[Synthesis and secretion of human atrial natriuretic factor in Escherichia coli cells]. / Sintez i sekretsiia predserdnogo natriiureticheskogo faktora chelovekav kletkakh Escherichia coli.

A recombinant plasmid providing for the synthesis and secretion of the human atrial natriuretic peptide (hANP) as a C-terminal hybrid with the St. aureus protein A was constructed. The level of secretion of the chimeric proteins and their proteolytic stability were shown to depend upon the genotype of the recipient strains and the cultivation conditions. The hybrid proteins were purified by chromatography on IgG Sepharose. The presence of peptides corresponding to the hANP in the acid hydrolysates of the secreted and affinity-purified proteins was confirmed by the enzyme-linked immunoassay and analytical HPLC.

[Expression of hybrid genes containing sequences coding for human adrenocorticotropic hormone in Escherichia coli strains]. / Izuchenie ékspressii gibridnykh genov, soderzhashchikh posledovatel'nost', kodiruiushchuiu adrenokortikotropnyi gormon cheloveka v shtammakh Escherichia coli.

E. coli strains producing a hybrid protein containing human adrenocorticotropic hormone (ACTH) and protein A of S. aureus were obtained. The sequence coding for ACTG was obtained from the bovine one using oligonucleotide-directed mutagenesis. The ACTG gene was linked with the protein A gene and its derivatives by synthetic adaptors. It was shown that each of the constructed plasmids directed the synthesis of hybrid protein in E. coli. This protein was purified on IgG-Sepharose. Then ACTH was obtained by HPLC after specific hydrolysis. The amino acid composition of purified sample was determined.

[The effects of a cardioactive hypothalamic neurohormone on the isolated rat heart]. / Issledovanie vliianii kardioaktivnogo neirogormona gipotalamusa na izolirovannoe serdtse krysy.

On the isolated perfused by Langendorf at heart the cardioactive hypothalamic neurohormone NG3a (2 x 10(-5) g/ml) significantly decreased the coronary flow rate; the effect persisted during 5-10 min, after that the second phase appeared which was expressed by the increase of coronary flow rate. The maximum effect was 122% compared with control. The second phase appeared in 30-45 min after infusion of NG3a. NG3a did not influence the heart rate, but sometimes (7.3% cases) during the maximum increase of coronary flow the bradyarrhythmia was registered. It has been concluded that NG3a influences directly the coronary vessels.

[The neuroendocrine heart and the hypothalamus--hormones of the integration and regulation of coronary vascular tonus]. / Neiroéndokrinnoe serdtse i gipotalamus--gormony integratsii i reguliatsii tonusa koronarnykh sosudov.

Neurosecretory hormone formation by atrial ganglionic cells plays a major role in self-regulation of the heart and its coronary circulation, as well as in integration of the atrium with endocrinal hypothalamus. New data on polypeptide hormones and their precursors is presented. Some atrial glycopeptides play the role of the liberins of hypothalamic cardio-active neurohormones thus carrying out the function of integration of endocrinal hypothalamus with the neuroendocrine heart. The problem of neural and humoral ways of the integration are discussed.

[Coronary-active bovine heart proteins induce relaxation in the rabbit aorta]. / Koronaroaktivnye belki serdtsa byka vyzyvaiut relaksatsiiu aorty krolika.

Seven cardioactive polypeptides have been identified in the precardiac and auricular regions of the bovine heart. Those polypeptides were studied for their action on the function of isolated preparations of the vessel. It has been found that compounds isolated from the precardiac region induce different (20-55%) relaxation of smooth muscles in a strip of the aorta of rabbit. The possibility of formation of endogenic cardioactive compounds in model experiments imitating processing is studied. The data obtained permit assuming and alternatively explaining existence of multiple forms of the mentioned compounds as a result of partial proteolytic splitting of larger molecules.

[Detection and identification of new cardioactive proteins in cattle heart]. / Obnaruzhenie i identifikatsiia v serdtse byka novykh kardioaktivnykh belkov.

Seven cardioactive polypeptides were identified in precardiac and auricular regions of bovine heart. Procedures used for purification of the polypeptides involved extraction of water-soluble proteins, fractionation by ammonium sulfate, gel filtration on Sephadex G-100, chromatography and rechromatography on DEAE cellulose. Some physico-chemical and biological properties were studied; amino acid composition, N-terminal amino acids, molecular mass and the effects on coronary blood vessels. The data obtained suggest that multiple forms of these proteins may exist either as a result of partial hydrolysis of large molecules or due to processing of precursors.

[Distribution of neurospecific cardioactive protein-hormone complex in the rat body during experimental myocardial ischemia]. / Raspredelenie neirospetsificheskogo kardioaktivnogo belok-gormonal'nogo kompleksa v organizme krysy pri éksperimental'noi ishemii miokarda.

