Dysregulation of the renin-angiotensin system during lung ischemia-reperfusion injury.

UNLABELLED: Aim/Purpose of the Study: Activation of the renin-angiotensin system leading to increased angiotensin-(1-7) (Ang-(1-7)) and decreased angiotensin 2 (Ang 2) levels may be a new therapeutic approach to reduce acute lung injury. Prolylcarboxypeptidase (PRCP) and prolyloligopeptidase (PREP) are capable of hydrolyzing Ang 2 into Ang-(1-7). However, their relation with circulating Ang 2 levels after lung ischemia-reperfusion injury (LIRI) has never been explored. This study determines whether the activity and expression of PRCP and PREP in plasma and lung tissue is related to circulating Ang 2 levels in a murine model of LIRI. MATERIALS AND METHODS: LIRI in Swiss mice (6 animals per group) was induced by temporary left lung hilar clamping (1 h) followed by 0, 1 or 24 h of reperfusion. Animals in the sham group received thoracotomy only. PRCP activity was measured via RP-HPLC, PREP activity using a fluorogenic substrate and plasma Ang 2 levels via ELISA. Western blotting was used to determine the PRCP and PREP protein expression profiles in left lung tissue. RESULTS: Plasma Ang 2 levels significantly rise after lung ischemia and remain increased after 1 h and 24 h of reperfusion compared to the sham group. While a significant decrease in plasma PREP activity was found after 24 h of reperfusion, a transient increase in plasma PRCP activity was observed after ischemia. However, no correlation with plasma Ang 2 levels could be demonstrated. The activity profiles of PRCP and PREP and the protein expression of PRCP in the lung tissues remained unchanged after LIRI. CONCLUSIONS: LIRI causes a dysregulation of circulating Ang 2 levels and plasma PREP activity, although no direct link between both phenomena could be shown. The activity profile of pulmonary PRCP and PREP was not significantly changed after LIRI, which implies a minor role for local PRCP and PREP in the ischemic lung itself.

Carboxypeptidase U (CPU, carboxypeptidase B2, activated thrombin-activatable fibrinolysis inhibitor) inhibition stimulates the fibrinolytic rate in different in vitro models.

Essentials AZD9684 is a potent inhibitor of carboxypeptidase U (CPU, TAFIa, CPB2). The effect of AZD9684 on fibrinolysis was investigated in four in vitro systems. The CPU system also attenuates fibrinolysis in more advanced hemostatic systems. The size of the observed effect on fibrinolysis is dependent on the exact experimental conditions. SUMMARY: Background Carboxypeptidase U (CPU, carboxypeptidase B2, activated thrombin-activatable fibrinolysis inhibitor) is a basic carboxypeptidase that attenuates fibrinolysis. This characteristic has raised interest in the scientific community and pharmaceutical industry for the development of inhibitors as profibrinolytic agents. Objectives Little is known about the contribution of CPU to clot resistance in more advanced hemostatic models, which include blood cells and shear stress. The aim of this study was to evaluate the effects of the CPU system in in vitro systems for fibrinolysis with different grades of complexity. Methods The contribution of the CPU system was evaluated in the following systems: (i) plasma clot lysis; (ii) rotational thromboelastometry (ROTEM) in whole blood; (iii) front lysis with confocal microscopy in platelet-free and platelet-rich plasma; and (iv) a microfluidic system with whole blood under arterial shear stress. Experiments were carried out in the presence or absence of AZD9684, a specific CPU inhibitor. Results During plasma clot lysis, addition of AZD9684 resulted in 33% faster lysis. In ROTEM, the lysis onset time was decreased by 38%. For both clot lysis and ROTEM, an AZD9684 dose-dependent response was observed. CPU inhibition in front lysis experiments resulted in 47% and 50% faster lysis for platelet-free plasma and platelet-rich plasma, respectively. Finally, a tendency for faster lysis was observed only in the microfluidic system when AZD9684 was added. Conclusions Overall, these experiments provide novel evidence that the CPU system can also modulate fibrinolysis in more advanced hemostatic systems. The extent of the effects appears to be dependent upon the exact experimental conditions.

Procarboxypeptidase U (proCPU, TAFI, proCPB2) in cerebrospinal fluid during ischemic stroke is associated with stroke progression, outcome and blood-brain barrier dysfunction.

