Recombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citri.

Xanthomonas citri subsp citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. The aim of this study was to describe the recombinant expression, purification, and characterization of a cysteine peptidase from Xac strain 306, which is a candidate for involvement in the pathogenicity of this bacterium. The gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ((HIS)CPXAC) recognized the purified recombinant cysteine peptidase (HIS)CPXAC, confirming the correct production of this protein in P. pastoris. The same antibody detected the protein in the culture supernatant of Xac grown in pathogenicity-inducing medium. Kinetic analysis revealed that (HIS)CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin substrate with a catalytic efficiency (k(cat)/K(m)) of 47 µM(-1)∙s(-1). The purified ((HIS))CPXAC displayed maximal catalytic activity at pH 5.5 and 30°C. The recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K(i) values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence in a secreted protein from Xanthomonas spp.

South American rattlesnake cationic polypeptide crotamine trafficking dynamic in Plasmodium falciparum-infected erythrocytes: Pharmacological inhibitors, parasite cycle and incubation time influences in uptake.

Malaria is a parasitic infectious disease caused by Plasmodium sp, which was responsible for about 409 thousand deaths only in 2019. The clinical manifestations in patients with malaria, which may include fever and anemia and that can occasionally lead to the death of the host, are mainly associated to the asexual blood stage of parasite. The discovery of novel compounds active against stages of the intraerythrocytic cell cycle has been the focus of many researches seeking for alternatives to the control of malaria. The antimalarial effect of a native cationic polypeptide from the venom of a South American rattlesnake named crotamine, with ability of targeting and disrupting the acidic compartments of Plasmodium falciparum parasite, was previously described by us. Herein, we extended our previous studies by investigating the internalization and trafficking of crotamine in P. falciparum-infected erythrocytes at different blood-stages of parasites and periods of incubation. In addition, the effects of several pharmacological inhibitors in the uptake of this snake polypeptide with cell-penetrating properties were also assessed, showing that crotamine internalization was dependent on ATP generated via glycolytic pathway. We show here that crotamine uptake is blocked by the glycolysis inhibitor 2-deoxy-D-glucose, and the most efficient internalization is observed at trophozoite stage of parasite after at least 30 min of incubation. The present data provide important insights into biochemical pathway and cellular features determined by the parasite cycle, which may be underlying the internalization and effects of cationic antimalarials as crotamine.

Extreme subcutaneous, intramuscular and inhaled insulin resistance treated by pancreas transplantation alone.

Diabetes mellitus with resistance to insulin administered subcutaneously or intramuscularly (DRIASM) is a rare syndrome and is usually treated with continuous intravenous insulin infusion. We present here two cases of DRIASM in 16 and 18 years female patients that were submitted to pancreas transplantation alone (PTA). Both were diagnosed with type 1 diabetes as young children and had labile glycemic control with recurrent episodes of diabetic ketoacidosis. They had prolonged periods of hospitalization and complications related to their central venous access. Exocrine and endocrine drainages were in the bladder and systemic, respectively. Both presented immediate graft function. In patient 1, enteric conversion was necessary due to reflux pancreatitis. Patient 2 developed mild postoperative hyperglycemia in spite of having normal pancreas allograft biopsy and that was attributed to her immunosuppressive regimen. Patient 1 died 9 months after PTA from septic shock related to pneumonia. In 8 months of follow-up, Patient 2 presented optimal glycemic control without the use of antidiabetic agents. In conclusion, PTA may be an alternative treatment for DRIASM patients.

The Candida albicans AAA ATPase homologue of Saccharomyces cerevisiae Rix7p (YLL034c) is essential for proper morphology, biofilm formation and activity of secreted aspartyl proteinases.