For more than 25 years the chemistry and the function of the protein-hormonal complexes (produced by magnocellular nuclei of human and same animals) have been studied. The methods of radioimmunological analyses (RIA) for the detection of new neuropecific cardioactive protein-hormone "K" (RHK) in rat organism with myocardial ischemia has been developed. Concentration of PHK in various regions of the brain by RIA a four days after the occlusion of the carotid artery has a sharp decrease. Particularly concentration of PHK decreases 100-fold in the cerebral cortex. At the same time the level of PHK content in the blood increased from 13 +/- 0.85 to 630 +/- 3.9 ng/ml. The maximum concentration of PHK shows a sharp rise in the spleen 200-fold of their original level. This distribution pattern implies that PHK may be of importance for peripheral tissues and to the scarring in heart neurosis zone.

[Expression in Escherichia coli of hybrid genes containing sequences coding the bovine adrenocorticotropic hormone]. / Ekspressiia v Escherichia coli gibridnykh genov, soderzhashchikh posledovatel'nosti, kodiruiushchie adrenokortikotropnyi gormon byka.

E. coli strains producing a hybrid protein, containing adrenocorticotropic hormone (ACTH) and protein A of S. aureus was obtained. The sequence coding for ACTH was obtained from the bovine proopiomelanocortin cDNA and, after the modification of the 5'- and 3'-terminal parts, was linked with the protein A gene and its derivatives due to synthetic adaptors. Three forms of ACTH gene, coding this hormone with differing N-terminal amino acid were used to construct the fusion gene. The hybrid proteins contain Asp-Pro or (Asp)4-Lys sequences for obtaining ACTH by acid or enterokinase treatment, respectively. It is shown that each of the constructed plasmids direct the synthesis of hybrid protein in E. coli. This protein was purified by the use of IgG-sepharose. The level of the expression of the hybrid protein is 4 mg/l of the bacterial culture. Most of the synthesized protein is secreted into the periplasmic space.

[Regional, cellular, subcellular distribution of neurospecific protein-hormonal complexes]. / Regional'noe, kletochnoe, subkletochnoe raspredelenie neirospetsificheskikh belok-gormonal'nykh kompleksov.

A sensitive radioimmunological assay (RIA) has been developed to detect the tissue specificity and subcellular localization of three specific protein-hormonal complexes (PHC) of the hypothalamus which have a regulatory function in the brain and visceral organs. Using the highly specific rabbit antisera to beef PHC an order of increasing immunoreactivity in different areas of CNS is as follows: hypothalamus, cerebellum, occipital cortex. The PHC-like immunoreactivity (IR) is found in the neurosecretory granules of the hypothalamo-neurohypophyseal system (about 30%) and in the synaptosomal fraction (70%). In the myelin, mitochondrial and nuclear fractions the PHC-like IR is not revealed. IR of PHC is 1000-fold higher in the brain than in visceral organs (heart, skeletal muscles, adrenals, pancreas and blood serum). A possible role of the PHC as markers of the neuroendocrine cells is discussed.

[p-Nitroanilides of amino acids and peptides and fluorescence peptide with inner fluorescence quenching as substrates for cathepsins H, B, D and high molecular weight aspartic peptidase in the brain]. / p-Nitroanilidy aminokislot i peptidov i fluorestsentnyi peptid s vnutrennim tusheniem fluorestsentsii kak substraty katepsinov H, B, D i vysokomolekuliarnoi aspartil'noi peptidazy golovnogo mozga.

p-Nitroanilides of amino acids and peptides were used to study the specificity of cathepsins H and B from human and bovine brain, respectively. The specific activity of cathepsin H decreased in the following order: Arg-pNa greater than or equal to Leu-pNa greater than Ala-pNa greater than or equal to Phe-pNa greater than Pro-pNa greater than Glu-pNa; Arg-pNa was split by the enzyme 12 times as fast as Bz-Arg-pNa. Among other oligopeptide p-nitroanilides tested (Ala-Ala, Ala-Leu, Ala-Ala-Ala, Ala-Ala-Leu, Gly-Gly-Leu, Gly-Gly-Phe, Gly-Leu-Phe, pGlu-Phe-Leu, pGlu-Phe-Ala, pGlu-Phe), PGlu-Phe-Leu and pGlu-Phe-Ala appeared to be the best substrates for cathepsin B; Km for hydrolysis were 0.1 mM and 0.165 mM, respectively, kcat were 5.1 and 8.3 s-1, respectively. A comparative study of substrate specificity of cathepsin D and high molecular weight aspartic peptidase with the use of fluorescent substrate with inner fluorescence quenching, Abz-Ala-Ala-Phe-Phe-pNa, revealed that both peptidases hydrolyzed the single bond between two phenylalanine residues, resulting in the increase of fluorescence (4.5-5-fold) of anthraniloyl tripeptide. The Km values for the substrate hydrolysis by cathepsin D and high molecular weight aspartic peptidase were 6.2 microM and 11.2 microM; kcat were 7.2 s-1 and 1.3 s-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Brain cathepsin as dipeptidylcarboxypeptidase transforming provasopressor, pro-opioid and model peptides]. / Katepsin golovnogo mozga kak dipeptidilkarboksipeptidaza, prevrashchaiushchaia provazopressornye, proopioidnye imodel'nye peptidy.