Essentials Little is known of procarboxypeptidase U (proCPU) in cerebrospinal fluid (CSF) of stroke patients. ProCPU levels were studied in CSF of controls and non-thrombolyzed acute ischemic stroke patients. ProCPU is elevated in CSF of stroke patients compared with controls. ProCPU in CSF correlates with stroke progression, outcome, and blood-brain barrier dysfunction. SUMMARY: Background Procarboxypeptidase U (proCPU, TAFI, proCPB2), the zymogen of CPU, which is a potent antifibrinolytic enzyme and a modulator of inflammation, has previously been investigated in plasma of stroke patients, but so far, no information on the proCPU levels in cerebrospinal fluid (CSF) during acute ischemic stroke (AIS) is available. Objectives This case-control observational study investigates proCPU in CSF of AIS patients compared with controls with an intact blood-brain barrier (BBB) and evaluates the relationship of CSF/plasma proCPU ratios with stroke parameters. Methods A sensitive HPLC-based enzymatic assay was used to determine proCPU levels in CSF of non-thrombolyzed patients in the hyperacute phase (< 24 h after onset) of AIS (n = 72). Individuals (n = 32) without stroke, an intact BBB and no apparent abnormalities in biochemical and microbiological tests, served as controls. Relations between the CSF/plasma proCPU ratio and (i) stroke severity, (ii) stroke progression/recurrence, (iii) stroke outcome and (iv) BBB dysfunction (CSF/serum albumin ratio) were assessed. Results Mean (SEM) proCPU levels were elevated in the CSF of stroke patients compared with controls (4.36 (0.23) U L-1 vs. 3.50 (0.23) U L-1 ). Higher median [IQR] CSF/plasma proCPU ratios were found in patients with stroke progression ((6.0 [4.2-6.9]) × 10-3 ) and poor outcome ((6.4 [3.9-7.0]) × 10-3 ) after 3 months (modified Rankin Scale; mRS > 3) compared with patients without progression ((3.9 [2.7-5.4]) × 10-3 ) or better outcome ((4.0 [2.8-5.0]) × 10-3 ). In stroke patients with a disrupted BBB, proCPU ratios were higher compared with stroke patients with an intact BBB ((6.4 [5.8-9.0]) × 10-3 vs. (3.7 [2.8-5.0]) × 10-3 ). Conclusions ProCPU is increased in CSF during hyperacute ischemic stroke and is associated with stroke progression and outcome after 3 months, most likely due to BBB dysfunction in the hyperacute phase of ischemic stroke.

Ligand-induced conformational changes in prolyl oligopeptidase: a kinetic approach.

Most kinetic studies of prolyl oligopeptidase (PREP) were performed with the porcine enzyme using modified peptide substrates. Yet recent biophysical studies used the human homolog. Therefore, the aim of this study was to compare the kinetic behavior of human and porcine PREP, as well as to find a suitable method to study enzyme kinetics with an unmodified biological substrate. It was found that human PREP behaves identically to the porcine homolog, displaying a double bell-shaped pH profile and a pH-dependent solvent kinetic isotope effect of the kcat/Km, features that set it apart from the related exopeptidase dipeptidyl peptidase IV (DPP IV). However, the empirical temperature coefficient Q10, describing the temperature dependency of the kinetic parameters and the non-linear Arrhenius plot of kcat/Km are common characteristics between PREP and DPP IV. The results also demonstrate the feasibility of microcalorimetry for measuring turn-over of proline containing peptides.

Rapid spectral scan and stopped-flow studies of carbon monoxide binding to bovine adrenocortical cytochrome P-450scc.

Carbon monoxide binding with both cholesterol-free (low-spin) and cholesterol-bound (high-spin) reduced forms of purified cytochrome P-450scc has been investigated by rapid-scan and stopped-flow spectrometry. CO binding occurs within 150 ms at 25 degrees C for both forms of P-450scc, with a typical absorption maximum at 450 nm. Isosbestic points occur at the following wavelengths: between reduced-CO and reduced cholesterol-free P-450scc at 434 and 471 nm; between reduced-CO and reduced cholesterol-bound P-450scc at 433 and 469 nm. Both the 'on' (k1) and 'off' rate constants (k-1) are found to be independent of pH between pH 5 and 9. The mean values of k1 for cholesterol-free (1.8 +/- 0.2) X 10(5) M-1 X s-1) and cholesterol-bound [1.9 +/- 0.1) X 10(5) M-1 X s-1) P-450scc are almost identical, while the mean value of k-1 for the former [2.3 +/- 0.3) X 10 s-1) is about double that of the latter [1.2 +/- 0.1) X 10 s-1). This suggests the instability of the reduced-CO complex in the absence of cholesterol.

