Biomedical subjects

M E Zupec

Publications and source records attributed to M E Zupec.

8 recordsLinked to original sources

PubMed1991

In vitro and in vivo effects of a peptide mimetic (SC-47643) of RGD as an antiplatelet and antithrombotic agent.

Platelet aggregation requires binding of fibrinogen (fgn) to activated platelets and inhibition of this binding blocks platelet aggregation. Synthetic peptides modeled after the platelet binding sequence on fgn block the platelet glycoprotein IIb/IIIa receptor and effectively inhibit aggregation. SC-47643 (SC) is a mimetic of the RGD-containing peptide sequence that is recognized by the platelet IIb/IIIa receptor. SC inhibited fgn binding to activated platelets (IC50: 1.0 x 10(-5) M) and prevented platelet aggregation in response to a variety of platelet agonists in both washed human platelets and platelet rich plasma (IC50's ranging from 4 x 10(-6) to 1 x 10(-5) M, respectively). SC inhibited collagen induced thrombocytopenia in the rat (ED50 0.07 mg/kg and t1/2 36 min). In dogs ex vivo collagen induced platelet aggregation was inhibited 50% after a bolus injection of 1.7 mg/kg. After a steady state infusion (2 hr), the ED50 was 0.03 mg/kg/min, with no effects on blood pressure, heart rate or platelet count. These data demonstrate that SC, a peptide mimetic of the natural fgn binding sequence, is capable of blocking platelet-fgn interactions and platelet aggregation.

Amino Acid Sequence↗

PubMed1991

Antiplatelet and antithrombotic effects of platelet glycoprotein IIb/IIIa (GPIIb/IIIa) inhibition by arginine-glycine-aspartic acid-serine (RGDS) and arginine-glycine-aspartic acid (RGD) (O-me)Y (SC-46749).

Arginine-glycine-aspartic acid (RGD) is the minimal sequence in fibrinogen that leads to recognition and binding to the glycoprotein IIb/IIIa platelet receptor during aggregation. Analogs of tetrapeptides containing the RGD sequence have been previously shown to block fibrinogen binding to activated platelets in vitro. SC-46749 is an analog of arginine-glycine-aspartic acid-phenylalanine in which the phenylalanine is replaced by O-methyltyrosine. In this study the biological activities of SC-46749 were examined and its actions compared with the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS), one of the natural sequences on the fibrinogen alpha chain that binds to platelets. In vitro, SC-46749 was more potent than RGDS in inhibiting fibrinogen binding (IC50: SC-46749, 27 microM; RGDS, 47 microM), in preventing ADP-induced aggregation in human platelet-rich plasma (IC50: SC-46749, 32 microM; RGDS, 95 microM) and in inhibiting thrombin-induced aggregation in washed human platelets (IC50: SC-46749, 23 microM; RGDS, 64 microM). In rats, SC-46749 prevented collagen-induced thrombocytopenia with an ED50 of 0.87 mg/kg whereas RGDS did not inhibit the response by 50% at doses up to 10 mg/kg. SC-46749 inhibited thrombus formation in an electrically damaged rat carotid artery in a dose-dependent fashion whereas the effects of RGDS were biphasic. RGDS appeared to delay thrombus formation at lower doses but had no effect at higher doses. When infused in dogs for 15 min, SC-46749 prevented ex vivo collagen-induced aggregation at 4 mg/kg/min. These data demonstrate that SC-46749 is a potent inhibitor of platelet aggregation and platelet-dependent thrombus formation.

Animals↗

PubMed1990

Substrate specificity of recombinant human renal renin: effect of histidine in the P2 subsite on pH dependence.

Steady-state kinetic analysis of human renin demonstrates the histidine proximal to the substrate scissile peptide bond contributes to the unique specificity and pH dependence of this aspartyl protease. Recombinant human renal renin purified from mammalian cell culture appears to be indistinguishable from renin isolated from human kidney with respect to specific activity (1000 Goldblatt units/mg). Recombinant renin contains carbohydrate covalently attached to asparagines at positions 5 and 75 (renin numbering) and disulfide linkages at Cys-51/Cys-58, Cys-217/Cys-221, and Cys-259/Cys-296. Renin pH dependence was evaluated between pH 4.0 and 8.0 by using a synthetic substrate identical with the amino terminus of porcine angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu*Leu-Val-Tyr-Ser, where the asterisk indicates the scissile peptide bond and the proximal histidine is in italics) and an analogous tetradecapeptide where the proximal histidine was substituted with glutamine. Comparison of the pH profiles shows the catalytic efficiency (V/Km) and maximal velocity (V) of renin are greater above pH 6.5 with the substrate containing histidine proximal to the scissile peptide bond, but below pH 5.0 these parameters are greater with the glutamine substrate analogue. Solvent isotope effects show that proton transfer contributes to the rate-limiting step in catalysis with both substrates and that the proximal histidine does not serve as a base in the catalytic mechanism. Molecular modeling indicates the substrate histidine could hydrogen bond to Asp-226 of the enzyme (renin numbering), thus perturbing the ionization of the catalytic aspartyl groups (Asp-38 and Asp-226).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Chemical synthesis of bovine transforming growth factor-alpha: synthesis, characterization and biological activity.

