The distribution of the glyprolines Pro-Gly-Pro and Thr-Lys-Pro-Arg-Pro-Gly-Pro (Selanc) was analyzed and compared in tissues of rat organs after different ways of their administration using the peptides uniformly labeled with tritium. Comparative data on changes in concentrations of the peptides in the rat organs after their intraperitoneal, intranasal, intragastric, and intravenous administration are given. The intranasal administration of both peptides was shown to be optimal for the delivery of glyproline molecules in the CNS. A high affinity of the studied glyprolines for gastric tissues was found for all the ways of their administration. We suggest that a high efficiency of action of glyprolines on homeostasis of the gastric mucous tunic was partially provided by accumulation of these peptides (to high concentrations) in gastric tissues.

Работа посвящена применению равномерно меченных тритием пептидов для изучения их биодеградации in vivo и in vitro. Реакцией высокотемпературного твердофазного каталитического изотопного обмена (ВТКИО) со спилловер-тритием получены равномерно меченные тритием биологически активные пептиды с молярной радиоактивностью 50-150 Ки/ммоль. Распределение изотопной метки по всем аминокислотам в этих пептидах позволяет определять практически все возможные продукты их ферментативного гидролиза одновременно. Разработанный метод анализа включает экстракцию органическими растворителями меченных тритием пептидов из тканей организма и хроматографиче-ское выделение индивидуальных меченых пептидов из смеси продуктов биодеградации. Концентрацию исследуемого пептида и продуктов его биодеградации рассчитывают с использованием жидкостного сцинтилляционного счета. С использованием описанного подхода исследованы пути биодеградации пептидов TKPRPGP (Селанк) и трипептида PGP в плазме крови. Исследована также фармакокинетика пептидного анксиолитика Селанка в тканях мозга при интраназальном введении in vivo. Определены концентрации меченых пептидов и показано, что основными продуктами биодеградации Селанка являются пентапептид TKPRP, трипептид ТКР, дипептиды RP и GP. Исследована биодеградация гептапептида MEHFPGP (Семакс) в присутствии нервных клеток и определено, что основными продуктами его биодеградации являются пентапептид HFPGP, трипептид PGP. Исследовано влияние на энкефалиназную активность плазмы крови с использованием равномерно меченного тритием [Lеu]энкефалина. Показано, что высокая ингибирующая способность Семакса на энкефалиназы плазмы крови связана с его действием на аминопептидазы. Предложенный метод, основанный на использовании равномерно меченных тритием пептидов, дает возможность как определять концентрацию пептидов в тканях in vitro и in vivo, так и оценивать активность ферментов, участвующих в их деградации.

A peptide acidic hydrolysate of collagen (PHC) was obtained under conditions (4 N HCl) ensuring the predominant formation of short peptides, glyprolines. They were separated and their antiulcer activity was studied. Thirty individual peptides with molecular masses of 174-420 amu were isolated from the PHC by HPLC. The PHC was shown to predominantly contain 2- to 4-aa peptides, including PG, GP, and PGP. Experiments on rats demonstrated that, on intragastric administration at a dose of 1 mg/kg, PHC enhances the stability of the gastric mucosa to the action of ulcerogenic factors, such as ethanol and stress, and exhibits a protecting antiulcer effect. Even a lesser dose (0.1 mg/kg), which reduced ulcer area twofold, was effective in the stress model of ulcer formation. The intraperitoneal and intragastric administration of PHC at a dose of 1 mg/kg was found to exhibit a therapeutic effect in the acetate model of ulcer formation.

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50-150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium-labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a tg scale of biological samples both in vitro and in vivo.

The new glyproline family was distinguished from the regulatory peptides recently. It includes the simplest proline- and glycine-containing peptides: PG, GP, PGP, and respective peptides with hydroxylated proline residues. Glyproline's bioactivity covers many important systems of the body including suppression of some reaction in the blood coagulation and platelet aggregation and gastric mucosal maintenance. It was shown that PGP, PG and GP have a wide spectrum of antiulcer activity with respect to gastric mucosal damages of various aetiology. GHyp and HypGP show also antiulcer action. In vivo glyprolines being fragments of collagen may be generated during synthesis and catabolism of collagen. It is well known that approximately 10-60% of newly synthesized collagen degrade intracellularly with succeeding secretion of small peptides composed of less than 5 aminoacid residues out of cells. Different simplest proline and hydroxyproline fragments of glyprolines are revealed in various type of collagen: GP, GHyp, PG, PPG, PGP, PHypG., GPHyp, GPP, GPG, GHypP, HypGP. It is possible that these fragments may be also secreted out of cells during the stage of degradation of newly synthesized collagen. We showed that the intragastric (per oral) introduction of hydrolyzed gelatin, having 20 small peptide fragments, including PGP and HypGP, also increase gastric stability showing protective and therapeutic antiulcer effect. The corresponding receptors for glyprolines are not completely identified yet but it may be supposed that PGP, GP and other glyprolines interact with the same receptors with which the III type collagen is binding with platelet's receptors. It is supposed that octapeptide sequence KPGGluPGPK of collagen is rather important for binding with receptor. When this sequence in the structure of collagen's molecule binds with the receptor, platelet aggregation is induced. Free octapeptide blocks the receptor and inhibits platelet aggregations. Qualitatve characteristics of parameters of inhibition with intact octapeptide and glyproline, as well as the receptor's structure--that's our concern for the nearest future.

