G.D. Kutuzova

Synthesis and pathway of Luciola mingrelica firefly luciferase in Xenopus laevis frog oocytes and in cell-free systems

Poly (A+)RNA was isolated from the lanterns of adult fireflies of L. mingrelica. The poly (A+)RNA was translated in a cell-free translation mixture from rabbit reticulocyte and from Krebs II mouse ascites cells and in Xenopus laevis frog oocytes. The synthesis of the enzymatically active firefly luciferase was demonstrated in all systems. In the cell-free systems a maximal quantity of luciferase is synthesized during the first 1-2 h, then the decreasing activity of luciferase is observed. The optimal concentration of mRNA for translation in the mouse ascites cell extract was found. The kinetics of the synthesis of the luciferase, its pathway and stability in X. laevis oocytes was studied. It was observed that luciferase is secreted from oocytes into the incubation medium.

FUNCTIONALLY IMPORTANT CARBOXYL GROUPS OF HORSERADISH-PEROXIDASE

The carboxyl groups of horseradish peroxidase (EC 1.11.1.7) were modified by the Koshland method using o-dianisidine as a nucleophilic agent (o-dianisidine, covalently bound to protein, has ϵ305 = 19.0 M−1·cm−1). At pH 5.0. only four COOH groups were accessible to modification. The modification of three of them decreased the peroxidase activity to the hydrogen donor, o-dianisidine, by 70% but did not alter its activity to the electron donor, potassium ferrocyanide. The constants, k3 and k4, of electron transfer from the hydrogen donor to the peroxidase compounds E1 and E2, decreased 10- and 1.5-fold, respectively. The modification of the fourth COOH group did not affect the enzyme activity. The hemin isolated from the o-dianisidine-modified peroxidase (M-hemin) had one modified propionate residue. The second propionate was inaccessible to modification. The activity of peroxidase reconstructed from M-hemin and native apoprotein was close to that of the native enzyme. It is concluded that the three functionally important COOH groups are of protein nature and are not directly involved in the enzyme-active site, but important for binding and oxidation of o-dianisidine.

Inhibition of horseradish peroxidase byN-ethylamide ofo-sulfobenzoylacetic acid

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence ofN-ethylamide ofo-sulfobenzoylacetic acid (EASBA) at pH 5.0–7.5. In the oxidation ofo-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases bothKm andVm in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.

The stress-related production of the active Photinus pyralis and Luciola mingrelica firefly luciferases in Escherichia coli

The kinetics ofPhotinus pyralis andLuciola mingrelica luciferase gene expression was studied on plasmids with the thermoinducible λPr promoter inEscherichia coli by SDS-gel electrophoresis of cell lysates to follow luciferase protein-synthesized, enzyme immunoassay (EIA) to follow native enzyme conformer, and the luciferase activity assay.E. coli cells were cultivated at temperature schemes 28–42–21°C or 28–21°C, or at alkali pH shift. In the cases of thermoinduction and pH shift, the luciferase expressions have similar features. The 3-h thermoinduction (42°C) followed by the incubation at 21°C, for 10 h resulted in the maximal amount of the luciferase protein of 4–5% of the total cell proteins. The yield did not change further. The amount of native luciferase conformer and the luciferase activity started to grow after incubation for 10 h at 21°C and reached the maximum after 50–60 h when the synthesized luciferase protein adopted the native-like conformation. At the same time, only 50% of the latter appeared to be catalytically active. An increase in the enzymatic activity correlates with an increase in the intracellular pH and ATP content. Intracellular metabolic reactions were shown to play a role in the conformational changes of the enzyme in a postthermoinduction period, and a possible mechanism of this effect is proposed.