using the microvariation of the reaction to determine the activity of saliva alpha-amylase, as well as to analyze the dependence of the enzyme activity on its concentration. Materials and methods. Saliva was obtained from 15 people with intact periodontal disease and the dentition, without somatic pathology. For in vitro studies, alpha-amylase solutions were prepared with an enzyme concentration of 10; five; 2.5; one; 0.5 and 0.25 mg / ml ex tempore. To save samples and reagents, the volume of the reaction participants was proportionally reduced. The further analysis procedure was carried out according to the instructions of the manufacturer of the «AMYLASE-VITAL» reagent kit to determine the activity of alpha-amylase. Statistical analysis of the results was performed using the Student’s t-test in the program Statistica 7.0. Results. The comparability of the results of determining the activity of alpha-amylase using the classical and microplate variants of the reaction is shown. With an increase in alpha-amylase concentration from 0 to 2.5 mg / ml, a directly proportional increase in enzyme activity is observed. In the case of an increase in the concentration of alpha-amylase above 2.5 mg / ml, a decrease in its activity is shown, which may be due to the precipitation of a part of the enzyme. The activity of the enzyme in saliva of practically healthy individuals using the microvariation of the reaction was 528.6 ± 2.4 U / l. In conclusion the use of a microvariant of the reaction for determining the activity of alpha-amylase may be justified for a large number of subjects. A linear dependence of the enzyme activity on its concentration in the range of 0-2.5 mg / ml is shown.

scholarly journals A RAPID METHOD FOR THE DETERMINATION OF PROTEOLYTIC ACTIVITIES OF ENZYME PREPARATIONS

1948 ◽  
Vol 32 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Bacon F. Chow ◽  
Mary-Ann Peticolas
Keyword(s):  
Enzyme Activity ◽  
Trichloroacetic Acid ◽  
Enzyme Concentration ◽  
Cent Solution ◽  
Enzyme Preparations ◽  
Proteolytic Activities ◽  
Activity Of Enzymes ◽  
Different Sources ◽  
Rate Of Activation

A method has been described for the determination of proteolytic activities of enzyme preparations using casein as substrate. The rate of digestion is proportional to the enzyme concentration used. This relationship is utilized as a measure of the enzyme activity. One unit of activity is defined as the amount which is required to digest casein in 15 minutes at 37.5°C. so that 50 per cent of the protein in 1 ml. of 0.25 per cent solution is not precipitable by trichloroacetic acid. This method has been used to determine the activity of enzymes from different sources and also used to follow the rate of activation of enzymes.

scholarly journals N-Terminal Domains of the Human Telomerase Catalytic Subunit Required for Enzyme Activity in Vivo

2001 ◽  
Vol 21 (22) ◽  
pp. 7775-7786 ◽  
Author(s):  
Blaine N. Armbruster ◽  
Soma S. R. Banik ◽  
Chuanhai Guo ◽  
Allyson C. Smith ◽  
Christopher M. Counter
Keyword(s):  
Enzyme Activity ◽  
Catalytic Activity ◽  
Cellular Proliferation ◽  
Telomere Maintenance ◽  
Biological Functions ◽  
Sequence Alignments ◽  
Telomere Elongation ◽  
Human Telomerase

ABSTRACT Most tumor cells depend upon activation of the ribonucleoprotein enzyme telomerase for telomere maintenance and continual proliferation. The catalytic activity of this enzyme can be reconstituted in vitro with the RNA (hTR) and catalytic (hTERT) subunits. However, catalytic activity alone is insufficient for the full in vivo function of the enzyme. In addition, the enzyme must localize to the nucleus, recognize chromosome ends, and orchestrate telomere elongation in a highly regulated fashion. To identify domains of hTERT involved in these biological functions, we introduced a panel of 90 N-terminal hTERT substitution mutants into telomerase-negative cells and assayed the resulting cells for catalytic activity and, as a marker of in vivo function, for cellular proliferation. We found four domains to be essential for in vitro and in vivo enzyme activity, two of which were required for hTR binding. These domains map to regions defined by sequence alignments and mutational analysis in yeast, indicating that the N terminus has also been functionally conserved throughout evolution. Additionally, we discovered a novel domain, DAT, that “dissociates activities of telomerase,” where mutations left the enzyme catalytically active, but was unable to function in vivo. Since mutations in this domain had no measurable effect on hTERT homomultimerization, hTR binding, or nuclear targeting, we propose that this domain is involved in other aspects of in vivo telomere elongation. The discovery of these domains provides the first step in dissecting the biological functions of human telomerase, with the ultimate goal of targeting this enzyme for the treatment of human cancers.