Bovine transforming growth factor-alpha (bTGF-alpha) is a 50 amino acid polypeptide with three disulfide linkages. In order to evaluate the biological function of this peptide, bTGF-alpha was synthesized via an automatic synthesizer and purified to homogeneity in high yield. The integrity of this synthetic peptide was confirmed by chemical analyses and bioassays. In a bovine liver radioreceptor assay, bTGF-alpha competes with radiolabeled EGF and has activity comparable to mEGF and hTGF-alpha. Compared to hEGF, bTGF-alpha elicits a greater response in a bovine mammary cell proliferation.

Amino Acid Sequence↗

PubMed1990

Multiple peptide synthesis using a single support (MPS3).

An automated multiple peptide synthesis method to synthesize, cleave, and purify several peptides simultaneously in a single batch has been developed. The technique is based on the synthesis of multiple peptides on a single solid phase support and is easily adapted to manual or to automated methods. The approach relies on coupling of amino acid mixtures to the resin and it has been found that DCC/HOBt gives the best coupling performance. Fast Atom Bombardment Mass Spectrometry (FAB-MS) was used to rapidly and efficiently identify the peptides in each synthetic mixture which significantly assisted the purification process by HPLC. The method has been successfully applied to the synthesis of magainin 2 and angiotensinogen peptides.

Amino Acid Sequence↗

PubMed1990

Isolation of genomic sequence encoding a biologically active bovine TGF-alpha protein.

Genomic clones encoding bovine TGF-alpha were identified by hybridization with probes derived from human TGF-alpha sequence. Nucleotide sequence of the clones predicts that mature bovine TGF-alpha is a 50 amino acid polypeptide which shares 96% and 92% homology with human and rat TGF-alpha, respectively. Bovine TGF-alpha with the predicted sequence was chemically synthesized and tested for activity. Synthetic bovine TGF-alpha competes in a radioreceptor assay with labelled mouse EGF with activity parallel to that of human TGF-alpha and mouse EGF. The mitogenic activity of bovine TGF-alpha is comparable to that of human EGF in causing proliferation of bovine mammary epithelial cells. An approximately 5.0 kilobase RNA transcript is observed in polyadenylated RNA from MDBK cells by Northern blot analysis. The polymerase chain reaction detects the presence of a TGF-alpha transcript in many bovine tissues. These data indicate that bovine TGF-alpha may be a normal regulator of cell growth in the bovine animal.

Amino Acid Sequence↗

PubMed1987

Phosphorylation of high- and low-molecular-mass atrial natriuretic peptide analogs by cyclic AMP-dependent protein kinase.

Synthetic high- and low-molecular-mass atrial peptides were phosphorylated in vitro by cyclic AMP-dependent protein kinase and [32P]ATP. From a series of atrial peptide analogs, it was deduced that the amino acid sequence, Arg101-Ser104 of atriopeptin was required for optimal phosphorylation. Phosphorylated AP(99-126) was less potent than the parent atriopeptin in vasorelaxant activity and receptor-binding properties. These results indicate that the presence of a phosphate group at the N-terminus of AP(99-126) decreases the interaction of the peptide with its receptor and, as a consequence, decreases bioactivity. These observations are in contrast to those of Rittenhouse et al. [(1986) J. Biol. Chem. 261, 7607-7610] who reported that phosphorylation of AP(101-126) enhanced the stimulation of Na/K/Cl cotransport in cultured vascular smooth muscle cells.

Animals↗

PubMed1985

Differential structure-activity relationships of atrial peptides as natriuretics and renal vasodilators in the dog.

Natriuretic-diuretic and vasodilator activities of synthetic atriopeptin (AP)-related peptides were examined in the anesthetized dog. We have selected, the naturally occurring, APIII as the reference compound for comparison with various related peptides. APIII is a 24 amino acid peptide with the sequence ser-ser-cys-phe-gly-gly-arg-ile-asp-arg-ile-gly-ala-gln-ser-gly-leu-gly- cys-asn-ser-phe-arg-tyr-OH. APII, another peptide isolated from atrial extracts, lacks the C-terminal arg- of APIII. N-terminal amino acid extensions on APIII or APII, exhibited enhanced natriuretic-diuretic effectiveness. Furthermore, the maximum response obtained by ser-leu-arg-arg-APIII and arg-arg-APIII were significantly higher and the dose-response curve was not parallel to that obtained with APIII. In contrast, there were no significant qualitative or quantitative differences between the renal blood flow responses produced by the N-terminal extended peptides and APII or APIII. These results suggest a heterogeneity of AP receptors in vascular and renal tubular tissues.

Amino Acid Sequence↗