Dipeptide-derived diphenyl phosphonate esters: mechanism-based inhibitors of dipeptidyl peptidase IV.

A number of dipeptide diphenyl phosphonate esters were studied as inhibitors of dipeptidyl peptidase IV, focusing on the role of the P2 residue in the inactivation process. The active compounds were slow irreversible inhibitors of the catalytic activity of the enzyme. With proline (or alanine) in the P1 position, the rate constants of inactivation correlated with the acylation rate constants reported for homologous dipeptide derived substrates. The kinetic data indicate that the mechanism of inhibition consists of the formation of a fairly weak initial complex, followed by a slow irreversible inactivation step. This indicates that, as in the case of trypsin-like proteinases, dipeptide diphenyl phosphonate esters form a covalent adduct with the catalytic site of DPP IV, even though this enzyme belongs to a completely distinct class of serine peptidases. Enantioselectivity and secondary specificity further support the evidence that diphenyl phosphonate esters are mechanism-based inhibitors. The dipeptide diphenyl phosphonate esters had a half-life of 3-10 h at 37 degrees C in Tris buffer. The inhibitors were degraded in human plasma, depending on the type of amino-terminal amino acid. The compound with proline in the P2 position was the most resistant to degradation in plasma. Due to their stability and the irreversible nature of the inhibition, the diphenyl phosphonate esters promise to be useful tools in the continuing investigation of the physiological function of dipeptidyl peptidase IV.

A prediction of DPP IV/CD26 domain structure from a physico-chemical investigation of dipeptidyl peptidase IV (CD26) from human seminal plasma.

Human DPP IV, isolated from seminal plasma by means of immobilised adenosine deaminase, occurs in different forms which are distinguishable by net charge and native molecular weight. Charge differences arise primarily from different degrees of glycosylation containing various amounts of sialic acid. The majority of DPP IV isolated from total seminal plasma consists of the extracellular part of the protein starting at Gly-31. It is a very stable protein resisting high concentrations of denaturant. Unfolding experiments under reducing conditions are indicative of the existence of at least two domains which function independently. One of these domains is highly stabilised by disulfide bonds. Disruption of the disulfide bonds does not affect the activity, the dimeric state nor the adenosine deaminase binding properties of the protein but renders it more susceptible to proteolysis. The low-angle X-ray scattering spectrum is consistent with a model for a protein containing two subunits, each composed of three domains linked by flexible regions with low average mass. The secondary structure composition, determined by FTIR spectrometry, indicates that 45% of the protein consists of beta-sheets, which is higher than expected from computed secondary structure predictions. Our results provide compelling experimental evidence for the three-domain structure of the extracellular part of DPP IV.

Plasma levels of carboxypeptidase U (CPU, CPB2 or TAFIa) are elevated in patients with acute myocardial infarction.

BACKGROUND: Two decades after its discovery, carboxypeptidase U (CPU, CPB2 or TAFIa) has become a compelling drug target in thrombosis research. However, given the difficulty of measuring CPU in the blood circulation and the demanding sample collecton requirements, previous clinical studies focused mainly on measuring its inactive precursor, proCPU (proCPB2 or TAFI). OBJECTIVES: Using a sensitive and specific enzymatic assay, we investigated plasma CPU levels in patients presenting with acute myocardial infarction (AMI) and in controls. METHODS: In this case-control study, peripheral arterial blood samples were collected from 45 patients with AMI (25 with ST segment elevation myocardial infarction [STEMI], 20 with non-ST segment elevation myocardial infarction [NSTEMI]) and 42 controls. Additionally, intracoronary blood samples were collected from 11 STEMI patients during thrombus aspiration. Subsequently, proCPU and CPU plasma concentrations in all samples were measured by means of an activity-based assay, using Bz-o-cyano-Phe-Arg as a selective substrate. RESULTS: CPU activity levels were higher in patients with AMI (median LOD-LOQ, range 0-1277 mU L(-1) ) than in controls (median < LOD, range 0-128 mU L(-1) ). No correlation was found between CPU levels and AMI type (NSTEMI [median between LOD-LOQ, range 0-465 mU L(-1) ] vs. STEMI [median between LOD-LOQ, range 0-1277 mU L(-1) ]). Intracoronary samples (median 109 mU L(-1) , range 0-759 mU L(-1) ) contained higher CPU levels than did peripheral samples (median between LOD-LOQ, range 0-107 mU L(-1) ), indicating increased local CPU generation. With regard to proCPU, we found lower levels in AMI patients (median 910 U L(-1) , range 706-1224 U L(-1) ) than in controls (median 1010 U L(-1) , range 753-1396 U L(-1) ). CONCLUSIONS: AMI patients have higher plasma CPU levels and lower proCPU levels than controls. This finding indicates in vivo generation of functional active CPU in patients with AMI.

Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion.

Dipeptidyl-peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady-state kinetics of DPPIV/CD26-mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase-activating peptide (PACAP27 and PACAP38), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C-terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.

The unique properties of dipeptidyl-peptidase IV (DPP IV / CD26) and the therapeutic potential of DPP IV inhibitors.

This review deals with the properties and functions of dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). This membrane anchored ecto-protease has been identified as the leukocyte antigen CD26. The following aspects of DPP IV/CD26 will be discussed : the structure of DPP IV and the new family of serine proteases to which it belongs, the substrate specificity, the distribution in the human body, specific DPP IV inhibitors and the role of CD26 in the intestinal and renal handling of proline containing peptides, in cell adhesion, in peptide metabolism, in the immune system and in HIV infection. Especially the latest developments in the search for new inhibitors will be reported as well as the discovery of new natural substrates for DPP IV such as the glucagon-like peptides and the chemokines. Finally the therapeutical perspectives for DPP IV inhibitors will be discussed.

Use of immobilized adenosine deaminase (EC 3.5.4.4) for the rapid purification of native human CD26/dipeptidyl peptidase IV (EC 3.4.14.5).

The leukocyte differentiation antigen CD26 identified as dipeptidyl peptidase IV.(EC 3.4.14.5), cleaves off N-terminal dipeptides from peptides when a proline or alanine is located at the penultimate position. Seminal plasma and especially prostasomes, prostate-derived organelles which occur freely in seminal plasma, contain high amounts of CD26/dipeptidyl peptidase IV and therefore are suitable sources for the purification of the protein. The use of adenosine deaminase (EC 3.5.4.4) affinity chromatography for its purification is described. CD26/dipeptidyl peptidase IV was purified from human seminal plasma and prostasomes by a two step procedure. Ion exchange chromatography on DEAE-Sepharose, followed by affinity chromatography on adenosine deaminase-Sepharose resulted in the pure, native protein with an overall yield ranging from 35 to 55%. The N-terminal sequence of the amphiphilic enzyme purified from human prostasomes was determined to be Met-Lys-Thr-Pro-Trp-Lys-Val-Leu. The preparation obtained was free of contaminating aminopeptidase activity and proved to be very stable (up to 1 month at 37 degrees C). The calf intestinal adenosine deaminase we used is commercially available and can be employed for the purification of human, bovine and rabbit CD26/dipeptidyl peptidase IV. High affinity binding of porcine dipeptidyl peptidase IV was not observed. The availability of a source with high specific activity and the introduction of adenosine deaminase affinity chromatography permits the rapid purification of milligram quantities of natural mammalian CD26/dipeptidyl peptidase IV.

Demonstration of glycosomes (microbodies) in the Bodonid flagellate Trypanoplasma borelli (Protozoa, Kinetoplastida).

Homogenates of Trypanoplasma borelli were subjected to subcellular fractionation by sequential differential and isopycnic centrifugation in sucrose. Glycerol-3-phosphate dehydrogenase and the glycolytic enzymes, glucosephosphate isomerase and triosephosphate isomerase, as well as the peroxisomal marker enzyme catalase were mainly, or in part, associated with sedimentable particles that had a buoyant density in sucrose of 1.22 g cm-3. Moreover, triosephosphate isomerase exhibited latency, both in total homogenates and in the particulate fraction. Electron microscopy of thin sections of T. borelli revealed the presence of microbodies that gave a positive reaction for catalase. Pyruvate kinase behaved as a typical soluble enzyme. It was stimulated by micromolar concentrations of fructose 2,6-bisphosphate and this stimulation was counteracted by inorganic phosphate in the millimolar range. The enzymes involved in the synthesis and degradation of fructose 2,6-bisphosphate, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase, were both present in T. borelli and behaved as soluble enzymes. We conclude that in T. borelli the glycolytic pathway is compartmentalized in a way similar to that found in another Kinetoplastid family, the Trypanosomatidae, where seven glycolytic enzymes and two enzymes of glycerol metabolism are associated with glycosomes. Apparently the presence of glycosomes is a characteristic of all Kinetoplastida.