scholarly journals The kinetic characteristics of human and trypanosomatid phosphofructokinases for the reverse reaction

2019 ◽  
Vol 476 (2) ◽  
pp. 179-191 ◽  
Author(s):  
Peter M. Fernandes ◽  
James Kinkead ◽  
Iain W. McNae ◽  
Frédéric Bringaud ◽  
Paul A.M. Michels ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Reverse Reaction ◽  
Kinetic Properties ◽  
Kinetic Characteristics ◽  
Inorganic Pyrophosphate ◽  
Physiological Relevance ◽  
Fructose 1,6 Bisphosphate

Abstract Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate to produce ADP and fructose 1,6-bisphosphate. The reverse reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in inorganic pyrophosphate-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. The evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the reverse reaction in vitro, allowing determination of kinetic properties. Additionally, the reverse reaction was shown possible for PFKs from three clinically important trypanosomatids; these enzymes are contained within glycosomes in vivo. This compartmentalisation may facilitate reversal, given the potential for trypanosomatids to have an altered ATP/ADP ratio in glycosomes compared with the cytosol. The kinetic properties of each trypanosomatid PFK were determined, including the response to natural and artificial modulators of enzyme activity. The possible physiological relevance of the reverse reaction in trypanosomatid and human PFKs is discussed.

scholarly journals Phosphorylation by protein kinase C decreases catalytic activity of avian phospholipase C-β

1999 ◽  
Vol 338 (2) ◽  
pp. 257-264 ◽  
Author(s):  
Theresa M. FILTZ ◽  
Michelle L. CUNNINGHAM ◽  
Kara J. STANIG ◽  
Andrew PATERSON ◽  
T. Kendall HARDEN
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase C ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Phospholipase C ◽  
G Protein ◽  
Erythrocyte Membranes ◽  
Basal Activity ◽  
Kinase C

The potential role of protein kinase C (PKC)-promoted phosphorylation has been examined in the G-protein-regulated inositol lipid signalling pathway. Incubation of [32P]Pi-labelled turkey erythrocytes with either the P2Y1 receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) or with PMA resulted in a marked increase in incorporation of 32P into the G-protein-activated phospholipase C PLC-βT. Purified PLC-βT also was phosphorylated by PKC in vitro to a stoichiometry (mean±S.E.M.) of 1.06±0.2 mol of phosphate/mol of PLC-βT. Phosphorylation by PKC was isoenzyme-specific because, under identical conditions, mammalian PLC-β2 also was phosphorylated to a stoichiometry near unity, whereas mammalian PLC-β1 was not phosphorylated by PKC. The effects of PKC-promoted phosphorylation on enzyme activity were assessed by reconstituting purified PLC-βT with turkey erythrocyte membranes devoid of endogenous PLC activity. Phosphorylation resulted in a decrease in basal activity, AlF4--stimulated activity, and activity stimulated by 2MeSATP plus guanosine 5´-[γ-thio]triphosphate in the reconstituted membranes. The decreases in enzyme activities were proportional to the extent of PKC-promoted phosphorylation. Catalytic activity assessed by using mixed detergent/phospholipid micelles also was decreased by up to 60% by phosphorylation. The effect of phosphorylation on Gqα-stimulated PLC-βT in reconstitution experiments with purified proteins was not greater than that observed on basal activity alone. Taken together, these results illustrate that PKC phosphorylates PLC-βT in vivo and to a physiologically relevant stoichiometry in vitro. Phosphorylation is accompanied by a concomitant loss of enzyme activity, reflected as a decrease in overall catalytic activity rather than as a specific modification of G-protein-regulated activity.

