scholarly journals A method for determining kinetic parameters at high enzyme concentrations

1982 ◽  
Vol 203 (1) ◽  
pp. 339-342 ◽  
Author(s):  
C J Halfman ◽  
F Marcus
Keyword(s):  
Enzyme Activity ◽  
Kinetic Parameters ◽  
Graphical Method ◽  
Free Ligand ◽  
Enzyme Concentration ◽  
Fructose 1,6 Bisphosphatase ◽  
High Enzyme ◽  
Substrate Inhibitor ◽  
The Relationship

A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.

A Study of the Colorimetric Dinitrophenylhydrazine Method for the Determination of Serum Glutamic Oxalacetic Transaminase Activity

1966 ◽  
Vol 12 (4) ◽  
pp. 217-225 ◽  
Author(s):  
J S Annino
Keyword(s):  
Enzyme Activity ◽  
Transaminase Activity ◽  
Reagent Blank ◽  
Glutamic Oxalacetic Transaminase ◽  
Color Development ◽  
High Enzyme ◽  
Serum Glutamic Oxalacetic Transaminase ◽  
The Relationship ◽  
High Enzyme Activity

Abstract Study of the colorimetric transaminase method of Reitman and Frankel for the determination of serum glutamic oxalacetic transaminase activity revealed the following: (1) although maximum absorption occurs at 444 mµ, absorbance readings at 505 mµ gave satisfactory results; (2) color development is immediate and the color is stable for at least 1 hr.; (3) a pyruvate calibration standard may be used; (4) sample blanks are not usually necessary; (5) a reagent blank should accompany each group of analyses and should be used as a photometric reference; (6) the relationship between dilution and enzyme activity is linear; and (7) although the relationship between incubation time and activity is not exactly linear, a factor has been determined to permit the use of a 12-min. incubation period with samples showing high enzyme activity.

scholarly journals The determination of binding parameters when the total and free substrate concentrations are not approximately equal

1979 ◽  
Vol 179 (3) ◽  
pp. 697-700 ◽  
Author(s):  
N Gains
Keyword(s):  
Substrate Concentration ◽  
Graphical Method ◽  
Enzyme Concentration ◽  
Binding Parameters ◽  
Free Concentration ◽  
Equilibrium Binding ◽  
Substrate Concentrations

By using a standard graphical method values of Km and V may be found that are independent of the conditions and assumptions that the total substrate concentration approximates to its free concentration and that Km is much larger than the enzyme concentration. The procedure is also applicable to the determination of equilibrium binding parameters of a ligand to a macromolecule.

scholarly journals Role of a Mutation at Position 167 of CTX-M-19 in Ceftazidime Hydrolysis

2004 ◽  
Vol 48 (5) ◽  
pp. 1454-1460 ◽  
Author(s):  
Soichiro Kimura ◽  
Masaji Ishiguro ◽  
Yoshikazu Ishii ◽  
Jimena Alba ◽  
Keizo Yamaguchi
Keyword(s):  
Crystal Structure ◽  
Kinetic Parameters ◽  
Catalytic Efficiency ◽  
Enzyme Concentration ◽  
Hydroxyl Group ◽  
Steric Interaction ◽  
Acidic Group ◽  
High Enzyme ◽  
High Enzyme Concentration

ABSTRACT CTX-M-19 is a recently identified ceftazidime-hydrolyzing extended-spectrum β-lactamase, which differs from the majority of CTX-M-type β-lactamases that preferentially hydrolyze cefotaxime but not ceftazidime. To elucidate the mechanism of ceftazidime hydrolysis by CTX-M-19, the β-lactam MICs of a CTX-M-19 producer, and the kinetic parameters of the enzyme were confirmed. We reconfirmed here that CTX-M-19 is also stable at a high enzyme concentration in the presence of bovine serum albumin (20 μg/ml). Under this condition, we obtained more accurate kinetic parameters and determined that cefotaxime (k cat /Km , 1.47 × 106 s−1 M−1), cefoxitin (k cat /Km , 62.2 s−1 M−1), and aztreonam (k cat /Km , 1.34 × 103 s−1 M−1) are good substrates and that imipenem (k +2 /K, 1.20 × 102 s−1 M−1) is a poor substrate. However, CTX-M-18 and CTX-M-19 exhibited too high a Km value (2.7 to 5.6 mM) against ceftazidime to obtain their catalytic activity (k cat). Comparison of the MICs with the catalytic efficiency (k cat /Km ) of these enzymes showed that some β-lactams, including cefotaxime, ceftazidime, and aztreonam showed a similar correlation. Using the previously reported crystal structure of the Toho-1 β-lactamase, which belongs to the CTX-M-type β-lactamase group, we have suggested characteristic interactions between the enzymes and the β-lactams ceftazidime, cefotaxime, and aztreonam by molecular modeling. Aminothiazole-bearing β-lactams require a displacement of the aminothiazole moiety due to a severe steric interaction with the hydroxyl group of Ser167 in CTX-M-19, and the displacement affects the interaction between Ser130 and the acidic group such as carboxylate and sulfonate of β-lactams.

