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Biomedical subjects

L Juliano

Publications and source records attributed to L Juliano.

158 records · Page 9Linked to original sources
PubMed1977

Conformations of synthetic tetradecapeptide renin substrate and of angiotensin I in aqueous solution.

The properties of aqueous solutions of synthetic renin substrate tetradecapeptide (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser) were examined through electrometric titrations, infrared and circular dichroism spectroscopy, and spectrofluorometry. Titration studies of angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) were also made, whose results indicated a flexible folded conformation similar to that previously proposed for the octapeptide angiotensin II, with a possible additional beta turn at the C terminus. The experimental results of the tetradecapeptide study, associated with Chou and Fasman calculations and with an analysis of structure-activity relationships in renin substrates and competitive inhibitors, led to the proposal of a beta turn involving the His-Pro-Phe-His sequence of the tetradecapeptide. This beta turn would be stabilized by beta-antiparallel interaction between residues 3-4 and 10-12 and by electrostatic attraction between the N-terminal ammonium and C-terminal carboxylate groups and would be destabilized below pH 5 by electrostatic repulsion between His6 and His9. The capacity to assume this conformation is related to structural requirements for renin substrates and competitive inhibitors.

Amino Acid Sequence↗
PubMed1977

Requirements for angiotensin tachyphylaxis in smooth muscles.

The ability of angiotensin II (AII) and of seven N-terminally modified peptide analogs to induce tachyphylaxis in isolated preparations of the rat uterus and the guinea-pig ileum was found to correlate well with the degree of amino group protonation. No correlation was found between tachyphylaxis and the affinity for the rat uterus receptors, as measured by the reciprocals of ED50 values. Dissociation between the tachyphylactic property and receptor affinity in the rat uterus was also evident by lowering the calcium concentration in the medium, which resulted in increased tachyphylaxis without significant changes in ED50 values. In the guinea-pig ileum, tachyphylaxis correlated with the reciprocal of ED50, but there was a better correlation with amino group protonation. [6-Pyrazolylalanine] AII was approximately as tachyphylactic as AII in both smooth muscles studied, indicating that protonation of the histidine side-chain may not be important for the manifestation of tachyphylaxis. The recovery of the guinea-pig ileum for AII tachyphylaxis was not affected by repeated treatments with BaCl2, but it was partially impaired by [Suc1]AII, des-Asp-AII, bradykinin and histamine.

Angiotensin II↗
PubMedDate not supplied

Characterization of a prolyl endopeptidase (kininase) from human urine using fluorogenic quenched substrates.

A prolyl endopeptidase (PE) was purified 83 times from human urine by DEAE-cellulose and Sepharose Mercurial chromatographies. In this work we studied the specificity of PE using different fluorogenics substrates. Further characterization of the enzyme was carried out using BK and it's analogue, Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp, for measure of enzymatic activity of prolyl endopeptidase (Abz=ortho-aminobenzoic acid; EDDnp=N-[2, 4-dinitrophenyl]ethylenediamine). The substrate Abz-FPQ-EDDnp was considered as specific for PE. The endopeptidase PE, with a molecular weight of 45 kDa, was inhibited 100% by EDTA and pOHMB and resistant to PMSF, thyorphan, E64 and phosphoramidon, when we used the mentioned substrates. These results suggest that PE is a metallo endopeptidase that contains a thiol group important for it's activity. It was also able to hydrolyze in Abz-RPPGFSPFRQ-EDDnp the F-R peptide bound, differing from those obtained upon BK molecule, where the enzyme prefer the peptide bound located after double proline. In the substrate Abz-FPQ-EDDnp PE hydrolyzes the P-Q peptide bound. Furthermore the urinary PE is particularly unable to hydrolyze peptides with single prolines such as substance P, neurotensin and LHRH. The determined K(m) for Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp were 0.74 and 0.65 uM, respectively. The optimum pH for the PE activity, using the substrate Abz-RPPGFSPFRQ-EDDnp was approximately 9.0, but using the specific substrate Abz-FPQ-EDDnp was 6.5 and 8.0. Endopeptidases, which are situated at brush border surface from proximal tubules, have an important role in kidney handling of many peptides, which are filtered by the glomerulus. The prolyl endopeptidase located at distal tubule could have an important physiological function in control of kinin formed in this portion. It's known that all components from kallicrein-kinin system like low molecular weigh kininogen and kallikrein are presents in this portion.

Bradykinin↗
PubMedDate not supplied

Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity.

To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S3 subsite and hydrophobic residues in S2; the most prevalent residue not being Phe but Val. The S1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S1'-S4' subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the kcat/KM's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO2)PMPPLCTSMK(Abz) (kcat/KM = 2109 (mM s)-1), Y(NO2)PYAVQSPQK(Abz) (kcat/KM = 1524 (mM s)-1), and Y(NO2)PVLRQQRSK(Abz) (kcat/KM = 1450 (mM s)-1). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft.

Amino Acid Sequence↗
PubMedDate not supplied

Synthesis and hydrolysis by arginyl-hydrolases of p-nitroanilide chromogenic substrates containing polyethylene glycol and D-gluconyl moieties.