Proper morphology is essential for the ability of Candida albicans to switch between yeast and hyphae and thereby sustain its virulence. Here we identified, by differential screening, a novel C. albicans AAA ATPase encoding gene, CaYLL34 (RIX7), with enhanced expression in hyphae. Phylogenetic analysis suggests that CaYLL34 belongs to a "VCP-like" subgroup of AAA ATPases essential for yeast viability and contains a bipartite nuclear localization signal. Inactivation of one copy of CaYLL34, by the URA-Blaster method, generated the heterozygous mutant strain M61. This strain has severe phenotypic alterations, such as a highly increased vacuole, abnormal cell shape and reduced growth in different conditions. Also, major pathogenicity factors are affected in M61, for instance, a significant decrease of hypha formation (>90%), surface biofilm adhesion (86%) and secreted aspartyl proteinase activity (76.5%). Our results show that the partial impairment of CaYll34p cellular levels is sufficient to affect the proper cellular morphology and pathogenicity factors and suggest that this protein is required for biogenesis of ribosomal subunits. Accordingly, we propose that the product of CaYLL34 could be tested as a novel target for antifungal drugs.

PnPP-19, a spider toxin peptide, induces peripheral antinociception through opioid and cannabinoid receptors and inhibition of neutral endopeptidase.

BACKGROUND AND PURPOSE: The synthetic peptide PnPP-19 has been studied as a new drug candidate to treat erectile dysfunction. However, PnTx2-6, the spider toxin from which the peptide was designed, induces hyperalgesia. Therefore, we intended to investigate the role of PnPP-19 in the nociceptive pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PnPP-19 was administered intraplantarly alone or with selective cannabinoid or opioid receptor antagonists. The hydrolysis of PnPP-19 by neutral endopeptidase (NEP) (EC 3.4.24.11), an enzyme that cleaves enkephalin, was monitored by HPLC and the cleavage sites were deduced by LC-MS. Inhibition by PnPP-19 and Leu-enkephalin of NEP enzyme activity was determined spectrofluorimetrically. KEY RESULTS: PnPP-19 (5, 10 and 20 µg per paw) induced peripheral antinociception in rats. Specific antagonists of µ opioid receptors (clocinnamox), δ opioid receptors (naltrindole) and CB1 receptors (AM251) partly inhibited the antinociceptive effect of PnPP-19. Inhibition of fatty acid amide hydrolase by MAFP or of anandamide uptake by VDM11 enhanced PnPP-19-induced antinociception. NEP cleaved PnPP-19 only after a long incubation, and Ki values of 35.6 ± 1.4 and 14.6 ± 0.44 µmol·L(-1) were determined for PnPP-19 and Leu-enkephalin respectively as inhibitors of NEP activity. CONCLUSIONS AND IMPLICATIONS: Antinociception induced by PnPP-19 appears to involve the inhibition of NEP and activation of CB1, µ and δ opioid receptors. Our data provide a greater understanding of the antinociceptive effects of PnPP-19. This peptide could be useful as a new antinociceptive drug candidate.

Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom.

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 µM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity.

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambda ex = 320 nm and lambda em = 420 nm) at 37 degrees C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 microM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 microM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 microM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Sex Hormones Promote Opposite Effects on ACE and ACE2 Activity, Hypertrophy and Cardiac Contractility in Spontaneously Hypertensive Rats.