Cathepsin B from brain exhibited both endopeptidase and dipeptidyl carboxypeptidase activity. Recently the factors, contributing to dipeptidyl carboxypeptidase properties of brain cathepsin B, were identified: I. occupation of the enzyme S3 subsite, 2. free C-terminal group of the substrate, 3. specific interaction between the split off dipeptide and the enzyme active site. The identification was carried out using angiotensin I, its C-end tripeptide and chromophore oligopeptides containing p-nitrophenylalanine residue. C-terminal dipeptide was split off in the proopioid peptides dynorphins 1-7 and 1-8, Met-enkephalin-Arg6-Phe7, Met-enkephalin-Arg6-Gly7-Leu8; the enzyme hydrolyzed also the C-terminal dipeptide bond in Leu- and Met-enkephalins without the subsequent hydrolysis of the remaining tripeptide. D-Ala2, D-Leu5-enkephalin were not hydrolyzed; the bond Arg9-Pro10 was resistant to proteolysis in dynorphin 1-11. Cathepsin B split off the C-terminal dipeptide in synthetic substrates Leu-Trp-Met-Arg-Phe-Ala and Trp-Met-Arg-Phe-Ala but not in Met-Arg-Phe-Ala. These results 06.08 M-15 demonstrated the essential role of branched-chain amino acid residue at the position of P2 and/or P3 of substrates for the enzyme dipeptidyl carboxypeptidase activity. The data obtained suggest that Arg residue at the position P2 (dynorphin 1-7) slowed down, D-amino acid at the position P2 (D-Ala2, D-Leu5-enkephalin) and Pro-Lys bond at the position P1-P2 (dynorphin 1-11) inhibited the cathepsin B dipeptidyl carboxypeptidase activity.

[Radioimmunological characteristics of the distribution of hypothalamic glycoprotein--a precursor of cardiotropic neurohormone C--in the brain and viscera]. / Radioimmunologicheskaia kharakteristika raspredeleniia gipotalamicheskogo glikoproteina--predshestvennika kardiotropnogo neirogormona C v mozge i vistseral'nykh organakh.

A radioimmunological procedure is described for estimation of cardiotropic glycoprotein (BNS), isolated from bovine hypothalamus, in various brain structures and some visceral tissues. The rabbit antiserum towards bovine BNS was used at a dilution 1:10,000. Concentration of BNS measured by the procedure in cat hypothalamus and cerebellum was equal to 1.5 ng and 0.84 ng/g of wet tissue, respectively. The immunoreactive activity of BNS was also found in adrenal glands, heart and skeletal muscles; it was not observed in kidney, lung and pancreas. Content of BNS in cat blood was 10 ng/ml. BNS appears to penetrate into circulating blood.

[Isoenzyme composition of cathepsin D from bovine hypothalamus]. / Izofermentnyi sostav katepsina D iz gipotalamusa byka.

The isoenzyme composition of cathepsin D from bovine hypothalamus was studied by isoelectric focusing. It was found that the soluble fraction of hypothalamic proteins contains five peaks of endopeptidase activity at pH 3.2. The properties studied allowed to identify these peaks of endopeptidase activity as isoenzyme forms of cathepsin D.

[Effect of neurohormone C and hexapeptide on incorporation of labeled amino acids into the proteins of different organs]. / Vliianie neirogormona C i geksapeptida na vkliuchenie mechenykh aminokislot v belki razlichnykh organov.

The influence of two coronarodilatatory substances (neurohormone "C" and hexapeptide) on the rate of protein synthesis was investigated. The stimulation of incorporation of labelled (14C- and 3H-) leucine into proteins of rat brain, heart and liver tissues characterized the hormonal regulation of the protein synthesis in these organs in vivo. Activation of the protein synthesis depended on the dose of the hormone administered. 5 mg of hexapeptide markedly inhibited the amino acid incorporation into proteins, while 0.5 mg produced the effect, which was contrary to the effect caused by 5 mg of the hormone. The data obtained suggest that hypothalamic neurohormonal preparations exhibit an important effect on protein metabolism in different organs.