The thermal stability of protein enzymes is determined in vitro by measuring the enzymatic activity during incubation at constant temperature. Refolding of thermal inactivated enzymes is carried out both in vitro and in vivo, in the presence of chaperones, usually at temperature optimal for the particular enzyme for the manifestation of enzymatic activity. In the present work thermal stability of enzymes in vitro (using purified preparations) and in vivo (directly in the bacterial cell) has been determined. Bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi and Photorhabdus luminescens as protein substrates have been used. It is shown that the thermal stability of the P. luminescens and P. leiognathi luciferases in vivo in the Escherichia coli MG1655 dnaK^(+) and PK202 ΔdnaKJ14 strains is considerable higher than the thermal stability of "cell-free extract" luciferases. When an uncoupler of oxidative phosphorylation the carbonyl-cyanide-3-chlorophenylhydrazone (CCCP) that reduce the intracellular concentration of ATP to a minimum level, and the volatile hydrophobic substance (-)-Limonene (C10H16) as an inhibitor of chaperone-dependent refolding are added to the medium, the thermal stability of luciferases reduces almost to the level which is characteristic for the purified protein preparation. It is shown that the ATP-dependent chaperones ClpA and ClpB are essential for the increase of thermostability of luciferases in bacterial cells. Also, it is shown that the DnaKJE-dependent refolding of thermoinactivated luciferases is practically absent if the protonophore СССР or the hydrophobic substance (-)-Limonene was added to the bacterial suspension. Taking the data presented in this paper into account, it is necessary to consider the presence in bacterial cells of two different groups of ATP-dependent chaperones: 1st group (DnaKJE, GroEL/ES) is able to conduct the refolding both at low temperature after protein thermal inactivation and at high temperature at which protein thermal inactivation occurs; 2nd group (ClpA,ClpB, and possibly still unknown chaperones) is unable to conduct the standard refolding (i.e. at low temperature), but capable due to the hydrolysis energy of ATP of maintaining nonequilibrium stabilization of protein native forms at high temperature.
Keywords: (-)-Limonene; ATP-dependent chaperone; bacterial luciferase; protonophore CCCP; refolding; thermal inactivation.