342ISSN 0003-6838, Applied Biochemistry and Microbiology, 2016, Vol. 52, No. 3, pp. 342–345. © Pleiades Publishing, Inc., 2016.Original Russian Text © D.I. Maltsev, V.P. Yamskova, A.P. Il’ina, B.B. Beresin, I.A. Yamskov, 2016, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2016, Vol. 52, No. 3,pp. 346–350.Bioregulator from Rat Liver TissueD. I. Maltsev a , V. P. Yamskova b , A. P. Il’ina a , B. B. Beresin a , and I. A. Yamskov aa Nesmeyanov Institute of Elemento-Organic Compounds of Russian Academy of Sciences, Moscow 119991b Koltsov Institute of Developmental Biology of Russian Academy of Sciences, Moscow, 119334 Russiae-mail: mal-dima@yandex.ru, yamskova-vp@yandex.ruReceived June 30, 2015Abstract—It has been shown that the membranotropic homeostatic tissue-specific bioregulator isolated from ratliver tissue contains a nanosized peptide-protein complex consisting of low-molecular peptides (1–6.5 kDa) anda protein from the serum albumin family. This bioregulator modulated the peptide biological activity and deter-mined the tissue specificity.Keywords: liver, bioregulators of mammalian tissuesDOI: 10.1134/S000368381603011XBioregulators previously found in different mamma-lian tissues, including the liver, were combined in agroup of membranotropic homeostatic tissue-specificbioregulators (MHTBs) due to the commonality oftheir physicochemical properties and biological activity.MHTBs in ultralow doses (ULD, 10 –8 –10 –15 mg/mL)affect basic biological processes in vivo (adhesion,migration, proliferation, differentiation, and apoptosis)and stimulate the repair and restoration of pathologi-cally changed tissues. Bioregulators have tissue specific-ity but not species specificity [1]. According to the exist-ing ideas, bioregulators of this group are extracellularnanosized peptide-protein complexes, with their nano-size being a key condition for activity in ultralow doses.It was previously shown that an MHTB-containingfraction isolated from rat liver extract via isoelectricfocusing activated pigmented newt liver cells, whichare responsible for the protective function in amphib-ians [2].The goal of the work was to study the structure ofMHTB isolated from rat liver tissue.EXPERIMENTALAfter the perfusion of rat liver through the portalvein, its fragments were treated with physiologicalsolution for 3 h. After centrifugation (2000 g, 30 min),the liver extract was studied or stored at –25°C for along time.The membranotropic activity of bioregulators wasevaluated by the adhesion method with a model of themultiple organotypic cultivation of the liver or lung ofhybrid F1 С57Bl/СВА male mice (18–22 g) from thevivarium of the Koltsov Institute of DevelopmentalBiology of the Russian Academy of Sciences [3].Ammonium sulfate was added to the extract of theliver tissue (to 100% saturation) with stirring, and themixture was incubated for 96 h at 4°C. Precipitatedproteins were separated by centrifugation (15000 g,30 min), and the resultant supernatant was dialyzedagainst a 50-fold excess of distilled water containingsodium azide for 10 days at 4°C with repeated waterchanges. Ammonium sulfate was then added to thedesalted supernatant (to 100% saturation), and theprecipitated proteins were separated by centrifugationand dialyzed as above. The resultant supernatant wasconcentrated in a rotary evaporator (37°C) and storedat –70°С.Electrophoresis was carried out by the Laemmlimethod [4] in denaturing 15% PAAG (8 × 10 cm, witha gel thickness of 0.75 mm) in the presence of sodiumdodecyl sulfate (SDS). The gel was stained byCoomassie G-250. Electrophoresis was also carriedout without SDS. The position of a protein withmolecular mass of 66–67 kDa was determined usingthe marker proteins. The desired protein was elutedfrom the gel by small amounts of saline. The resultanteluates were combined, dialyzed against saline, andconcentrated in SpeedVac.Circular dichroism spectra in the UV region (195–260 nm) were recorded at 20°C on a Jasco 720 CDspectrometer (Japan) in quartz cuvettes with an opticalpath length of 1 mm. The scanning rate was 50 nm/minat intervals of 1 nm with accumulation for 2 s; the pro-tein concentration was 100 μg/mL. The final spectrumwas obtained by averaging the results of three scans andsubtracting the spectrum of the base line (control). Theelemental contents of the secondary structure was eval-uated by the CDNN program (Germany).The hydrodynamic radius of the particles in thesolution was determined by the dynamic light scatter-