BACKGROUND: There is growing interest in sex differences and RAS components. However, whether gender influences cardiac angiotensin I-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activity is still unknown. In the present work, we determined the relationship between ACE and ACE2 activity, left ventricular function and gender in spontaneously hypertensive rats (SHRs). METHODOLOGY/PRINCIPAL FINDINGS: Twelve-week-old female (F) and male (M) SHRs were divided into 2 experimental groups (n = 7 in each group): sham (S) and gonadectomized (G). Fifty days after gonadectomy, we measured positive and negative first derivatives (dP/dt maximum left ventricle (LV) and dP/dt minimum LV, respectively), hypertrophy (morphometric analysis) and ACE and ACE2 catalytic activity (fluorimetrically). Expression of calcium handling proteins was measured by western blot. Male rats exhibited higher cardiac ACE and ACE2 activity as well as hypertrophy compared to female rats. Orchiectomy decreased the activity of these enzymes and hypertrophy, while ovariectomy increased hypertrophy and ACE2, but did not change ACE activity. For cardiac function, the male sham group had a lower +dP/dt than the female sham group. After gonadectomy, the +dP/dt increased in males and reduced in females. The male sham group had a lower -dP/dt than the female group. After gonadectomy, the -dP/dt increased in the male and decreased in the female groups when compared to the sham group. No difference was observed among the groups in SERCA2a protein expression. Gonadectomy increased protein expression of PLB (phospholamban) and the PLB to SERCA2a ratio in female rats, but did not change in male rats. CONCLUSION: Ovariectomy leads to increased cardiac hypertrophy, ACE2 activity, PLB expression and PLB to SERCA2a ratio, and worsening of hemodynamic variables, whereas in males the removal of testosterone has the opposite effects on RAS components.

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.

Specificity comparison of a serine endopeptidase (SH1) and a serine thiol endopeptidase (STH2) purified from human urine.

In this study, we compared the properties of a serine endopeptidase H1 (SH1) and a serine thiol endopeptidase (STH2) purified from human urine by DEAE-cellulose followed by a Bio Gel A0.5 m or Sepharose Mercurial chromatographs. These enzymes differ in their action upon different hormone peptides. We used fluorogenic substrates to further characterize the enzyme. The substrate specificity of urinary SH1 was studied using different internally quenched fluorescent peptides, and AbzFGQEDDnp was hydrolyzed by SH1. Other enzymes present in urine, such as serine endopeptidase H2, prolyl endopeptidase, neutral endopeptidase like and angiotensin-I converting enzyme, were not able to hydrolyze this substrate. SH1 is 100% inhibited by PMSF and resistant to EDTA, OPA, thiorphan, E64, pOHMB and phosphoramidon. Endopeptidase STH2 is completely inhibited by PMSF, E64 and pOHMB. Enzyme SH1 hydrolyzes the peptide bound F5-S6 at bradykinin (BK: RPPGFSPFR) molecule and R-Q at AbzBKQEDDnp. When studying enzyme STH2, the cleavage sites determined to the related substrates were F5-S6 using BK as substrate and F-R using AbzBKQEDDnp. The kilometers value obtained for AbzBKQEDDnp and AbzFGQEDDnp were 1.18 and 0.007 uM, respectively. Kininases from kidney and urine can hydrolyze peptide bounds from components of the kallikrein-kinin system, the angiotensin-renin system and the neuropeptides system, straight contributing in kidney homeostasis. SH1 was located at the distal tubule [Casarini et al., 1999a, Am. J. Physiol. 277, F66] and can have an important function in the control of kinin found in this portion, since is known that all components of the kallikrein-kinin system were found in this portion. The physiological role of SHT2 could be related to the inter-relation between the kallikrein-kinin system and neuropeptides in the control of the water electrolyte balance [Braz. J. Med. Biol. Res. 25 (3) (1992) 219].

Angiotensin I-converting enzyme isoforms (high and low molecular weight) in urine of premature and full-term infants.