A possibility of dipeptide derivatization by alkyl chloroformate-alkanol (Alk = Me, Et, Pr, Bu) mixed reactants for their determination and differentiation of isomers in mixtures by gas chromatography/mass spectrometry with electron and chemical ionization and collisional activation was studied. Prospects for using derivatization by mixtures of methyl chloroformate with 2,2,2-trifluoroethanol, 2,2,3,3,3-pentafluoropropanol, and 2,2,3,3,4,4,4-heptafluorobutanol were shown. Diagnostic ions that make it possible to determine the amino acid sequence in dipeptides and distinguish residues of isomeric leucine and isoleucine were revealed.

Разработан метод анализа энкефалиназной активности плазмы крови, основанный на использовании [Leu]энкефалина, меченного тритием по всем аминокислотным остаткам и позволяющий одновременно оценивать активность нескольких пептидаз в микроколичестве ткани. Методом твердофазного каталитического изотопного обмена получен [G-3H, Leu]энкефалин (120 Ки/ммоль), который подвергали протеолизу под действием плазмы крови, а затем радиоактивные метаболиты разделяли методом ВЭЖХ в присутствии смеси немеченых фрагментов [Leu]энкефалина как внутренних стандартов. Показано, что около 80% энкефалиндеградирующей активности в плазме крови человека приходится на аминопептидазы, 2% - дипептидиламинопептидазы, 10% - дипептидилкар-боксипептидазы. Обнаружен новый путь деградации [Leu]энкефалина карбоксипептидазой, обеспечивающий около 6% энкефалиндеградирующей активности. Показано, что бестатин ингибирует преимущественно аминопептидазы и карбоксипептидазы, а Селанк более специфичен к карбокси-и дикарбоксипептидазам.A method of analysis of enkephalinase activity in blood plasma based on the application of

A method of analysis of enkephalinase activity in blood plasma based on the application of Leu-enkephalin generally labeled with tritium at all its amino acid residues was developed. The method allows the simultaneous estimation of activity of several peptidases in microquantities of tissues. [G-3H]Leu-enkephalin was prepared by the method of solid phase catalytic isotope exchange (120 Ci/mmol) and subjected to proteolysis by the treatment with blood plasma. The resulting radioactive metabolites were separated by HPLC in the presence of the mixture of unlabeled fragments of Leu-enkephalin as internal standards. It was shown that aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases respond for approximately 80%, 2%, and 10% of the total enzymatic activity, respectively. The new pathway of degradation of Leu-enkephalin by carboxypeptidase that provides for approximately 6% of the total enkephalin-degrading activity was discovered. Bestatin was shown to predominantly inhibit aminopeptidases and carboxypeptidases, whereas Selank is more specific for carboxypeptidases and dicarboxypeptidases. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Main pathways of degradation of PGP peptide possessing antiulcer and antithrombotic activities were studied after its intraperitoneal, intragastric, and intraintestinal administration. In experiments on rabbits we showed by HPLC that unmodified PGP is released into the blood after administration by all three routes and is detected in the plasma over 3-5 h. PG dipeptide is a more stable PGP metabolite presumably determining (together with tripeptide) its pharmacological properties.

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue if less than four negatively charged amino acid residues are in positions P2–P5 of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR ↓ VYIHPF) and the Hb(2–8) (LTAEEK ↓ A) and Hb(1–9) (MLTAEEK ↓ AA) peptides of the cattle hemoglobin β-chain. The Km values for all the substrates (≈10–3 M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the –DDDDK– full-size linker (Km ≈ 10–4 M). The kcat values for AT and Hb(2–8) were also close and low (≈ 30 min–1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2–8) peptide by one amino acid residue from both its N- and C-termini results in a dramatic increase in the catalytic efficiency of the hydrolysis: the kcat value for Hb(1–9) is 1510 min–1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.