scholarly journals In vitro Anti-Arthritic activity of Pseudarthria viscida against Protein Denaturation and Proteinase enzyme

2020 ◽  
Vol 11 (2) ◽  
pp. 284-288
Author(s):  
Pandian P
Keyword(s):  
Enzyme Activity ◽  
Aqueous Extract ◽  
Protein Denaturation ◽  
Bone Erosion ◽  
Diclofenac Sodium ◽  
Ethanol Extract ◽  
Maximum Inhibition

Arthritis is an autoimmune with chronic inflammatory, the patient has very painful due deformities and bone erosion which is caused by damage of the joints. The plant Pseudarthria viscida was collected from the Thirunelveli district and extracted with aqueous and ethanol solvent. The two method was used for determination of invitro anti-arthritic activity. The Inhibition of Protein Denaturation Method shows the anti-arthritic activity with the value from 40.46±0.72 to 78.36±0.64 for aqueous extract and 48.62±0.86 to 84.42±0.86 for ethanol extract and Inhibition of Proteinase Enzyme Activity shows 38.62±0.32 to 72.58±0.58 in aqueous extract and 46.28±0.58 to 80.52±0.56 in ethanol extract. Diclofenac sodium were used as standard, the concentration is 100, 200, 300, 400, and 500. In both the method the concentration of 500Microgram per milliliters shows maximum inhibition and compare to both extract the ethanol shows better activity than aqueous extract.

scholarly journals Fluorescence-based determination of enzyme activity of recombinant CYP51b1 (sterol 14α-demethylase) with coumarin derivatives

2010 ◽  
Vol 56 (1) ◽  
pp. 132-137 ◽  
Author(s):  
N.A. Petushkova ◽  
A.V. Lisitsa ◽  
V.F. Pozdnev ◽  
I.I. Karuzina
Keyword(s):  
Enzyme Activity ◽  
Acetic Acid ◽  
Electron Donor ◽  
Model System ◽  
Coumarin Derivatives ◽  
Current Investigation ◽  
Soluble Model ◽  
Highly Sensitive

The current investigation was undertaken with the aim to carry out an in vitro evaluation of the ability of coumarin derivatives as probe substrates to predict the activity of CYP51b1. The results obtained indicate that 7-aminocoumarin-4-acetic acid (ACAC) can be used to determine the recombinant CYP51b1 activity. Determination of CYP51b1 activity with ACAC is based on the direct registration of fluorescence increasing at 30°C. It was found also that BMR in a simple soluble model system can be used as an electron donor for CYP51B1. Fluorescence-based assay is highly sensitive and can be used for the screening of sterol 14alpha-demethylase inhibitors.

In Vitro Determination of 6PGD Enzyme Activity Purified from Lake Van Fish (Chalcalburnus tarichii Pallas, 1811) Liver Exposed to Pesticides

2013 ◽  
Vol 91 (5) ◽  
pp. 560-564 ◽  
Author(s):  
Muhammet Guler ◽  
M. Riza Kivanc ◽  
Vedat Turkoglu ◽  
Zehra Basi ◽  
Hilal Kivrak
Keyword(s):  
Enzyme Activity ◽  
Lake Van

scholarly journals Chromate Reduction by Cell-Free Extract of Bacillus firmus KUCr1

2010 ◽  
Vol 59 (3) ◽  
pp. 185-190 ◽  
Author(s):  
GOPI BALLAV SAU ◽  
SWAGATA CHATTERJEE ◽  
SAMIR KUMAR MUKHERJEE
Keyword(s):  
Enzyme Activity ◽  
Reductase Activity ◽  
Enzyme Concentration ◽  
Metabolic Inhibitor ◽  
Chromate Reduction ◽  
Cell Free Extract ◽  
Bacillus Firmus ◽  
Soluble Cell ◽  
Enzymatic Reduction

