Prolidase activity assays. A survey of the reported literature methodologies.

Prolidase (EC.3.4.13.9) is a dipeptidase known nowadays to play a pivotal role in several physiological and pathological processes. More in particular, this enzyme is involved in the cleavage of proline- and hydroxyproline-containing dipeptides (imidodipeptides), thus finely regulating the homeostasis of free proline and hydroxyproline. Abnormally high or low levels of prolidase have been found in numerous acute and chronic syndromes affecting humans (chronic liver fibrosis, viral and acute hepatitis, cancer, neurological disorders, inflammation, skin diseases, intellectual disability, respiratory infection, and others) for which the content of proline is well recognized as a clinical marker. As a consequence, the accurate analytical determination of prolidase activity is of greatly significant importance in clinical diagnosis and therapy. Apart from the Chinard's assay, some other more sensitive and well validated methodologies have been published. These include colorimetric and spectrophotometric determinations of free proline produced by enzymatic reactions, capillary electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, electrochemoluminescence, thin layer chromatography, and HPLC. The aim of this comprehensive review is to make a detailed survey of the in so far reported analytical techniques, highlighting their general features, as well as their advantages and possible drawbacks, providing in the meantime suggestions to stimulate further research in this intriguing field.

Prolidase expression in knee osteoarthritis and healthy controls: Observational study.

ABSTRACT: Prolidase enzyme activity is important for collagen resynthesis. In late stages of osteoarthritis (OA) its activity is decreased.To evaluate prolidase expression in knees of patients undergoing total arthroplasty for OA, and compare with young people undergoing knee arthroscopy due to traumatic injuries.In this cross-sectional study we included 20 patients with OA grade IV who underwent total knee arthroplasty and 20 controls of young patients who underwent arthroscopy for another reason besides OA. All participants were evaluated by knee ultrasound before the procedure. During the procedure, synovial tissue biopsies were taken and analyzed by immunofluorescence to search inflammation. Measures of central tendency, dispersion measures and position measures were used for the case of quantitative variables. Student t test or Mann-Whitney U test, and the logistic regression of Cox, was used.Prolidase expression in the synovial biopsy was significantly lower in the OA group than in the controls (0.017 ±â€Š0.009 vs 0.062 ±â€Š0.094, P < .05). Power Doppler (PD) signal was present in the synovitis of all knee recesses of the OA group in grayscale and in 17 (85%) of knees. The mean of the micro-vessel count in patients with OA was significantly higher vs controls (11 + 5.3 vs 4 + 2.1, P = .001). The neovascularization correlated significantly with the presence of PD signal in patients with OA (1.16, 95% CI, 1.02-1.34, P = .02).The prolidase expression in the synovial membrane evaluated by immunofluorescence, in patients with late stages of knee OA, is low, which may be interpreted as an evidence of decreased collagen resynthesis.

Prolidase enzyme is required for extracellular matrix integrity and impacts on postnatal cerebellar cortex development.

The extracellular matrix is essential for brain development, lamination, and synaptogenesis. In particular, the basement membrane below the pial meninx (pBM) is required for correct cortical development. The last step in the catabolism of the most abundant protein in pBM, collagen Type IV, requires prolidase, an exopeptidase cleaving the imidodipeptides containing pro or hyp at the C-terminal end. Mutations impairing prolidase activity lead in humans to the rare disease prolidase deficiency characterized by severe skin ulcers and mental impairment. Thus, the dark-like (dal) mouse, in which the prolidase is knocked-out, was used to investigate whether the deficiency of prolidase affects the neuronal maturation during development of a brain cortex area. Focusing on the cerebellar cortex, thinner collagen fibers and disorganized pBM were found. Aberrant cortical granule cell proliferation and migration occurred, associated to defects in brain lamination, and in particular in maturation of Purkinje neurons and formation of synaptic contacts. This study deeply elucidates a link between prolidase activity and neuronal maturation shedding new light on the molecular basis of functional aspects in the prolidase deficiency.

Rectal application of argan oil improves healing of colorectal anastomosis in rats1.