The most stable regulatory peptides (RP) including the new family of RP (glyprolines) and derivatives of hybrid peptide MEHFPGP are characterized. High ability of glyprolines to penetrate into the blood-stream through the gastrointestinal tract is demonstrated. Antiulcer, antithrombotic and antidiabetic activities of glyprolines were discovered in experiments on white rats. The activity of oligopeptides PGP, PG and GP is compared. Mechanisms of glycoprolines activities and feasibility of their administration with connective tissue food proteins are discussed. Thus, glyprolines are perspective drugs for treatment of gastric ulcer, correction of hemostasis and thrombosis suppression prepared for preclinical trial.

Реакцией высокотемпературного твердофазного каталитического изотопного обмена (ВТКИО) водорода на тритий при температуре 150°С получен [3Н]даларгин с молярной радиоактивностью 52 Ки/ммоль. Меченный тритием пептид полностью сохранил биологическую активность, которую оценивали по способности связываться с опиоидными рецепторами (ОР) головного мозга крыс. Определена величина константы диссоциации комплексов даларгина с ОР, составляющая 4.3 нМ. Для этого пептида была определена зависимость химического выхода и молярной радиоактивности от длительности и температуры реакции ВТКИО. Рассчитана энергия активации реакции ВТКИО для пептида, составляющая 32 ккал/моль. Аминокислотным анализом [3Н]даларгина получены данные по распределению изотопной метки в аминокислотных остатках пептида. При реакции ВТКИО тритиевая метка включается во все аминокислотные остатки пептида, причем с ростом температуры, наряду со значительным увеличением общего включения трития, увеличивается доля активности, находящейся в ароматических остатках.A [3H]

A [3H]Dalargin preparation with a molar radioactivity of 52 Ci/mmol was obtained by the high temperature solid-state catalytic isotope exchange (HSCIE) of tritium for hydrogen at 150°C. This tritium-labeled peptide was shown to completely retain its biological activity in the test of binding to opioid receptors from rat brain. The dissociation constant of the Dalargin-opioid receptor complex was found to be 4.3 nM. The dependences of the chemical yield and the molar radioactivity on the reaction time and temperature of HSCIE were determined. The activation energy of the HSCIE reaction for the peptide was calculated to be 32 kcal/mol. The amino acid analysis showed that tritium is distributed between all the amino acid residues of [3H]Dalargin at the HSCIE reaction, with the temperature growth significantly increasing the total tritium incorporation and, especially, enhancing the radioactivity incorporation into aromatic residues.

A [3H]Dalargin preparation with a molar radioactivity of 52 Ci/mmol was obtained by the high temperature solid-state catalytic isotope exchange (HSCIE) of tritium for hydrogen at 150 degrees C. This tritium-labeled peptide was shown to completely retain its biological activity in the test of binding to opioid receptors from rat brain. The dissociation constant of the Dalargin-opioid receptor complex was found to be 4.3 nM. The dependencies of the chemical yield and the molar radioactivity on the reaction time and temperature of HSCIE were determined. The activation energy of the HSCIE reaction for the peptide was calculated to be 32 kcal/mol. The amino acid analysis showed that tritium is distributed between all the amino acid residues of [3H]Dalargin at the HSCIE reaction, with the temperature growth significantly increasing the total tritium incorporation and, especially, enhancing the radioactivity incorporation into aromatic residues.

More than 20 peptides have been isolated and sequenced from the brain of hibernating ground squirrel Citellus undulatus. Some of the isolated peptides were chemically synthesized and investigated for the spectrum of biological activity. One of the isolated peptides, neokyotorphin (Thr-Ser-Lys-Tyr-Arg), earlier known as a weak analgetic, is found to have a cardiotropic and thermoregulatory activity. Neokyotorphin activates in vitro voltage-dependent calcium and blocks ATP-dependent potassium currents in the frog atrial fibres. Intraperitoneal injection of this peptide in hibernating ground squirrels speeds up the arousal of animals increasing sharply the heart rate and oxygen consumption. Intraperitoneal and intranasal administrations of neokyotorphin in rats raises body temperature in thermoneutral conditions (26-28 degrees C) exerting no effects at low (4-6 degrees C) and high (31-32 degrees C) environmental temperatures. Another isolated peptide, Asp-Tyr, blocks the inward voltage-dependent calcium current in the frog atrial fibres and slightly increases the outward potassium current. Sulfated analogue of this dipeptide (aspartyl-O-sulfate-tyrosine) more effectively blocks the inward voltage-dependent calcium current in the frog atrial fibres and have no effects on the outward potassium current.

Dipeptide ASP-TYR extracted from the brain of hibernating squirrels was found to potentiate vagal effect on the cardiac rhythm, whereas pentapeptide neokyotorphin--to diminish it. In both cases the effect was due to an alteration of the tonic component's strength in the vagal chronotropic effect. The selective peptidergic modulation of the vagal effect on the cardiac rhythm suggests different physiological roles of the tonic and synchronizing components in regulation of the heart.