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Biomedical subjects

L Juliano

Publications and source records attributed to L Juliano.

At least 127 records · Page 7Linked to original sources
PubMed1995

Calcium channel blockers as inhibitors of angiotensin I-converting enzyme.

Using ion-exchange chromatography of dialyzed human urine from healthy and hypertensive patients, we detected two peaks of angiotensin I-converting enzyme (ACE) activity on hippuryl-His-Leu eluted at ionic strengths of 0.7 (F1 peak) and 1.25 (F2 peak) mS. These hydrolytic activities decreased gradually in the urine of patients submitted to isradipine treatment, F2 and F1 disappearing after 12 and 24 hours, respectively. By Western blot analysis, the urine fractions corresponding to both peaks from healthy and untreated patients presenting ACE activity and from treated patients (24 hours) without this activity were recognized by an ACE-specific antibody. These results indicated that ACE was present but inhibited in the urine of isradipine-treated patients. In vitro assays with ACE isolated from human urine and guinea pig plasma demonstrated that the enzyme is inhibited by isradipine and other commercially available calcium channel blockers, such as felodipine, nifedipine, and verapamil. A noncompetitive inhibition was observed with all calcium channel blockers studied. In conclusion, these results suggest that besides the primary effect on calcium channels, the more commonly used calcium channel blockers are also ACE inhibitors. The development of efficient calcium channel blockers with higher ACE inhibitory activity could result in interesting bifunctional antihypertensive drugs.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1995

A new fluorometric assay for neutral endopeptidase (EC 3.4.24.11).

An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, Abz-GGDFLRRV-EDDnp, was selectively hydrolyzed at the R-V bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 microM, kcat = 127/min and kcat/Km = 42/min microM) similar to those of Leu-enkephalin. The specificity of the assay for NEP was demonstrated by incubating Abz-GGDFLRRV-EDDnp with a kidney homogenate and with crude membrane preparations of brain and lung. For all three homogenates the complementary fragment Abz-GGDFLRR and V-EDDnp accounted for more than 95% of the products which are totally inhibited by 1 microM thiorphan, a highly specific NEP inhibitor. A continuous fluorometric assay for only 15 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified NEP or the equivalent enzymatic activity in crude tissue preparations.

Animals↗
PubMed1994

Structural requirements of bioactive peptides for interaction with endopeptidase 22.19.

A series of biologically active peptides and related compounds (opioid peptides, neurotensin, and bradykinin) were used as substrates or competitive inhibitors to study the structural requirements for peptide interaction with endopeptidase 22.19. The kinetics of hydrolysis of these peptides indicated that, in contrast to other proteases, the substrate specificity of endopeptidase 22.19 is not determined by the amino acids flanking the sensitive bonds of the substrates. The competition between bioactive peptide analogues and the quenched fluorescence substrate of endopeptidase 22.19 indicated that their length and their flexibility may be the dominant factors to explain their binding specificities. These peculiar features of endopeptidase 22.19 may be of importance to understand the physiological processes of conversion and inactivation of biologically active peptides.

Amino Acid Sequence↗
PubMed1994

New substrates of papain, based on the conserved sequence of natural inhibitors of the cystatin family.

A series of peptide substrates with different fluorogenic leaving groups has been synthesized. The peptide moiety in these substrates mimics a highly conserved sequence (QVVAG) in the natural reversible inhibitors of cysteine proteinases, the cystatins, that participates to the tight binding of target proteinases. This sequence is invariably cleaved at the A-G bond when synthetic peptides containing it were incubated with papain. AEC and AMC fluorophores were therefore attached to the Ala residue to construct new substrates for cysteine proteinases. The solubility of the resulting substrates was improved by attaching a N-terminal gluconoyl group, or by introducing an arginyl residue at P5 (nomenclature of Schechter I, Berger A (1967) Biochem Biophys Res Commun 27, 157-162). Neither induced significant changes in the kcat/Km values with papain. Those values were all in the 10(5) M-1 s-1 range. The kcat/Km was increased 10-50-fold by using substrates with intramolecularly quenched fluorescence. With these, the enzyme specificity on both sides of the scissile bond can be investigated. The substrate Abz-QVVAGA-EDDnp is among the most sensitive papain substrates ever reported, with a kcat/Km value of 29 10(6) M-1 s-1. The positioning and conformation of the bound QVVA moiety within the active site of papain were predicted by molecular modelling using the X-ray coordinates of a peptide inhibitor-papain complex.

