Imobilizacija Na,K-ATPpaze izolovane iz sinaptičkih plazma-membrana mozga pacova

Rat brain Na,K-ATPase partially purified by SDS from synaptic plasma membranes (SPM) was immobilized by adsorption on nitrocellulose (NC), polyvinylidene fluoride (PVDF) and glass fiber (GF) membranes, Partial SDS solubilization increased the enzyme activity by 40%. With regard to the presentation of the enzyme activity. nitrocellulose Was shown to be the optimal support for die immobilization, The enzyme showed the highest percentage activity (14%) after 30 min of SPM adsorption. at 20 degreesC under the vaccum. with 25 mug of proteins per NC disc filter. In addition, adsorption on NC stabilizes the Na,K-ATPase, since the activity was substantial 72 h after adsorption at 20 degreesC, After adsorption. the sensitivity of the enzyme to HgCl2 and CdCl2 inhibition was higher, The results show that immobilized Na,K-NTPase SPM can be used as a practical model for the detection of metal ions in different samples.Delimično prečišćena Na,K-ATPaza sinaptičkih plazma–membrana (SPM) mozga pacova imobilizovana je adsorpcijom na nitrocelulozne (NC) poliviniliden-fluorid (PVDF) membrane i membrane od staklenih vlakana (SV). Aktivnost enzima delimično prečišćenog solubilizacijom SDS-om povećana je oko 40%. Najveći procenat aktivnosti (14%) enzim zadržava posle 30 minuta adsorpcije SPM na 20ºC, pod vakuumom, sa 25 μg proteina po nitroceluloznom disku. Na,K-ATPaza imobilizovana na nitroceluloznoj membrani stabilna je 72 sata na 20ºC. Adsorpcijom, osetljivost enzima na inhibiciju Hg2+ i Cd2+ se povećava. Rezultati pokazuju da se imobilizovana Na,K-ATPaza SPM može koristiti za detekciju toksičnih metalnih jona u različitim uzorcima

Pharmacological Aspects of Vipera xantina palestinae Venom

In Israel, Vipera xantina palestinae (V.x.p.) is the most common venomous snake, accounting for several hundred cases of envenomation in humans and domestic animals every year, with a mortality rate of 0.5 to 2%. In this review we will briefly address the research developments relevant to our present understanding of the structure and function of V.x.p. venom with emphasis on venom disintegrins. Venom proteomics indicated the presence of four families of pharmacologically active compounds: (i) neurotoxins; (ii) hemorrhagins; (iii) angioneurin growth factors; and (iv) different types of integrin inhibitors. Viperistatin, a α1β1selective KTS disintegrin and VP12, a α2β1 selective C-type lectin were discovered. These snake venom proteins represent promising tools for research and development of novel collagen receptor selective drugs. These discoveries are also relevant for future improvement of antivenom therapy towards V.x.p. envenomation

Vipegitide: a folded peptidomimetic partial antagonist of α2β1 integrin with antiplatelet aggregation activity

Tatjana Momic,1 Jehoshua Katzhendler,1 Ela Shai,2 Efrat Noy,3 Hanoch Senderowitz,3 Johannes A Eble,4 Cezary Marcinkiewicz,5 David Varon,2 Philip Lazarovici11School of Pharmacy Institute for Drug Research, Faculty of Medicine, The Hebrew University of Jerusalem, 2Department of Hematology, Coagulation Unit, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 3Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel; 4Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany; 5Department of Bioengineering, College of Engineering, Temple University, Philadelphia, PA, USAAbstract: Linear peptides containing the sequence WKTSRTSHY were used as lead compounds to synthesize a novel peptidomimetic antagonist of α2β1 integrin, with platelet aggregation-inhibiting activity, named Vipegitide. Vipegitide is a 13-amino acid, folded peptidomimetic molecule, containing two α-aminoisobutyric acid residues at positions 6 and 8 and not stable in human serum. Substitution of glycine and tryptophan residues at positions 1 and 2, respectively, with a unit of two polyethylene glycol (PEG) molecules yielded peptidomimetic Vipegitide-PEG2, stable in human serum for over 3 hours. Vipegitide and Vipegitide-PEG2 showed high potency (7×10-10 M and 1.5×10-10 M, respectively) and intermediate efficacy (40% and 35%, respectively) as well as selectivity toward α2 integrin in inhibition of adhesion of α1/α2 integrin overexpressing cells toward respective collagens. Interaction of both peptidomimetics with extracellular active domain of α2 integrin was confirmed in cell-free binding assay with recombinant α2 A-domain. Integrin α2β1 receptor is found on the platelet membrane and triggers collagen-induced platelet aggregation. Vipegitide and Vipegitide-PEG2 inhibited α2β1 integrin-mediated adhesion of human and murine platelets under the flow condition, by 50%. They efficiently blocked adenosine diphosphate- and collagen I-induced platelet aggregation in platelet rich plasma and whole human blood. Higher potency of Vipegitide than Vipegitide-PEG2 is consistent with results of computer modeling of the molecules in water. These peptidomimetic molecules were acutely tolerated in mice upon intravenous bolus injection of 50 mg/kg. These results underline the potency of Vipegitide and Vipegitide-PEG2 molecules as platelet aggregation-inhibiting drug lead compounds in antithrombotic therapy.Keywords: adhesion, collagen I, platelets, integrin antagonist, peptidomimeti

Vixapatin (VP12), a C-Type Lectin-Protein from Vipera xantina palestinae Venom: Characterization as a Novel Anti-angiogenic Compound

A C-type lectin-like protein (CTL), originally identified as VP12 and lately named Vixapatin, was isolated and characterized from Israeli viper Vipera xantina palestinae snake venom. This CTL was characterized as a selective α2β1 integrin inhibitor with anti-melanoma metastatic activity. The major aim of the present study was to prove the possibility that this protein is also a potent novel anti-angiogenic compound. Using an adhesion assay, we demonstrated that Vixapatin selectively and potently inhibited the α2 mediated adhesion of K562 over-expressing cells, with IC50 of 3 nM. 3 nM Vixapatin blocked proliferation of human dermal microvascular endothelial cells (HDMEC); 25 nM inhibited collagen I induced migration of human fibrosarcoma HT-1080 cells; and 50 nM rat C6 glioma and human breast carcinoma MDA-MB-231 cells. 1 µM Vixapatin reduced HDMEC tube formation by 75% in a Matrigel assay. Furthermore, 1 µM Vixapatin decreased by 70% bFGF-induced physiological angiogenesis, and by 94% C6 glioma-induced pathological angiogenesis, in shell-less embryonic quail chorioallantoic membrane assay. Vixapatin’s ability to inhibit all steps of the angiogenesis process suggest that it is a novel pharmacological tool for studying α2β1 integrin mediated angiogenesis and a lead compound for the development of a novel anti-angiogenic/angiostatic/anti-cancer drug