Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania.

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.

The use of dacron plates for DOT enzyme-linked immunosorbent assay (DOT-ELISA)

Dacron (polyethylenetherephthalate) is proposed as a matrix for dot-ELISA procedures, as an alternative to nitrocellulose. Plates of dacron were partially hydrazinolyzed and hydrazide groups introduced were converted to azide groups. The derivative dacron-antigen was covalently linked on to the plates through these azide groups. The derivative dacron-antigen was exaustively washed according to CROOK and antigen was still fixed onto the plates. Protein F1A purified from Yersinia pestis was used as a model. Triration of sera from immunized and non immunized rabbits against this protein was carried out by employing the dot-ELISA method. No significant difference was observed using dacron-antigen and nitrocellulose-antigen preparations. However, both procedures showed to have a significant better performance in comparasion with the passive hemagglutination method. The specificity and reproductibility of the dot-ELISA assay using both preparations showed a similar behaviour. Nitrocellulose preparation was stable at 4ºC, 28ºC and -20ºC for 90 days, whereas the dacron-antigen derivative was stable only when stored at 4ºC. Dacron-antigen derivative could be re-used when the spot developing was proceeded using 4-chloro-1-naphtol as substrate.

Pharmacokinetics of angiotensin converting enzyme inhibitors.

1. The pharmacokinetics of most ACE inhibitors have been evaluated indirectly by the measurements of plasma ACE activity and circulating levels of angiotensin I and II. 2. Although plasma ACE activity is very useful to study the degree and the time-course of ACE inhibition, one has to be aware that very different results can be obtained depending on the substrate employed in the assay. It is therefore impossible to compare the results of different inhibitors unless an identical methodology is used. 3. A clear dissociation between plasma angiotensin II levels and the antihypertensive effects of ACE inhibitors has been reported. This observation is in part linked to problems with the measurement of angiotensin II. New methods of determination of plasma angiotensin II have now allowed demonstration of the complete disappearance of plasma angiotensin II following acute ACE inhibition. During chronic treatment, however, angiotensin II generation is effectively blocked only during part of the day, but blood pressure remains controlled permanently. 4. Among the different pharmacokinetic characteristics of ACE inhibitors presently available, the route of excretion and to a lesser degree the half-life appear to be the most clinically relevant. However, the importance of the ability of ACE inhibitors to inhibit tissue renin-angiotensin systems remains to be defined.

Granzyme A is an interleukin 1 beta-converting enzyme.

Apoptosis is critically dependent on the presence of the ced-3 gene in Caenorhabditis elegans, which encodes a protein homologous to the mammalian interleukin (IL)-1 beta-converting enzyme (ICE). Overexpression of ICE or ced-3 promotes apoptosis. Cytotoxic T lymphocyte-mediated rapid apoptosis is induced by the proteases granzyme A and B. ICE and granzyme B share the rare substrate site of aspartic acid, after which amino acid cleavage of precursor IL-1 beta (pIL-1 beta) occurs. Here we show that granzyme A, but not granzyme B, converts pIL-1 beta to its 17-kD mature form. Major cleavage occurs at Arg120, four amino acids downstream of the authentic processing site, Asp116. IL-1 beta generated by granzyme A is biologically active. When pIL-1 beta processing is monitored in lipopolysaccharide-activated macrophage target cells attacked by cytotoxic T lymphocytes, intracellular conversion precedes lysis. Prior granzyme inactivation blocks this processing. We conclude that the apoptosis-inducing granzyme A and ICE share at least one downstream target substrate, i.e., pIL-1 beta. This suggests that lymphocytes, by means of their own converting enzyme, could initiate a local inflammatory response independent of the presence of ICE.

Isochorismate synthase (PchA), the first and rate-limiting enzyme in salicylate biosynthesis of Pseudomonas aeruginosa.

In Pseudomonas aeruginosa the extracellular metabolite and siderophore pyochelin is synthesized from two major precursors, chorismate and l-cysteine via salicylate as an intermediate. The regulatory role of isochorismate synthase, the first enzyme in the pyochelin biosynthetic pathway, was studied. This enzyme is encoded by pchA, the last gene in the pchDCBA operon. The PchA protein was purified to apparent electrophoretic homogeneity from a PchA-overexpressing P. aeruginosa strain. The native enzyme was a 52-kDa monomer in solution, and its activity strictly depended on Mg(2+). At pH 7.0, the optimum, a K(m) = 4.5 microm and a k(cat) = 43.1 min(-1) were determined for chorismate. No feedback inhibitors or other allosteric effectors were found. The intracellular PchA concentration critically determined the rate of salicylate formation both in vitro and in vivo. In cultures grown in iron-limiting media to high cell densities, overexpression of the pchA gene resulted in overproduction of salicylate as well as in enhanced pyochelin formation. From this work and earlier studies, it is proposed that one important factor influencing the flux through the pyochelin biosynthetic pathway is the PchA concentration, which is determined at a transcriptional level, with pyochelin acting as a positive signal and iron as a negative signal.