Microbial enzymatic reduction of a toxic form of chromium [Cr(VI)] has been considered as an effective method for bioremediation of this metal. This study reports on the in vitro reduction of Cr(VI) using cell-free extracts from a Cr(VI) reducing Bacillus firmus KUCr1 strain. Chromium reductase was found to be constitutive and its activity was observed both in soluble cell fractions (S12 and S150 and membrane cell fraction (P150). The reductase activity of S12 fraction was found to be optimal at 40 microM Cr(VI) with enzyme concentration equivalent to 0.493 mg protein/ml. Enzyme activity was dependent on NADH or NADPH as electron donor; optimal temperature and pH for better enzyme activity were 70 degrees C and 5.6, respectively. The Km value of the reductase was 58.33 microM chromate having a V(max) of 11.42 microM/min/mg protein. The metabolic inhibitor like sodium azide inhibited reductase activity of membrane fraction of the cell-free extract. Metal ions like Cu2+, Co2+, Ni2+ and As3+ stimulated the enzyme but others, such as Ag+, Hg2+, Zn2+, Mn2+, Cd2+ and Pb2+, inhibited Cr(VI) reductase activity.

Determination of lipase catalytic activity in two reference materials: BCR 693 and BCR 694 by titrimetry at constant pH

2004 ◽  
Vol 42 (1) ◽  
Author(s):  
Jean-Marc Lessinger ◽  
Stella Parashou ◽  
Panteleimon Arzoglou ◽  
Paul Ramos ◽  
Catherine Chapus ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Reference Materials ◽  
Pancreatic Lipase ◽  
Measurement Procedure ◽  
Constant Ph ◽  
In Vitro Diagnostic ◽  
Standardized Measurement ◽  
Correct Implementation

AbstractBecause routine assays for pancreatic lipase catalytic activity are not yet standardized, between-method comparability is very poor. This is mainly due to the lack of reference materials (RMs). The aim of this study was to assign values of catalytic concentration to two human pancreatic lipase RMs, one prepared from human pancreatic juice (BCR 693), the other obtained by recombinant technology (BCR 694). Lipase catalytic activity was assayed in five experienced laboratories, using aliquots from the same lot of triolein emulsion and a standardized titrimetric procedure, optimized with regard to substrate, cofactors and pH. The accepted sets of data (n=4) gave a mean ± the corresponding uncertainty expressed as the 0.95 confidence interval of 1732±72 U/l and 1043±60 U/l for BCR 693 and 694, respectively. Transferability of the whole operating procedure proved to be quite satisfactory. The authors conclude that both RMs can be used to verify the correct implementation of the standardized measurement procedure and to assign values to secondary lipase materials (commercial calibrators, control products) which, in turn, ensures traceability to the standardized procedure in this study, and contributes to the harmonization of laboratory results according to the Directive for in vitro Diagnostic Medical Devices.

An Automated Method for Measuring Creatine Phosphokinase Activity in Serum

1970 ◽  
Vol 16 (4) ◽  
pp. 294-299 ◽  
Author(s):  
Ronald K Wright ◽  
Roy L Alexander
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Creatine Phosphokinase ◽  
Creatine Phosphate ◽  
Enzyme Concentration ◽  
Magnesium Ion ◽  
Manual Method ◽  
Automated Method ◽  
Automated Procedures

Abstract We describe a procedure for automating the determination of creatine phosphokinase (CPK) activity in serum by use of the AutoAnalyzer. Enzyme activity is determined by measuring the creatine phosphate formed from the CPK-catalyzed reaction of creatine with adenosine triphosphate (ATP). The sensitivity of the automated procedure was comparable to that of the manual method. By use of the most favorable concentrations of creatine, ATP, and magnesium ion in the substrate, a linear relationship was obtained between enzyme concentration and enzyme activities up to 600 mU, representing a sixfold improvement over that obtained by the manual method. The degree of correlation between results obtained by the manual and automated procedures is shown.

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