PURPOSE: To investigate the possible effects of argan oil on the healing of colorectal anastomoses. METHODS: n Group 1 (sham), laparotomy was performed and the colon was mobilized. In the control (Group 2) and argan oil (Group 3) groups, colonic resection and anastomosis were applied. To the control and sham groups, 2 mL of 0.9% NaCl was administred rectally, and in the argan oil group, 2 mL/day argan oil was applied rectally for 7 days. RESULTS: The mean bursting pressures of the argan oil and sham groups were significantly higher than the values in the control group. A significant difference was determined between the tissue hydroxyproline and prolidase levels of control group and other groups. Histopathologically, argan oil showed significant beneficial effects on colonic wound healing. In the argan oil and sham groups, the tissue malondialdehyde and fluorescent oxidation product levels were found to be lower and total sulfhydryl levels were higher than the control group. CONCLUSIONS: The rectally administered argan oil was observed to have significantly ameliorated wound healing parameters and exerted a significant antioxidant effect. This is the first study in the literature about the beneficial effects of argan oil on colorectal anastomoses.

Rectal application of argan oil improves healing of colorectal anastomosis in rats

Abstract Purpose: To investigate the possible effects of argan oil on the healing of colorectal anastomoses. Methods: I n Group 1 (sham), laparotomy was performed and the colon was mobilized. In the control (Group 2) and argan oil (Group 3) groups, colonic resection and anastomosis were applied. To the control and sham groups, 2 mL of 0.9% NaCl was administred rectally, and in the argan oil group, 2 mL/day argan oil was applied rectally for 7 days. Results: The mean bursting pressures of the argan oil and sham groups were significantly higher than the values in the control group. A significant difference was determined between the tissue hydroxyproline and prolidase levels of control group and other groups. Histopathologically, argan oil showed significant beneficial effects on colonic wound healing. In the argan oil and sham groups, the tissue malondialdehyde and fluorescent oxidation product levels were found to be lower and total sulfhydryl levels were higher than the control group. Conclusions: The rectally administered argan oil was observed to have significantly ameliorated wound healing parameters and exerted a significant antioxidant effect. This is the first study in the literature about the beneficial effects of argan oil on colorectal anastomoses.

Glutamine dipeptide supplementation improves clinical responses in patients with diabetic foot syndrome

ABSTRACT The effect of glutamine dipeptide (GDP) supplementation in patients with diabetic foot syndrome was evaluated. A total of 22 patients took part in the study. GDP was supplied in 10 g sachets, and was dissolved in water immediately before use, with ingestion once a day, after lunch or after dinner (20 g/day) over a period of 30 days. Quantification of foot insensitive areas, oxidative stress, blood cytokines, and biochemical, hematological and toxicological parameters was performed before and after GDP supplementation. We observed an increase in blood levels of interferon-α (P=0.023), interferon-γ (P=0.038), interleukin-4 (P=0.003), interleukin-6 (P=0.0025), interleukin-7 (P=0.028), interleukin-12 p40 (P=0.017), interleukin-13 (P=0.001), leukocytes (P=0.037), eosinophils (P=0.049), and typical lymphocytes (P<0.001) due to GDP administration. In addition, we observed a reduced number (P=0.048) of insensitive areas on the foot, and reduction (P=0.047) of fasting hyperglycemia. Patients also showed increased blood high density lipoprotein (P<0.01) and protein thiol groups (P=0.004). These favorable results were associated with the absence of renal and hepatic toxicity. These results are of clinical relevance, since supplementation with GDP over 30 days improved clinical responses in patients with diabetic foot syndrome.

Evaluation of antioxidative/oxidative status and prolidase parameters in cases of inguinal hernia with joint hypermobility syndrome.

PURPOSE: Most previous reports have shown that the basic mechanism of inguinal hernia involves insufficient collagen strength and metabolism. The aim of this study was to evaluate whether joint hypermobility is involved in the development of inguinal hernia in children and to investigate oxidative stress parameters and prolidase activity in tissue samples from children with inguinal hernia. METHODS: This cross-sectional study involving 41 patients (age, 6.36 ± 2.96 years) with inguinal hernia treated in the pediatric surgery department of our institution and 40 age- and sex-matched controls (age, 6.02 ± 3.13 years) was performed from May to December 2011. Joint hypermobility was assessed using the Beighton criteria in all patients. Hernia sacs were analyzed with respect to the total antioxidative/oxidative status and prolidase activity. The patients were divided into two groups (inguinal hernia with and without hypermobility) according to a Beighton score cut-off of ≥6. RESULTS: A total of 81 subjects aged 3-10 years participated. The ratio of joint hypermobility was significantly higher in patients than in controls (p = 0.01). The prolidase activity, total oxidant status, and oxidative stress index were higher in tissue samples from patients with joint hypermobility (p < 0.001). CONCLUSIONS: Our results show that joint hypermobility syndrome is associated with inguinal hernia in children and that increased prolidase activity and oxidative stress in tissue samples from patients with joint hypermobility syndrome are related to collagen tissue damage and turnover.