Angiotensin I-converting enzyme (ACE) isoforms in urine from healthy and mildly hypertensive untreated patients have been described in the literature. Healthy subjects have high- and low-molecular-weight ACEs (170 and 65 kDa), whereas mildly hypertensive untreated patients have only low-molecular-weight ACEs (90 and 65 kDa), both of which resemble ACE from the N-terminal domain. Previous studies have shown that ACE is regulated during development, and renal tubules of premature human infants are not completely mature, given that nephrogenesis is not complete until the 36th week of gestation. The aim of the present study was to purify and characterize ACE isoforms from urine of premature and full-term infants and to detect the presence of the N-domain form of ACE during prenatal development. Urine from premature and full-term infants was concentrated in an Amicon concentrator, dialyzed in the same equipment against 50 mmol/L Tris-HCl buffer (pH 8.0) that contained 150 mmol/L NaCl, and submitted to gel filtration on an AcA-34 column equilibrated with the buffer described above. Two peaks (P1 and P2 for premature infants; TP1 and TP2 for full-term infants) with ACE activity on hippuryl-His-Leu (K(m), 3 mmol/L) were detected. All enzymes were Cl(-) dependent and inhibited by captopril and EDTA. The peptides angiotensin-(1-7) and N-acetyl-Ser-Asp-Lys-Pro, described as specific for N-domain ACE, were hydrolyzed by P2 and TP2, which suggests that both enzymes are N-domain ACE. In premature infants, P1 activity with hippuryl-His-Leu was 12-fold lower than P2 activity, but in full-term infants, the difference between TP1 and TP2 was 1.6-fold. Chromatography profiles of urine from premature infants were analyzed on days 1, 3, 7, 14, 21, and 30 after birth. The P1 of ACE was detected around the 21st and 30th days, whereas P2 was detected from day 1. These results suggest that ACE activity is related to renal development and that N-domain ACE as well as full-length ACE is present in urine from premature infants. This may indicate that healthy subjects produce and secrete the N-domain form of ACE even before term development.

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.

Characterization of two cysteine proteinases secreted by Fasciola hepatica and demonstration of their kininogenase activity.

We have isolated and purified two cysteine proteinases of molecular weights 25 and 26 kDa, secreted by Fasciola hepatica adult worm. Their 15 N-terminal residues were found to be identical to those of earlier described cathepsin L-like enzymes, isolated from the same source, reported as CL1 and CL2. Radioimmunoassay experiments have shown that these CL1- (25 kDa) and CL2-like (26 kDa) cysteine proteinases mediated kinin release from high molecular weight kininogen (HMWK). Lys-bradykinin (KRPPGFSPFR) was characterized as the kinin released from a synthetic fragment of HMWK from Leu373 to Ile393 (Abz-LGMISLMKRPPGFSPFRSSRI-NH2) labeled with the fluorescent group Abz (ortho-aminobenzoic acid). We examined the activity of CL1- and CL2-like on internally quenched fluorescent peptides containing HMWK sequences, in which Met379-Lys380 or Arg389-Ser390 bonds were present in the middle of the molecules. These peptides were flanked by the fluorescent donor-acceptor pair Abz and EDDnp (N-[2,4-dinitrophenyl] ethylenediamine). Peptidyl-methylcoumarin amides (MCA) were used to study the substrate specificity requirements. The enzymes presented significantly lower Km values at pH 8.0. The inverse was observed with the kcat values, which were higher at pH 5.0.

Internally quenched fluorogenic substrates for angiotensin I-converting enzyme.

OBJECTIVE: Development of internally quenched fluorogenic substrates for sensitive and continuous assays of angiotensin I-converting enzyme (ACE). DESIGN: We synthesized internally quenched fluorogenic bradykinin-related peptides introducing Abz (ortho-aminobenzoic acid) and EDDnp (N-[2,4-dinitrophenyl]-ethylenediamine) at their N- and C-terminal groups, respectively, and these were assayed as ACE substrates. We examined two series of peptides, Abz-GFSPFRX-EDDnp and Abz-GFSPFXQ-EDDnp (X, various amino acids). METHODS: Hydrolysis of the fluorogenic substrates by ACE was followed by continuous recording of the rising fluorescence (lambda(em) = 420 nm and lambda(ex) = 320 nm). The peptides were obtained by solid-phase synthesis or by classical solution methods. RESULTS: Despite of the blocked C-terminal sequences, the internally quenched bradykinin-related peptides were hydrolysed by ACE. The best substrates for plasma guinea pig ACE were Abz-GFSPFRA-EDDnp and Abz-GFSPFFQ-EDDnp, in which the fluorescence appeared after the first cleavage that occurred at R-A and F-Q bond, respectively. This ACE activity was sensitive to NaCl concentration and the optimum pH is greater than 8.0. Measurements of ACE activity with Hip-His-Leu and Abz-GFSPFFQ-EDDnp in the serum of 20 healthy patients correlated closely (r = 0.959). Complete inhibition of the hydrolysis of Abz-GFSPFFQ-EDDnp by human serum was observed with captopril and lisinopril. CONCLUSIONS: We describe internally quenched fluorogenic substrates for ACE devoid of free C-terminal carboxyl group. They are convenient tools for ACE studies as they permit continuous fluorimetric measurements of the enzymatic activity, even in human serum.