Invertase Production by Fungi, Characterization of Enzyme Activity and Kinetic Parameters

2017 ◽  
Vol 68 (10) ◽  
pp. 2205-2208 ◽  
Author(s):  
Gabi Mirela Matei ◽  
Sorin Matei ◽  
Maria Pele ◽  
Flavia Dumitrescu ◽  
Adrian Matei
Keyword(s):  
Aspergillus Niger ◽  
Kinetic Parameters ◽  
Invertase Activity ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Aspergillus Wentii ◽  
Penicillium Aurantiogriseum ◽  
The Relationship ◽  
Menten Constant

The present paper presents the results of research carried out on 14 fungal isolates of various origins aiming to select new efficient sources for invertase production for further biotechnological application. Aspergillus flavus presented the highest protein content and Aspergillus niger, the most intense invertase activity. The relationship between enzyme concentration and enzymatic activity at 0.25 mM mL-1sucrose as substrate assayed for successive decimal dilutions of Aspergillus niger enzyme ranging from 0.1 to 1mlLrevealed a linear correspondence between 0.1 and 0.5mL. The kinetic parameters Michaelis-Menten constant (Km) and maximal velocity (Vmax) for invertase activity of Aspergillus niger, Penicillium aurantiogriseum, Aspergillus wentii and Rhizopus stolonifer were calculated. The determination coefficients R2 calculated from Lineweaver-Burk plots presented values very close or equal to 1.

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.

Extending the linear range for kinetic reactions by considering the relationship between enzyme activity of the reagent and measurement intervals.

1986 ◽  
Vol 32 (11) ◽  
pp. 2021-2025 ◽  
Author(s):  
M P Goren ◽  
J E Davis
Keyword(s):  
Experimental Data ◽  
Enzyme Activity ◽  
Linear Range ◽  
Concentration Curve ◽  
Measurement Interval ◽  
Km Value ◽  
The Relationship ◽  
Substrate Concentrations ◽  
Selection Of

Abstract We show that the rate-concentration curve is sigmoidal for enzyme-catalyzed procedures that are commonly applied to rapid automated analyzers. Linear data can be obtained by judicious selection of the reagent enzyme activity (Vm) and the measurement interval (t1 to t2). For determination of substrate concentrations much less than Km, conditions that balance linearity with sensitivity and accuracy are obtained when Vm/Km = [ln (t2/t1)]/(t2-t1). We also present theoretical and experimental data that show the linear range can be extended to concentrations exceeding the Km value. We illustrate the application of theoretically appropriate conditions by analysis of procedures reported to be optimized. Familiarity with these concepts can obviate laborious and potentially misleading experimentation.

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.

scholarly journals Isolation and Activity Determination of Enzyme Phosphatase Secreted by Aspergillus niger

2019 ◽  
Vol 9 (1) ◽  
pp. 41-45
Author(s):  
Tafadzwa Zharare ◽  
Rumbidzai Mangoyi
Keyword(s):  
Enzyme Activity ◽  
Aspergillus Niger ◽  
Recombinant Dna ◽  
Extracellular Enzyme ◽  
Enzyme Concentration ◽  
Recombinant Dna Technology ◽  
Activity Determination ◽  
Pure Cultures ◽  
Industrial Use

The use of enzymes on industrial scale saves a lot of energy and avoids pollution, thus holding a promise for green and economically sustainable alternative strategies in industrial transformations.  Generally, the fungi Aspergillus niger secretes enzymes which can be used in different industries. Thus, coming up with these enzymes in large amounts will definitely result in reduced costs encountered in importing them for industrial use.  This study focussed on isolation and activity determination of an enzyme phosphatase secreted by Aspergillus niger.  This enzyme can be of great importance in molecular biology industries, particularly for recombinant DNA technology.  For this study, pure cultures of Aspergillus niger were used.  Aspergillus niger was resuscitated on potato dextrose agar and then subcultured in Adam’s medium, a medium specific for the production of phosphatase.  Cells were centrifuged and the filtrate was collected whilst the residue was discarded. The filtrate was expected to contain the crude enzyme phosphatase since Aspergillus niger secretes the extracellular enzyme into the medium. Disodium phenyl phosphate was used as a substrate for the determination of the phosphatase activity. The enzyme activity was determined spectrophotometrically by reading absorbance of phenol formed in the presence of enzyme and the substrate. The concentration of phenol liberated was then used to calculate the enzyme activity expressed in King Armstrong Units (KAU).  Further work on enzyme activity determination was done by varying enzyme and substrate concentrations.  Results showed that the isolated alkaline phosphatase had activity of 4.0 KAU and 4.5 KAU at 25 ºC and 37 ºC respectively. Acidic phosphatase had activity of 5 KAU and 7 KAU at 25 ºC and 37 ºC respectively. Rate of activity increased upon increasing enzyme concentration and substrate.  Thus, Aspergillus niger produces the enzyme phosphatase, however, there is need to induce the production of these enzymes for industrial use.

Effects of Ca, Mg, and EDTA on creatine kinase activity in cerebrospinal fluid.

1979 ◽  
Vol 25 (1) ◽  
pp. 147-150 ◽  
Author(s):  
P Urdal ◽  
J H Strømme
Keyword(s):  
Cerebrospinal Fluid ◽  
Creatine Kinase ◽  
Enzyme Activity ◽  
Kinase Activity ◽  
Creatine Kinase Activity ◽  
Precise Estimate ◽  
The Relationship ◽  
Sample Fraction ◽  
Sensitive Method

Abstract For one to obtain a precise estimate of creatine kinase (CK) activity in cerebrospinal fluid, the sample fraction is increased by about 10-fold over that used for serum. This increases the concentration of interfering substances, Ca being especially important. Therefore, the relationship between Ca, Mg, and EDTA was examined. Enzyme activity was maximal with 15 mmol of Mg per liter in the presence of 3 mmol of EDTA per liter, otherwise according to the (Scandinavian) recommended conditions for determination of CK activity in serum. These modifications increased the activity of CK by 35% for CK-MM and by 60% for CK-BB. Counteraction of Ca-induced inhibition was the main reason to this increase. We describe a practical and sensitive method for determining CK in cerebrospinal fluid.

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.

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