ABSTRACT
The relationship between the structures of 39 natural flavonoids (including flavones, flavonols, flavonones, and isoflavones) and their inhibitory and positive inotropic effects have been found. It is shown that flavonoids considerably inhibit the activity of the purified preparation of pig kidney medulla Na+,K(+)-ATPase with Ki = 1.4-22.2 microM and some of the flavonoids studied exert positive inotropic effect on the papillary muscle. Flavones and flavonols, containing hydroxyl groups inside the phenyl radical in ortho- and vicinal positions, exhibit high inhibitory and positive inotropic effects. The inhibitory and positive inotropic effects of flavonoid glycosides and methoxyflavonoids are less pronounced. The presence of dimethylallyl groups in flavonones leads to the increase in the inhibitory and positive activities. Isoflavones produced virtually no effect on the enzyme and the isolated heart muscle. The effect of quercetin on the structure of Na+,K(+)-ATPase was studied by the fluorescence method.
Subject(s)
Cardiotonic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Kidney Medulla/drug effects , Myocardial Contraction/drug effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Animals , Cardiotonic Agents/chemistry , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Flavonoids/chemistry , Kidney Medulla/enzymology , Kinetics , Molecular Structure , Muscle Proteins/antagonists & inhibitors , Papillary Muscles/drug effects , Rats , Structure-Activity Relationship , SwineABSTRACT
Localization of the PCMB-R spin label and benzocarboline probe bound with the purified preparation of pig kidney-Na+, K(+)-ATPase relative to active site of the enzyme was studied by EPR method. The number of Mn2+ ions in active site of the enzyme as well as that bound with lipids was determined from EPR spectra of paramagnetic manganese ions replacing magnesium ions were measured in frozen protein samples of Na2+, K(+)-ATPase at 77 K. It has been found that sulfhydryl group of the enzyme modified by PCMB-R and benzocarboline probe are placed at distances 38 A and 50 A, respectively, from Mn2+ ions in the active site of Na+, K(+)-ATPase. Evaluation of the immersion depth of the nitroxyl radical into protein globule showed that benzocarboline probe was immobilized near the macromolecular protein surface; there are two bound probe sites, distinguished by accessibility of ferricyanide ions.
Subject(s)
Carbolines/chemistry , Chloromercuribenzoates/chemistry , Kidney/enzymology , Sodium-Potassium-Exchanging ATPase/chemistry , Spin Labels , Animals , Binding Sites , Electron Spin Resonance Spectroscopy , Manganese/chemistry , Molecular Probes , Swine , p-Chloromercuribenzoic AcidABSTRACT
Possibility of registration of protein interactions in the membranes was demonstrated. The membrane preparation of Na+, K+ ATPase was used in the investigations. The Na+, K+ ATPase was bound with 4-acetoamido-4'-isothiocyanatostilbene-2,2' disullfonic acid (SITS) and erythrosinisothiocyanate (ERITC). The label/Na+,K+ATPase (M/M) ratio was equal to 1:1 for SITS and changed from 1:1 to 1:5 for ERITC. The cis-trans isomerization of SITS was initiated by triplet-triplet energy transfer from light excited ERITC to SITS. The kinetics of isomerization was recorded by the SITS fluorescence measurements. The rate constant of triplet-triplet energy transfer (kT) from ERITC to cis isomer of SITS, (3 divided by 7) X 10(3) M-1 s-1 was determined at 25 degrees C. The kT value of the energy transfer between loose molecules of erythrosine and SITS in buffer solution equaled to 7 X 10(7) M-1 s-1. This drop of kT in the membrane at 10(4) reflected the decrease in the frequency of label collisions caused by the increase in the media viscosity and steric hindrances.
Subject(s)
Membrane Proteins/analysis , Photochemistry , Animals , Cell Membrane/enzymology , Sodium-Potassium-Exchanging ATPase/analysis , SwineABSTRACT
The transformed steroids containing delta 5-3 beta-hydroxy- or 3 beta, 5 alpha-dihydroxy-6-keto groups in the A/B rings and an additional cycle E (17,20-dihydroxy-delta-lactone, 16,23-pyranone or delta 20(22)-16 alpha, 17 alpha-dihydroxy-23-carbethoxy-side chain) (1 . 10(-5) M) inhibit Na+,K+-ATPase from pig kidney medulla or from ox brain. The steroid structure has a noticeable effect on ATPase inhibition varying from 3 to 26%. The data obtained suggest that the uneven distribution of polar substituents in the steroid molecule is essential for ATPase inhibition by steroid aglycons.