Inhibitory domain-specific antibodies to cytochrome P-450scc

AbstractHighly specific antibodies to cytochrome P-450scc and its F1 and F2 fragments, representing N- and C-terminal sequences of the hemeprotein respectively, were raised in rabbits. These antibodies were found to be inhibitory (up to 50–90%) for the cholesterol transformation into pregnenolone in the reconstituted system, indicating the involvement of both F1 and F2 domains formed by the respective fragments in monooxygenase catalysis. Cytochrome P-450scc in mitoplasts is not accessible for trypsin as revealed by immunological techniques. However, the treatment of submitochondrial particles with trypsin results in two main fragments identified by immunoblotting in the presence of the monospecific antibodies as F1 and F2 fragments. This indicates that the trypsin sensitive 250–257 region in cytochrome P-450scc molecule connecting both domains is exposed to the matrix side of the inner mitochondrial membrane

Sequence variation in CYP51A from the Y strain of Trypanosoma cruzi alters its sensitivity to inhibition

CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.Fil: Cherkesova, Tatiana S.. National Academy of Sciences of Belarus. Institute of Bioorganic Chemistry; BielorrusiaFil: Hargrove, Tatiana Y.. Vanderbilt University; Estados UnidosFil: Vanrell, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ges, Igor. Vanderbilt University; Estados UnidosFil: Usanov, Sergey A.. National Academy of Sciences of Belarus. Institute of Bioorganic Chemistry; BielorrusiaFil: Romano, Patricia Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Lepesheva, Galina I.. Vanderbilt University; Estados Unido

Resonances in the "post with gap—adjacent piston" waveguide system

The electrodynamic system composed of a metal post with a gap and a short-circuiting piston located nearby is investigated theoretically and experimentally. It is shown that such a system can be used successfully in designing small-size microwave devices, whose characteristics are controlled by magnetic field

A rectangular resonator with its characteristics controlled by magnetic field

The paper shows that in microwave systems made of nonmagnetic materials, in which the conditions for resonance are established, we can observe a strong dependence of their electrodynamic characteristics on the applied magnetic field. The dependence arises from the Hall component of the microwave current flowing in the internal semiconductor insertions and in metal walls of the resonator

A high-Q low-dimensional resonator with electrically tunable frequency

A low-dimensional high-Q resonator has been proposed and its frequency-response characteristics have been investigated. The specified resonator features the frequency-response characteristics that are electrically tunable by using a semiconductor diode with variable capacitance

Experimental Study on the Interaction of an Impulse Water Jet with Molten Metal

The impingement of a short-duration water jet on a pool of molten Rose’s metal is studied experimentally herein. Short-duration water jet impacting on the free surface of a molten metal pool with a temperature of 300 °C are generated with a pneumatic water delivery system, with two-camera high-speed video registration. A total of 14 experimental series, each containing 5 repeated tests, are performed for a water volume of 0.2–1 mL and a jet impact velocity of 4.1–9.0 m/s. The cavity development in the melt layer is studied, with the main stages described herein. Despite the significantly higher density of melt in comparison with water, the cavity can reach the melt pool bottom; furthermore, its further collapse results in the formation of a central jet rising to the height of a few centimeters. The maximum height of the central jet is shown to depend linearly on the total momentum of the water jet, and a semi-logarithmic correlation is found for the maximum diameter of the cavity. Repeatability analysis is performed within each experimental series, and the relative standard deviation for the melt splash height is shown to be from 8.8% to 26.8%. The effects of the pool depth, the vessel shape, and the water temperature are weaker in the range of the experimental parameters used here

Magnetic frequency tuning of the microwave Gunn diode oscillator

It has been found experimentally that the oscillation frequency of a Gunn diode placed in the low-dimensional resonance system “metal pin–closely set short-circuiter” can be effectively controlled by magnetic field applied in the normal direction with respect to the waveguide wide wall

Radiothermometric Study of the Effect of Amino Acid Mutation on the Characteristics of the Enzymatic System

The radiothermometry (RTM) study of a cytochrome-containing system (CYP102 A1) has been conducted in order to demonstrate the applicability of RTM for monitoring changes in the functional activity of an enzyme in case of its point mutation. The study has been performed with the example of the wild-type cytochrome (WT) and its mutant type A264K. CYP102 A1 is a nanoscale protein-enzymatic system of about 10 nm in size. RTM uses a radio detector and can record the corresponding brightness temperature (Tbr) of the nanoscale enzyme solution within the 3.4–4.2 GHz frequency range during enzyme functioning. It was found that the enzymatic reaction during the lauric acid hydroxylation at the wild-type CYP102 A1 (WT) concentration of ~10−9 M is accompanied by Tbr fluctuations of ~0.5–1 °C. At the same time, no Tbr fluctuations are observed for the mutated forms of the enzyme CYP102 A1 (A264K), where one amino acid was replaced. We know that the activity of CYP102 A1 (WT) is ~4 orders of magnitude higher than that of CYP102 A1 (A264K). We therefore concluded that the disappearance of the fluctuation of Tbr CYP102 A1 (A264K) is associated with a decrease in the activity of the enzyme. This effect can be used to develop new methods for testing the activity of the enzyme that do not require additional labels and expensive equipment, in comparison with calorimetry and spectral methods. The RTM is beginning to find application in the diagnosis of oncological diseases and for the analysis of biochemical processes