Amino Acid Sequence↗
PubMed1993

Dynorphin-derived peptides reveal the presence of a critical cysteine for the activity of brain endo-oligopeptidase A.

Brain endo-oligopeptidase A, a neuropeptide-metabolizing endopeptidase, has been considered a cysteine-endopeptidase because it is activated by thiols and inhibited by phydroxymercuribenzoate or 5,5'-dithiobis-(2-nitrobenzoic acid). The understanding of the unique specificity of endo-oligopeptidase A was useful for the synthesis of affinity labeling compounds containing as a thiol reactive group the Cys-(3-nitro-2-pyridinesulfenyl) group into dynorphin-derived peptides which are among the best substrates and competitive inhibitor of endopeptidase 22.19. Of the ten compounds tested, only peptides containing 8 to 13 amino acid residues caused irreversible inhibition. The fact that the most effective inhibitors had the reactive group either at the P'1 or at P'3 position [nomenclature of Schechter and Berger] would seem to argue that the reactive cysteine is in the vicinity of the active site, or actually involved in the catalytic step.

Amino Acid Sequence↗
PubMed1993

A new, sensitive fluorogenic substrate for papain based on the sequence of the cystatin inhibitory site.

We have designed and tested a new papain substrate with intramolecularly quenched fluorescence. It is based on a highly conserved sequence in all members of the cystatin superfamily that participates in the inhibition of cysteine proteinases. This substrate, O-aminobenzoyl (Abz)-QVVAGA-ethylenediamine-2-4-dinitrophenyl (EDDnp) is very sensitive to papain with a second-order rate constant kcat/Km of 3.1 10(7) M-1S-1. It is also efficiently hydrolyzed by cathepsin L, although the kcat/Km for this proteinase is about 60-fold lower than that for papain. This change is due to a decrease in kcat, the Km's are almost identical. This allows clear functional discrimination between these two proteinases, and may lead to the development of selective inhibitors for individual cysteine proteinases. Unlike most commonly used papain substrates, Abz-QVVAGA-EDDnp is not hydrolyzed by trypsin. The papain cleavage site was identified as the A-G bond by N-terminal amino acid sequencing. The use of sensitive and specific substrates such as the one described here will prove invaluable for investigating cysteine proteinase activities in parasite infections. The close interaction between papain or cathepsin L with Abz-QVVAGA-EDDnp is compared to that with cystatin inhibitors, which all include a QxVxG consensus segment in their structure.

Amino Acid Sequence↗
PubMed1992

Substrate specificities of tissue kallikrein and T-kininogenase: their possible role in kininogen processing.

The present studies demonstrate the importance of subsite interactions in determining the cleavage specificities of kallikrein gene family proteinases. The effect of substrate amino acid residues in positions P3-P'3 on the catalytic efficiency of tissue kallikreins (rat, pig, and horse) and T-kininogenase was studied using peptidyl-pNA and intramolecularly quenched fluorogenic peptides as substrates. Kinetic analyses show the different effects of D-amino acid residues at P3, Pro at P'2, and Arg at either P'1 or P'3 on the hydrolysis of substrates by tissue kallikreins from rat and from horse or pig. T-Kininogenase was shown to differ from tissue kallikrein in its interactions at subsites S2, S'1, and S'2. As a result of these differences, Abz-FRSR-EDDnp with Arg at P'2 is a good substrate for tissue kallikreins from horse, pig, and rat but not for T-kininogenase. Abz-FRRP-EDDnp and Abz-FRAPR-EDDnp with Pro at P'2 (rat high molecular weight kininogen sequence) are susceptible to rat tissue kallikrein but not to tissue kallikreins from horse and pig. Arg at P'3 increased the susceptibility of the Arg-Ala bond to rat tissue kallikrein. These data explain the release of bradykinin by rat tissue kallikrein and of kallidin by tissue kallikreins from other animal species. Abz-FRLV-EDDnp and Abz-FRLVR-EDDnp (T-kininogen sequence) are good substrates for T-kininogenase but not for tissue kallikrein. Arg at the leaving group (at either P'1, P'2, or P'3) lowers the Km values of T-kininogenase while Val at P'2 increases its kcat values.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Intramolecularly quenched fluorogenic peptide substrates for human renin.