Purification and characterization of a neutral endopeptidase-like enzyme from human urine.

OBJECTIVE: The aims of this study were to purify and characterize a neutral endopeptidase-like enzyme (NEP-like) in human urine and propose a rapid, sensitive and specific assay for this enzyme using the fluorogenic substrate Abz-FDQ-EDDnp, where Abz = O-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine. METHODS: Soluble urinary NEP was purified from human urine using a DEAE-cellulose Cellex D column and gel filtration on an AcA-44 column. NEP-like activity was assayed by its ability to hydrolyse bradykinin (BK) and the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FDQ-EDDnp. The Km was determined using Abz-FDQ-EDDnp as a substrate. The hydrolysis products of BK and Abz-FDQ-EDDnp were analysed by high-performance liquid chromatography (HPLC). The mol. wt was estimated by polyacrylamide gel electrophoresis and the enzyme analysed by Western blot using the antibody obtained from purified recombinant NEP expressed in Pichia pastoris yeast. RESULTS: The NEP-like was purified from human urine until homogeneity and presented a mol. wt of 94000. The substrate Abz-FDQ-EDDnp was selectively hydrolysed at the F-D bond by NEP-like and by recombinant NEP. For this substrate, the NEP-like activity was maximal at pH 7.0, although a small peak of activity was observed at pH 8.0, and the determined Km was 14 microM. The enzymatic activity was inhibited by thiorphan and phosphoramidon. In Western blot analysis, NEP-like reacted strongly with a polyclonal antibody for NEP. CONCLUSION: A NEP-like enzyme was purified from human urine. Based on the mol. wt of the isolated NEP-like enzyme, it was concluded that this enzyme was produced in the kidney. In the kidney, this enzyme may cleave the kinins filtered through the glomerulus and also the kinins produced in the distal nephron. An internally quenched fluorogenic peptide, Abz-FDQ-EDDnp, was selectively hydrolysed by NEP-like and by recombinant NEP.

Purification and characterization of angiotensin I-converting enzymes from mesangial cells in culture.

OBJECTIVE: Previous analysis of the angiotensin I-converting enzyme (ACE) gene in this laboratory showed that primary mesangial cells in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on glomerular microcirculation. The aim of this study was to purify and characterize the secreted and intracellular ACE forms from mesangial cells in culture. METHODS AND RESULTS: Medium from Wistar rats mesangial cells was collected (third passage), incubated for 20 h with RPMI without fetal bovine serum and concentrated 29 times in an Amicon concentrator. The concentrated medium was submitted to gel filtration on an AcA-34 column and two peaks (ACE1, mol. wt 130 000 and ACE2, 60000) with ACE on activity Hippuryl-His-Leu and Z-Phe-His-Leu were separated. The mesangial cells were collected and ACE enzyme was extracted using Triton X-114, followed by centrifugation and concentration. The supernatant was submitted to the same chromatography as described above and two peaks with ACE activity (ACEInt1, mol. wt 130000 and ACEInt2, 68000) were separated. The purified ACE were inhibited by enalaprilat and captopril, two potent competitive inhibitors of ACE and by EDTA, using Hippuryl-His-Leu as a substrate. The Km values were 2 mM for ACE1 and ACE2 and 3 mM for ACEInt1 and ACEInt2. The enzymes ACE1 and ACE2 presented an optimum pH of 8.0 and ACEInt1 and ACEInt2 an optimum pH of 7.5. CONCLUSION: The activities of full-length wild-type and N-domain ACE were characterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Leu, which was 1 and 4, respectively. The ratios found for ACE1, ACE2, ACEInt1 and ACEInt2 in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of intracellular ACE, are able to secret both full-length wild-type ACE and N-domain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hormone-releasing hormone and Met-enkephalin to interfere with glomerular haemodynamics and with the renal microcirculation.

