Tag Archives: kinetics
Kinetics of casein hydrolysis by peptidase from Bacillus thuringiensis var. israelensis
O. V. Sevastyanov1, Yu. A. Shesterenko1, A. A. Ryzhak1,
I. I. Romanovska1, N. A. Dziubliuk2, L. D. Varbanets2
1A. V. Bogatsky Physico-Chemical Institute, National Academy of Sciences of Ukraine, Odesa;
2Danylo Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: romairina@gmail.com
Received: 25 October 2018; Accepted: 14 March 2019
The kinetics of enzyme reaction is generally studied using the Michaelis-Menten equation and various methods of its linearization. Each method has its advantages and drawbacks, so their comparison for determining the kinetics of new enzymes action is topical. The aim of this work was to study the kinetics of casein hydrolysis catalyzed by new peptidase from Bacillus thuringiensis var. israelensis IMB B-7465 using several methods of enzyme activity assessment and Michaelis-Menten equation linearization. The satisfactory agreement between kinetic constants values obtained by the methods of Lineweaver-Burk, Hanes, Eadie-Hofstee, Cornish-Bowden-Eisenthal was established. The Lineweaver-Burk method was shown to be optimal for determining Km and Vmax of casein hydrolysis. Estimation of caseinolytic activity with the use of ortho-phthalic dialdehyde allowed more accurate Vmax determination compared to the use of Anson and Kunitz methods.
Influence of Са(2+) on kinetic parameters of pancreatic acinar mitochondria in situ respiration
B. O. Manko, V. V. Manko
Ivan Franko National University of Lviv, Ukraine;
e-mail: mankobo@gmail.com
The dependence of respiration rate of rat permeabilized acinar pancreacytes on oxidative substrates concentration was studied at various [Ca2+] – 10-8–10-6 M. Pancreacytes were permeabilized with 50 µg of digitonin per 1 million cells. Respiration rate was measured polarographically using the Clark electrode at oxidation of succinate or pyruvate either glutamate in the presence of malate. Parameters of Michaelis-Menten equation were calculated by the method of Cornish-Bowden or using Idi-Hofsti coordinates and parameters of Hill equation – using coordinates {v; v/[S]h}. In the studied range of [Ca2+] the kinetic dependence of respiration at pyruvate oxidation is described by the Michaelis-Menten equation, and at oxidation of succinate or glutamate – by Hill equation with h = 1.11–1.43 and 0.50–0.85, respectively. The apparent constant of respiration half-activation (K0.5) did not significantly change in the studied range of [Ca2+] while at 10-7 M Ca2+ it was 0.90 ± 0.06 mM for succinate, 0.096 ± 0.007 mM for pyruvate and 0.34 ± 0.03 mM for glutamate. Maximum respiration rate Vmax at pyruvate oxidation increased from 0.077 ± 0.002 to 0.119 ± 0.002 and 0.140 ± 0.002 nmol O2/(s·million cells) due to the increase of [Ca2+] from 10-7 to 5×10-7 or 10-6 M, respectively. At oxidation of succinate or glutamate Ca2+ did not significantly affect Vmax. Thus, the increase of [Ca2+] stimulates respiration of mitochondria in situ of acinar pancreacytes at oxidation of exogenous pyruvate (obviously due to pyruvate dehydrogenase activation), but not at succinate or glutamate oxidation.