Stable complexes involving acetylcholinesterase and amyloid-ß-peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer's fibrils

Brain acetylcholinesterase (AChE) forms stable complexes with amyloid-beta peptide (Abeta) during its assembly into filaments, in agreement with its colocalization with the Abeta deposits of Alzheimer's brain. The association of the enzyme with nascent Abeta aggregates occurs as early as after 30 min of incubation. Analysis of the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with senile plaques, which includes a resistance to low pH, high substrate concentrations, and lower sensitivity to AChE inhibitors. Furthermore, the toxicity of the AChE-amyloid complexes is higher than that of the Abeta aggregates alone. Thus, in addition to its possible role as a heterogeneous nucleator during amyloid formation, AChE, by forming such stable complexes, may increase the neurotoxicity of Abeta fibrils and thus may determine the selective neuronal loss observed in Alzheimer's brain.

Charge dependent substrate activity of C3' and N3 functionalized, organometallic technetium and rhenium-labeled thymidine derivatives toward human thymidine kinase 1.

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for the noninvasive imaging of cancer cell proliferation using radiolabeled thymidine analogues such as [(18)F]3'-fluoro-3'-deoxythymidine ([(18)F]FLT). A thymidine analogue for single photon emission computed tomography (SPECT), which incorporates the readily available and inexpensive nuclide technetium-99m, would be of considerable practical interest. hTK1 is known to accommodate modification of the structure of the natural substrate thymidine at the positions N3 and C3' and, to a lesser extent, C5. In this work, we used the copper-catalyzed azide-alkyne cycloaddition to synthesize two series of derivatives in which thymidine is functionalized at either the C3' or N3 position with chelating systems suitable for the M(CO)(3) core (M = (99m)Tc, Re). The click chemistry approach enabled complexes with different structures and overall charges to be synthesized from a common precursor. Using this strategy, the first organometallic hTK1 substrates in which thymidine is modified at the C3' position were identified. Phosphorylation of the organometallic derivatives was measured relative to thymidine. We have shown that the influence of the overall charge of the derivatives is dependent on the position of functionalization. In the case of the C3'-functionalized derivatives, neutral and anionic substrates were most readily phosphorylated (20-28% of the value for the parent ligand thymidine), whereas for the N3-functionalized derivatives, cationic and neutral complexes were apparently better substrates for the enzyme (14-18%) than anionic derivatives (9%).

Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2.

Regulation of the epithelial Na(+) channel (ENaC) by ubiquitylation is controlled by the activity of two counteracting enzymes, the E3 ubiquitin-protein ligase Nedd4-2 (mouse ortholog of human Nedd4L) and the ubiquitin-specific protease Usp2-45. Previously, Usp2-45 was shown to decrease ubiquitylation and to increase surface function of ENaC in Xenopus laevis oocytes, whereas the splice variant Usp2-69, which has a different N-terminal domain, was inactive toward ENaC. It is shown here that the catalytic core of Usp2 lacking the N-terminal domain has a reduced ability relative to Usp2-45 to enhance ENaC activity in Xenopus oocytes. In contrast, its catalytic activity toward the artificial substrate ubiquitin-AMC is fully maintained. The interaction of Usp2-45 with ENaC exogenously expressed in HEK293 cells was tested by coimmunoprecipitation. The data indicate that different combinations of ENaC subunits, as well as the α-ENaC cytoplasmic N-terminal but not C-terminal domain, coprecipitate with Usp2-45. This interaction is decreased but not abolished when the cytoplasmic ubiquitylation sites of ENaC are mutated. Importantly, coimmunoprecipitation in HEK293 cells and GST pull-down of purified recombinant proteins show that both the catalytic domain and the N-terminal tail of Usp2-45 physically interact with the HECT domain of Nedd4-2. Taken together, the data support the conclusion that Usp2-45 action on ENaC is promoted by various interactions, including through binding to Nedd4-2 that is suggested to position Usp2-45 favorably for ENaC deubiquitylation.

A fluorescent peptide substrate for the surface metalloprotease of Leishmania.

A fluorescent oligopeptide substrate for the promastigote surface protease (PSP) of Leishmania was designed using the data reported for the substrate specificity of the enzyme (Bouvier, J., Schneider, P., Etges, R. J., and Bordier, C. 1990. Biochemistry 29, 10113-10119). The indole fluorescence of the tryptophan residue was efficiently quenched through resonance energy transfer by an N-terminal dansyl group located five amino acid residues away. The heptapeptide, dansyl-A-Y-L-K-K-W-V-NH2, was cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 8.8 x 10(6) M-1sec-1. Hydrolysis by the enzyme results in a time-dependent increase of fluorescence intensity of 3.7-fold. Assays can be designed based on the tryptophan fluorescence at 360 nm or by individual product analyses using thin-layer chromatography. The synthetic substrate is readily cleaved by the metalloprotease at the surface of fixed promastigotes. The specificity and sensitivity of such internally quenched fluorescent peptide substrate will facilitate the identification of novel inhibitors for the enzyme and aid in detailed studies on its enzymology.