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe8 to His13, His6 to His13, and Tyr4 to His13 of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC50 = 7.8 x 10(-9) M), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707-2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 microGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.

Amino Acid Sequence↗
PubMed1992

Determinants of tissue kallikrein cleavage specificity in the limited proteolysis of kininogens.

Further evidence for interactions at tissue kallikrein extended binding sites, as determinants of the kininogen cleavage specificities is presented. Differences in the cleavage sites in kininogen hydrolysis by rat and other tissue kallikreins is related to subsite S1' specificity, while the low susceptibility of rat kininogen to horse tissue kallikrein is explained by the difference in their subsite S3'.

Amino Acid Sequence↗
PubMed1992

Comparative studies on P2 specificity of wild-type rat tissue kallikrein, Y99H:W215G mutant and tonin.

To probe residues responsible for P2 specificity, we have recently created a mutant enzyme from rat tissue kallikrein with Tyr99 to His and Trp215 to Gly exchange (Y99H:W215G) using site-specific mutagenesis. In the present study, additional characterization of substrate specificities of both wild-type tissue kallikrein, Y99H:W215G mutant and native tonin was performed using synthetic Ac-X-Arg-pNA substrates especially designed for testing P2 specificity. Kinetic analyses of Km and kcat demonstrate a clear correlation between dramatically reduced affinity for hydrophobic P2 side-chain and the loss of the Tyr99-Trp215 hydrophobic pair. Analyses of rat tonin reveal a correlation between increased reaction rate and P2 hydrophilicity although tonin displays similar pattern in P2 affinity as compared with tissue kallikrein, suggesting a less hydrophobic environment in the substrate-binding pocket of rat tonin. The results strongly support the hypothesis that Tyr99-Trp215 interaction is the major determinant for P2 specificity and that the presence of a hydrophobic side-chain in P2 position substantially facilitates substrate hydrolysis of tissue kallikrein-like enzymes.

Amino Acid Sequence↗
PubMed1992

Inhibitors of the major cysteinyl proteinase (GP57/51) impair host cell invasion and arrest the intracellular development of Trypanosoma cruzi in vitro.

Peptidyl diazomethane (PDAM) derivatives, a class of irreversible inhibitors for cysteine proteinase, were screened for the ability to impair Trypanosoma cruzi invasion and intracellular development in primary cultures of heart muscle cells (HMC). T. cruzi GP57/51, a purified cysteinyl proteinase, and the substrate Z-Phe-Arg-NHMec were used to determine inhibition rate constants (k'+2) by continuous kinetic assays. The k'+2 values ranged from 25,400 to 2,800. The best inhibitors of GP57/51 had bulky hydrophobic residues in the P1 position (in addition to P2), the S1 sub-site specificity of the enzyme being thus similar to mammalian cathepsin L. The effects of these PDAM on parasite infectivity were then investigated. The ability to invade HMC was markedly impaired when trypomastigotes were briefly exposed to 10 microM of Z-(S-Bzl)Cys-Phe-CHN2. Striking effects were observed when PDAM were added to HMC cultures that had been previously infected with trypomastigotes: Z-(S-Bzl)Cys-Phe-CHN2 with an IC50 of 0.4 microM, and less markedly Z-Phe-Phe-CHN2 and Z-Tyr-Phe-CHN2 (or Z-Phe-Tyr-CHN2) blocked amastigote replication as well as their transformation into trypomastigotes, thereby arresting intracellular development. Bz-Phe-Gly-CHN2, in contrast, failed to display antiparasite activity. Direct characterization of the target cysteinyl proteinase was sought, by incubating viable amastigotes or infected HMC with Z-[125I]Tyr-Phe-CHN2. Affinity labeling implicated GP57/51 as the major cysteinyl proteinase target for this probe. We propose that T. cruzi intracellular development is critically dependent on GP57/51 (cruzipain). Selective inhibitors for this cysteinyl proteinase may have therapeutic potential.

Animals↗
PubMed1991

Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins.

Five intramolecularly quenched fluorogenic substrates for arginyl hydrolases with the sequence Abz-Phe-Arg-X-Y-EDDnp (X = Arg or Ser; Y = Val, Pro, or Arg) were synthesized by classical solution methods. Kinetics of their hydrolysis by tissue and plasma kallikreins, trypsin, and thrombin characterized Abz-Phe-Arg-Ser-Arg-EDDnp as a specific and sensitive substrate for the continuous assay of tissue kallikreins while Abz-Phe-Arg-Arg-Pro-EDDnp was the best substrate for human plasma kallikrein. The five peptides were poor substrates for trypsin and resistant to thrombin.

