Proteolysis Coupled to ATP Hydrolysis: Regulation of the Activity of Proteolytic Sites of Lon Protease from Escherichia coli

Regulation of activity of the proteolytic sites of Lon protease was studied. It was found that ATP–Mg has the properties of a noncompetitive activator of peptidase sites. The processive mechanism of the hydrolysis of protein substrates by Lon protease was experimentally confirmed under the conditions of ATP hydrolysis. It was shown that the oligomeric state of the enzyme is the necessary prerequisite for the processive proteolysis by native Lon protease. The study of the properties of the mixed mutant Lon-K362Q/S679A confirmed the existence of intra- and intersubunit pathways of signal transduction from the ATPase to proteolytic sites. The mutual influence of substrates of Lon protease was studied, and the existence of cooperative interactions between the peptidase sites in the oligomeric enzyme was suggested.     

Coupling of Proteolysis to ATP Hydrolysis upon Escherichia coliLon Protease Functioning: II. Hydrolysis of ATP and Activity of the Enzyme Peptide Hydrolase Sites

The absence of direct correlation between the efficiency of functioning of ATPase and peptide hydrolase sites of Lon protease was revealed. It was shown that Lon protease is an allosteric enzyme, in which the catalytic activity of peptide hydrolase sites is provided by the binding of nucleotides, their magnesium complexes, and free magnesium ions in the enzyme ATPase sites. It was revealed that the ADP–Mg complex, an inhibitor of the native enzyme, is an activator of the Lon-K362Q (the Lon protease mutant in the ATPase site). Variants of functional contacts between different sites of the enzyme are considered. It was established that two ways of signal transduction from the ATPase sites to peptide hydrolase ones exist in the Lon protease oligomer--intra- and intersubunit ways. The enzyme ATPase sites are suggested to be located in the areas of the complementary surfaces of subunits. It is hypothesized that upon degradation of protein substrates by the E. coliLon protease in vivoATP hydrolysis acts as a factor of limitation of the enzyme degrading activity.     

The summation properties of the somatosensory system demonstrated via a study of the tactile sensations evoked by an electrical current and by focused ultrasound

Sensational thresholds of the skin of fingers, palm and forearm have been determined in 5 healthy subjects ageing from 20 to 49 years. About 100 summation curves were obtained. Critical duration of the electrical stimuli remained constant in various areas. Critical duration of ultrasonic stimulation increased from fingers to forearm. The data obtained together with those found in literature suggest that critical duration of the stimulus is associated with the diameter of nonmyelin nervous fibers which are affected by the stimulus increasing with the increase in fiber diameter.     

Coupling of proteolysis with ATP hydrolysis by Escherichia coli Lon proteinase. I. Kinetic aspects of ATP hydrolysis

Some aspects of the ATPase function of the Escherichia coli Lon protease were studied around the optimum pH value. It was revealed that, in the absence of the protein substrate, the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg2+ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)2- inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg2+ ions is caused by the formation of the "ADP-Mg-form" of the ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)2- complex to the enzyme, by the elimination of the inhibiting action of free Mg2+ ions without altering the efficiency of catalysis of ATP hydrolysis (based on the kcat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)- complex (without changing the Ki value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.     

Functional characteristics of the skin receptor structures in animals and man

Threshold electrical reactions of single fibers from the ischiadic nerve of rats to mechanical stimulation and rectangle impulses of focused ultrasound have been compared with respect to the parameters of stimulation to the effect of focused ultrasound on the skin of human fingers evoking different sensations. It was concluded that low-threshold fibers may be associated with tactile reception, mean-threshold ones--with tactile and thermal, whereas high-threshold fibers may be referred to reception of specific skin pain.