Development of irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator.

In this letter we report the synthesis and biochemical evaluation of selective, irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator (uPA). A diphenyl phosphonate group was introduced on the substratelike peptide Z-d-Ser-Ala-Arg, and modification of the guanidine side chain was investigated. A guanylated benzyl group appeared the most promising side chain modification. A k(app) value in the 10(3) M(-1) s(-1) range for uPA was obtained, together with a selectivity index higher than 240 toward other trypsin-like proteases such as tPA, thrombin, plasmin, and FXa.

Structure-activity relationship of diaryl phosphonate esters as potent irreversible dipeptidyl peptidase IV inhibitors.

The previously reported diphenyl 1-(S)-prolylpyrrolidine-2(R, S)-phosphonate (5) was used as a lead compound for the development of potent and irreversible inhibitors of dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). The synthesis of a series of diaryl 1-(S)-prolylpyrrolidine-2(R,S)-phosphonates with different substituents on the aryl rings (hydroxyl, methoxy, acylamino, sulfonylamino, ureyl, methoxycarbonyl, and alkylaminocarbonyl) started from the corresponding phosphites. A good correlation was found between the electronic properties of the substituent and the inhibitory activity and stability. The most striking divergence of this correlation was the high potency combined with a high stability of the 4-acetylamino-substituted derivative 11e. This compound shows low cytotoxicity in human peripheral blood mononuclear cells and also has favorable properties in vivo. Therefore bis(4-acetamidophenyl) 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate (11e) is considered as a major improvement and will be a highly valuable DPP IV inhibitor for further studies on the biological function of the enzyme and the therapeutic value of its inhibition.

Characterisation of a novel series of aprotinin-derived anticoagulants. II. Comparative antithrombotic effects on primary thrombus formation in vivo.

Upon vascular damage platelet activation and blood coagulation are initiated. Interference at the initial level of the activation of the coagulation cascade can result in effective inhibition of thrombus formation. The in vivo antithrombotic properties of a series of bovine pancreatic trypsin inhibitor mutants (BPTI, aprotinin) 4C2, 7L22, 5L15, 5L15-PEG, 6L15 and 5L84, as described in the accompanying paper, with a combined inhibitory activity on factor Xa, factor VIIa-tissue factor complex, factor XIa and plasma kallikrein were compared to rTAP, r-hirudin, heparin and enoxaparin in a platelet rich thrombosis model in hamsters. Platelet dependent thrombus deposition was quantified by dedicated image analysis after transillumination of the femoral vein to which a standardised vascular trauma was applied. After increasing intravenous bolus injections all tested agents, except for aprotinin, induced a dose dependent decrease of thrombus formation and a concomitant prolongation of the aPTT. From the linear correlation between these two parameters it was found that 5 out of the 6 tested aprotinin analogues, rTAP and r-hirudin completely inhibited thrombus formation at a therapeutical (2- to 3-fold) aPTT prolongation while 4C2, heparin and enoxaparin only inhibited thrombus formation for 40 to 50 percent at a 2-fold aPTT prolongation. Based on the calculated IC50 values for thrombus formation rTAP was found to be the most active compound in this model. It is concluded that acceptable interference at the initial level of the blood coagulation, e.g. within a therapeutical aPTT prolongation, can significantly inhibit platelet deposition at a site of vascular injury.

Characterisation of a novel series of aprotinin-derived anticoagulants. I. In vitro and pharmacological properties.