Animals↗
PubMed1991

Synthesis of human angiotensinogen (1-17) containing one of the putative glycosylation binding sites and its hydrolysis by human renin and porcine pepsin.

The N-terminal heptadecapeptide of human angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn-Glu-Ser-Thr-NH2 ), with the C-terminal carboxyl group amidated, was synthesized in order to study the role of Asn-Glu-Ser, a putative carbohydrate binding site, on the hydrolysis by human renin. The synthesis was performed by fragment condensation using the Honzl and Rudinger azide procedure. In our conditions for azide segment condensation, histidine racemization was demonstrated to be negligible for most of the condensation reactions. Human renin liberates angiotensin I from h-angiotensinogen (1-17)-NH2 with a Km value of 3.4 x 10(-5) M, at pH 7.3 and 37 degrees being similar to h-angiotensinogen (1-13), an analog without the carbohydrate binding site. However, the Vmax value of 4.1 x 10(-9) mol/G.U. min is one order of magnitude higher. Porcine pepsin was demonstrated to cleave preferentially Leu10-Val11 bond and, surprisingly, His9-Leu10 as well.

Amino Acid Sequence↗
PubMed1990

A selective assay for endooligopeptidase A based on the cleavage of fluorogenic substrate structurally related to enkephalin.

A novel quenched fluorescence substrate, QF-ERP7 (Abz-G-G-F-L-R-R-V-EDDn), structurally related to enkephalins, proved to be suitable for assaying the endooligopeptidase A (E.C.3.4.22.19) activity. The enzyme only splits the L-R bond (Km 1.75 microM, Kcat 8.25 s-1), a reaction efficiently blocked by anti-endooligopeptidase A antibodies and by inhibitor and alternative substrates of the enzyme. Evidences based on the action of inhibitors and on the analysis of QF-ERP7 fragments demonstrated that endooligopeptidase A contributes with 100% of the QF-ERP7 cleaving activity found in the cytosol of rabbit brain homogenates and with 85% of that recovered in the membrane fraction. Homologous substrates, Abz-G-G-F-L-R-R-EDDn and Abz-G-G-F-L-R-EDDn, were resistant to hydrolysis. The convenience and sensitivity of the fluorimetric assay based on the QF-ERP7 moiety offers several advantages compared with previously described painstaking procedures for endooligopeptidase A activity measurements, what will certainly contribute to further our understanding of the role of this enzyme on the peptide hormone metabolism.

Amino Acid Sequence↗
PubMed1990

The anti-leishmanial activity of dipeptide esters on Leishmania amazonensis amastigotes.

L-Amino acid esters, such as L-Leu-OMe, kill Leishmania amazonensis amastigotes by a mechanism which appears to involve ester hydrolysis by cysteine proteinases located in the parasite megasomes. We have examined the killing of isolated amastigotes by L-dipeptide esters and derived some structure-activity correlations. Toxicity of the compounds for the parasites was measured by a tetrazolium (MTT) reduction assay. The results show that active dipeptide esters contained at least 1 hydrophobic amino acid (Leu, Ile, Val, Phe or Trp). The activity of homodipeptide methyl esters depended on the nature of the amino acid, as indicated by the following series: Phe-Phe-OMe greater than Val-Val-OMe greater than Leu-Leu-OMe greater than Trp-Trp-OMe greater than Ile-Ile-OMe. The nature of the amino acids in Leu-X-OMe and X-Leu-OMe was relatively unimportant when X was Phe, Trp or Val. However, when X was Ala or Gly, Leu-X-OMe was several-fold more active than X-Leu-OMe. A similar preference for the more hydrophobic residue in the amino terminal position was also found in esters containing a single phenylalanine or valine. Protection of the amino group by benzyloxycarbonyl (Z) or t-butyloxycarbonyl (BOC) substituents markedly enhanced the activity of the esters. An-mPhe-Gly-OEt, a retro-inverso analogue of Bz-Phe-Gly-OEt, was several-fold more active than the parent compound. Selected esters were assayed on infected macrophages and concentrations that induced minimal toxicity to the host cells were estimated. The ED50s for intracellular parasites were 1.5 to 5-fold higher than those for isolated amastigotes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