D-Gluconic acid and alpha-carboxymethyl-polyethylene-glycol-omega-methyl ether (PEG) (mol wt 550) were covalently bound at N alpha-amino group of H-Phe-Arg-pNa to study the effect on hydrolysis by arginyl-hydrolases of chromogenic substrates containing high hydrophilic and amphiphilic groups. For comparison, epsilon-aminocaproyl-, sarcosyl- and succinyl-Phe-Arg-pNa were also synthesized. The obtained compounds were assayed as substrates for porcine pancreatic kallikrein, horse urinary kallikrein, tonin and beta-trypsin. Both PEG- and gluconyl-Phe-Arg-pNa had kcat values of hydrolysis 2-4 times higher than the N-acetyl derivative for all the studied enzymes. epsilon-NH2caproyl-Phe-Arg-pNa resulted in the best chromogenic substrate described for the two tissue kallikreins. The PEG-derivative and D-gluconyl groups were also introduced in the N alpha-amino group of H-Arg-pNa and assayed as beta-trypsin substrates. In comparison with benzoyl-Arg-pNa, the D-gluconyl group had no effect on Km but reduced the kcat value more than 15 times; however, PEG-Arg-pNa was hydrolyzed with similar Km but with kcat 5 times higher. The presence of D-gluconyl and PEG groups in the chromogenic substrate molecules increased their water solubility significantly.

Amino Acid Sequence↗
PubMedDate not supplied

[Antithrombin III dosage using the chromogenic substrate Tos-Gly-Pro-Arg-NAN, in several pathological situations].

UNLABELLED: Functional methods for ATIII determination are essential for the diagnosis of deficiencies of this important thrombin inhibitor. PURPOSE: The aim of this work was standardize the method for ATIII level determination in plasma, in microplates. METHOD: ATIII levels were measured, using the chromogenic substrate Tos-Gly-Pro-Arg-NAN, which is specific for thrombin, and which has been sinthesized at the Biophysical Department of the Escola Paulista de Medicina of the Federal University of São Paulo, Brazil. RESULTS: Among 21 patients with deep venous thrombosis (DVT), 20 had ATIII levels above 70% (113 +/- 22%). A 22 year-old female patient, who had recurrent DVT and a familiar DVT antecedent, had a congenital ATIII deficiency (56%). ATIII levels were normal in 6 patients with von Willebrand disease (109 +/- 28%), as expected. In 20 patients with severe hepatic failure, it has been found reduced ATIII levels (42 +/- 19%), since this inhibitor is produced by the liver. In 3 patients with sepsis and DIC, ATIII levels have also been reduced (45 +/- 5%) owing to consumption during blood coagulation activation. There was a significant correlation between ATIII levels and the prothrombin time, as well as the factor V levels, and both are good parameters to assess hepatic function and to monitor DIC. There was a significant correlation between ATIII levels measured using the chromogenic substrate Tos-Gly-Pro-Arg-NAN and those measured using S-2238, produced by Kabi Laboratories. CONCLUSIONS: This method for ATIII determination in plasma is easy to perform and it can detect ATIII deficiency in patients with hepatic failure, disseminated intravascular coagulation and thrombophilia.

Adult↗
PubMedDate not supplied

Studies on lactam formation during coupling procedures of N alpha-N omega-protected arginine derivatives.

We evaluated the quantity of delta-lactam generated during the synthesis of arginine-containing dipeptides using Z-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Fmoc-Arg(Boc)2-OH and Fmoc-Arg(Pmc)-OH and assayed several carboxyl-activating procedures for coupling the protected arginines to different amino components. We observed significant amounts of delta-lactam during the synthesis of Z-Arg(Tos)-methyl ester and Z-Arg(Tos)-amide, as well as of Boc-Arg(Tos)-chloromethyl ketone. The mixed anhydride coupling procedure and the di-Boc-protecting guanidino group induced more delta-lactam formation than any other coupling or NG-protection method. The amide, benzyl, 4-(NO2)-benzyl and methyl alpha-carboxyl-protected amino acids generated more delta-lactam than did those protected by tertbutyl or N2H2-Boc. So far it has not been possible to propose a general mechanism for delta-lactam formation or a process that completely abolishes it. Therefore, this side reaction should be considered almost inevitable. Its minimization requires examination of arginine-containing peptides in each specific synthesis.

Arginine↗
PubMedDate not supplied

Synthesis of N alpha-protected aminoacyl 7-amino-4-methyl-coumarin amide by phosphorous oxychloride and preparation of specific fluorogenic substrates for papain.

We report an improved procedure for the synthesis of fully protected aminoacyl 7-amino-4-methylcoumarin amide (MCA) employing the phosphorous oxychloride anhydride method. Seven Boc-X-MCA [where X = Arg(NG Tos), Cys(S-Bzl), Thr(O-Bzl), Ser(O-Bzl), Phe, Leu and Gly] and Z-Tyr(O-Me) were synthesized using this procedure, with yields ranging from 50% to 75%. These aminoacyl-MCA derivatives were employed for the synthesis of epsilon-NH2-caproyl-Leu-X-MCA, a fluorescent peptide series, which were assayed as papain substrates. All of them were completely hydrolyzed by papain, indicating that all of the Boc-X-MCA derivatives obtained were practically free of racemization. Since epsilon-NH2-Caproyl-Leu-(S-Bzl)Cys-MCA is very susceptible to hydrolysis by papain, quite resistant to hydrolysis by chymotrypsin and not hydrolyzed by trypsin, it is recommended for assays of thiol-proteinases in which specificity is required.

Amino Acids↗