Carbonic anhydrase activity in primary sensory neurons. II. Influence of environmental factors on the phenotypic expression of the enzyme in dissociated cultures of chicken dorsal root ganglion cells.

Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors.

Effects of Cytochrome P450 Enzyme Inhibitors on the Pharmacokinetics of Nonsteroidal Anti-inflammatory Drugs and Venlafaxine

Cytochrome P450 (CYP) enzymes play a pivotal role in the metabolism of many drugs. Inhibition of CYP enzymes usually increases the plasma concentrations of their substrate drugs and can thus alter the safety and efficacy of these drugs. The metabolism of many widely used nonsteroidal antiinflammatory drugs (NSAIDs) as well as the metabolism of the antidepressant venlafaxine is nown to be catalyzed by CYP enzymes. In the present studies, the effect of CYP inhibition on the armacokinetics and pharmacodynamics of NSAIDs and venlafaxine was studied in clinical trials with healthy volunteers and with a crossover design, by using different antifungal agents as CYP inhibitors. The results of these studies demonstrate that the inhibition of CYP enzymes leads to increased concentrations of NSAIDs. In most cases, the exposure to ibuprofen, diclofenac, etoricoxib, and meloxicam was increased 1.5to 2 fold when they were used concomitantly with antifungal agents. CYP2D6 inhibitor, terbinafine, substantially increased the concentration of parent venlafaxine, whereas the concentration of active moiety of venlafaxine (parent drug plus active metabolite) was only slightly increased. Voriconazole, an inhibitor of the minor metabolic pathway of venlafaxine, produced only minor changes in the pharmacokinetics of venlafaxine. These studies show that an evident increase in the concentrations of NSAIDs may be expected, if they are used concomitantly with CYP inhibitors. However, as NSAIDs are generally well tolerated, use of single doses of NSAIDs concomitantly with CYP inhibitors is not likely to adversely affect patient safety, whereas clinical relevance of longterm concomitant use of NSAIDs with CYP inhibitors needs further investigation. CYP2D6 inhibitors considerably affect the pharmacokinetics of venlafaxine, but the clinical significance of this interaction remains unclear.

Enzyme loading dependence of cellulose hydrolysis of sugarcane bagasse

The enzymatic hydrolysis of steam-pretreated sugarcane bagasse, either delignified or non-delignified, was studied as a function of enzyme loading. Hydrolysis experiments were carried out using five enzyme loadings (2.5 to 20 FPU/g cellulose) and the concentration of solids was 2% for both materials. Alkaline delignification improved cellulose hydrolysis by increasing surface area. For both materials, glucose concentrations increased with enzyme loading. On the other hand, enzyme loadings higher than 15 FPU/g did not result in any increase in the initial rate, since the excess of enzyme adsorbed onto the substrate restricted the diffusion process through the structure.

Increased angiotensin-converting enzyme activity in the left ventricle after infarction

An increase in angiotensin-converting enzyme (ACE) activity has been observed in the heart after myocardial infarction (MI). Since most studies have been conducted in chronically infarcted individuals exhibiting variable degrees of heart failure, the present study was designed to determine ACE activity in an earlier phase of MI, before heart failure development. MI was produced in 3-month old male Wistar rats by ligation of the anterior branches of the left coronary artery, control rats underwent sham surgery and the animals were studied 7 or 15 days later. Hemodynamic data obtained for the anesthetized animals showed normal values of arterial blood pressure and of end-diastolic pressure in the right and left ventricular cavities of MI rats. Right and left ventricular (RV, LV) muscle and scar tissue homogenates were prepared to determine ACE activity in vitro by measuring the velocity of His-Leu release from the synthetic substrate Hyp-His-Leu. ACE activity was corrected to the tissue wet weight and is reported as nmol His-Leu g-1 min-1. No significant change in ACE activity in the RV homogenates was demonstrable. A small nonsignificant increase of ACE activity (11 &plusmn; 9%; P0.05) was observed 7 days after MI in the surviving left ventricular muscle. Two weeks after surgery, however, ACE activity was 46 &plusmn; 11% (P<0.05) higher in infarcted rats compared to sham-operated rats. The highest ACE activity was demonstrable in the scar tissue homogenate. In rats studied two weeks after surgery, ACE activity in the LV muscle increased from 105 &plusmn; 7 nmol His-Leu g-1 min-1 in control hearts to 153 &plusmn; 11 nmol His-Leu g-1 min-1 (P<0.05) in the remaining LV muscle of MI rats and to 1051 &plusmn; 208 nmol His-Leu g-1 min-1 (P<0.001) in the fibrous scar. These data indicate that ACE activity increased in the heart after infarction before heart failure was demonstrable by hemodynamic measurements. Since the blood vessels of the scar drain to the remaining LV myocardium, the high ACE activity present in the fibrous scar may increase the angiotensin II concentration and decrease bradykinin in the cardiac tissues surrounding the infarcted area. The increased angiotensin II in the fibrous scar may contribute to the reactive fibrosis and hypertrophy in the left ventricular muscle surviving infarction

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.