Inhibition of angiotensin converting enzyme and potentiation of bradykinin by retro-inverso analogues of short peptides and sequences related to angiotensin I and bradykinin.

There is pharmacological evidence indicating that, in addition to the inhibition of angiotensin converting enzyme (ACE; EC 3.4.15.1), the potentiation of bradykinin (BK) responses may also involve the BK receptor or some binding site in the structures involved in the contractile response to this peptide. Dipeptides such as Val-Trp and some of its analogues as well as tripeptide homologues, including total and partial retro-inverso peptides, were synthesized and assayed for their ability to inhibit purified guinea pig plasma ACE and to potentiate the action of BK on the isolated ileum of the same species. The peptides containing the P2-P1, P1-P'1, and P'1-P'2 inverted amide bonds inhibited ACE, were resistant to hydrolysis, and, depending on the amino acid composition, some of them potentiated the contractile response to BK while others did not. Des-[Arg1]-BK, which has an intrinsic activity at concentrations higher than 10(-5) M, and the very dissimilar angiotensin I (AI) analogue [Cys5-Cys10]-angiotensin-I-(5-10)-amide, which has no detectable contractile activity, were able to inhibit ACE and potentiate BK. In contrast to these peptides, BPP5a and BPP9a from Bothrops jararaca venom, and Potentiators B and C from Agkistrodon halys blomhoffi venom were more effective as BK potentiators than as ACE inhibitors. In conclusion, we have synthesized and assayed compounds that preferentially inhibit ACE, e.g. retro-inverso tripeptides, or potentiate the response of smooth muscle to BK, e.g. snake venom peptides.

Thimet oligopeptidase EC 3.4.24.15 is a major liver kininase.

Bradykinin (BK) is a potent hepato-portal hypertensive agent although it is efficiently inactivated by the liver. The organ converts angiotensin I to AII, but at a much slower rate than it inactivates BK. We had previously identified EC 3.4.24.15 as an hepatic bradykinin inactivating endopeptidase that hydrolyzes BK at the F5-F6 bond. The aim of this study was to determine the relative importance of BIE, as compared to other kininases, in normal, cirrhotic or inflamed rat livers, as well as in samples of human liver. Using specific substrates and inhibitors we showed that: 1) purified BIE preparation hydrolyzed BK and a BK analogue (BK-Q) with similar efficacy; BK-Q was functionally active since it caused an increase in hepato-portal pressure, as did BK itself. 2) BK degradation in rat serum was performed by ACE since BIE and prolylendopeptidase (PEP) activities were negligible. 3) normal rat liver homogenate contained a large amount of BIE activity which was eliminated by a specific EC 3.4.24.15 inhibitor; ACE and PEP activities were negligible. 4) There was no difference (p>0.05) in BIE activity in the liver homogenates from rats with normal, inflamed or cirrhotic livers. 5) BIE activity was efficiently removed from livers (normal, inflamed or cirrhotic) that were perfused with TritonX-100.6) Human liver had an similar enzymatic pattern although ACE activity was detected. We concluded that in normal, inflamed or cirrhotic rat livers, as well as in the human liver, the bradykinin inactivating endopeptidase (EC 3.4.24.15), and not ACE, is the major hepatic kininase.

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp.

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37 degrees C, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors.

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 micro/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 microM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 microM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 microM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).