Previous investigations have indicated that interference with the initial level of the blood coagulation may lead to effective antithrombotic therapy. Recently a series of potential coagulation inhibitors derived from bovine pancreatic trypsin inhibitor (BPTI, aprotinin) was described. We have determined their inhibition constants, effects on coagulation assays, effects in an in vitro human thrombosis model and pharmacological profiles in hamsters. The aprotinin-derived analogues (4C2, 7L22, 5L15, 6L15, 5L84) showed significantly increased inhibitory activity towards factor Xa, factor VIIa-tissue factor (TF) complex, factor XIa and plasma kallikrein or a combination of them, and a significantly decreased plasmin inhibition as compared to aprotinin. In the coagulation assays, 4C2 and 7L22 mainly inhibited factor Xa, 5L15 and 6L15 inhibited factor VIIa-TF complex and 5L84 inhibited factor Xa, factor VIIa-TF complex and the contact activation. In flow chamber experiments with human blood 7L22, 5L15, 6L15, 5L84 and rTAP significantly inhibited fibrin formation and platelet deposition on extracellular matrix from phorbol ester stimulated human endothelial cells both under high and low shear stress and in the presence of low molecular weight heparin. The pharmacological profiles of the aprotinin analogues and rTAP with a mean residence time of 64 to 140 min were not significantly different. Modification of an aprotinin analogue with PEG (5L15-PEG) resulted in a 10-fold decrease of the inhibition constant for the factor VIIa-TF complex and in a significant prolongation of the secondary half-life, while the initial half-life was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo inhibition of dipeptidyl peptidase IV activity by pro-pro-diphenyl-phosphonate (Prodipine).

Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), also known as CD26, is a membrane-bound serine protease that cleaves off aminoterminal dipeptides from peptides with a penultimate proline (or, at a much slower rate, a penultimate alanine). Recently, we synthesized and characterized a number of dipeptide-derived diphenylphosphonates. Out of the resulting series of slow-binding irreversible inhibitors of DPP IV, diphenyl 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate hydrochloride (Pro-Pro-diphenylphosphonate or Prodipine) was selected for further study. We investigated the in vivo applicability of Prodipine. Male rabbits weighing 3-4 kg received a single intravenous injection with 10 mg Prodipine or saline. After 1 hr, plasma DPP IV activity had decreased to less than 20% of the preinjection value and remained unchanged during a 24-hr observation period. In a next step, we aimed to study (i) the dose dependency and (ii) the duration of the effect after a single intravenous dose of Prodipine. A profound and long-lasting inhibition of plasma DPP IV activity was observed in the treated animals (1, 5 or 10 mg). It took 5 to 8 days to reach half of the pretreatment DPP IV activity and generally more than 20 days for a complete recovery. Systemic treatment with Prodipine not only led to inhibition of plasma DPP IV activity but also decreased tissue DPP IV activity in circulating mononuclear cells, kidney cortex, thymus, spleen, lung, and liver. No differences in activities of the related peptidases aminopeptidase P (APP, EC 3.4.11.9), prolyl oligopeptidase (PO, EC 3.4.21.26), or aminopeptidase M (mAAP, EC 3.4.11.2) were detected between Prodipine-treated and control rabbits. The in vivo applicability of this chemically stable, irreversible inhibitor of DPP IV opens new possibilities, not only to further unravel the biological functions of this intriguing ectopeptidase, but also to explore this enzyme as a new target in various fields of pharmacological research.

Study of the enzymatic degradation of vasostatin I and II and their precursor chromogranin A by dipeptidyl peptidase IV using high-performance liquid chromatography/electrospray mass spectrometry.

The interaction of dipeptidyl peptidase IV with structurally related proteins differing in chain length, namely vasostatin I and II and their precursor protein chromogranin A, was examined using high-performance liquid chromatography in combination with electrospray mass spectrometry. Suitable analytical procedures were developed involving the use of reversed-phase high-performance liquid chromatography for purification of the enzymatic degradation products and a peptide mapping procedure for evaluating the enzymatic degradation of the large precursor protein chromogranin A. While vasostatin I was found to be a substrate for dipeptidyl peptidase IV, no N-terminal cleavage of Leu-Pro could be noted for chromogranin A. With respect to vasostatin II, N-terminal degradation was only observed after degradation in the C-terminal domain to proteins containing < or = 78 amino acids. The specificity of the N-terminal release of Leu-Pro was proved by addition of a DPP IV specific inhibitor.

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase.

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.

A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease.

BACKGROUND AND PURPOSE: The aggregation of α-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on α-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human α-syn and in the brains of two A30P α-synuclein transgenic mouse strains. EXPERIMENTAL APPROACH: Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 × 3 mg·kg(-1) a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble α-synuclein protein levels were measured by Western blot. α-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and α-synuclein,and the effect of KYP-2047 on cell viability were also investigated. KEY RESULTS: In cell lines, oxidative stress induced a robust aggregation of α-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with α-synuclein inclusions while abolishing the colocalization of α-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated α-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of α-synuclein immunoreactivity and soluble α-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS: The results suggest that the PREP may play a role in brain accumulation and aggregation of α-synuclein, while KYP-2047 seems to effectively prevent these processes.