Serum prolidase activity, oxidative stress, and nitric oxide levels in patients with bladder cancer.

OBJECTIVES: Prolidase is a member of the matrix metalloproteinase family. It plays a major role in collagen turnover, matrix remodeling and cell growth. Nitric oxide (NO) regulates many processes such as collagen synthesis and matrix remodeling. Thus, NO may augment angiogenesis, tumor invasion, and metastasis. The aim of this study was to investigate total antioxidant status (TAS), malondialdehyde (MDA) and NO levels in patients with bladder cancer and to determine their relationship with prolidase activity. DESIGN AND METHODS: Thirty-five patients with bladder cancer and 32 controls were enrolled. Serum TAS, MDA, prolidase activity and NO levels were determined. RESULTS: Serum prolidase activity, NO levels and MDA levels were significantly higher in bladder cancer than controls (all, P < 0.05), while TAS levels were significantly lower (P < 0.05). CONCLUSIONS: Our results show that increased prolidase seems to be associated with increased NO levels and oxidative stress along with decreased antioxidant levels in bladder cancer.

A preliminary study pointing out the role of serum prolidase activity and oxidative-antioxidative status parameters during the treatment process of patients with idiopathic clubfoot.

BACKGROUND: We aimed to investigate serum prolidase activity and to find out its association with oxidative-antioxidative status in patients with idiopathic clubfoot and during the course of the disease. MATERIAL AND METHODS: Oxidative status parameters, including total free sulfhydryl groups (-SH), total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI), as well as serum prolidase activity were assessed at the beginning of the treatment in patients with idiopathic clubfoot (n = 38), at the end of 3 months during the treatment of the disease and in healthy controls (n = 40). All patients were managed with the Ponseti method and severity of the foot deformity was evaluated according to the Pirani Severity Score. RESULTS: Serum prolidase activity, TOS and OSI values of the patients at the beginning of the treatment were found to be significantly higher but -SH and TAC values were found to be significantly lower as compared to controls. In the treatment process, a significant decrease in serum prolidase activity, TOS and OSI values and Pirani Severity Score of the patients was observed, however a significant increase in -SH and TAC values of the patients was observed at the end of 3 months during the treatment of the disease as compared to the beginning of the treatment. CONCLUSION: Elevated levels of serum prolidase activity, TOS and OSI, and decreased levels of -SH and TAC may be associated with idiopathic clubfoot, and that these parameters may be useful adjunctive tools for follow-up in patients with idiopathic clubfoot.

Activity of prolidase isoenzymes in the rat brain: subcellular and regional distribution during development.

Prolidase deficiency is characterized by chronic ulcerative dermatitis, mental retardation, and frequent infections. In the present study we examined the characteristics of rat brain prolidase isoenzymes. Prolidase isoenzymes (PD I and PD II) were isolated from the rat brain using DEAE cellulose column chromatography. PD I showed higher activity against seryl-proline and alanyl-proline, while PD II was particularly active against methionyl-proline. Prolidase activity in the whole brain and in the different brain regions showed higher activity against methionyl-proline and seryl-proline. PD II activity was highest in the hippocampus, followed by the cerebellum, cerebral cortex, caudatum, and the midbrain. The most rapid changes in the activities of PD I and PD II occurred perinatally, with a peak at three days before birth and a nadir at two days after birth, which then gradually increased until 21 days. N-benzyloxycarbonyl-l-proline inhibited PD I activity against various substrates in a dose-dependent manner. In contrast, there was no inhibition of PD II activity against methionyl-proline at low concentrations. In summary, these data suggest that maintenance of levels of proline, other amino acids and peptides containing proline in the rat brain is regulated by prolidase isoenzymes. The age-related alterations in PD I and PD II also may help to elucidate the fundation of prolidase isoenzymes in brain nervous system.

Screening, purification, and identification of the enzyme producing N-(L-alpha-L-aspartyl)-L-phenylalanine methyl ester from l-isoasparagine and L-phenylalanine methyl ester.

Screening was carried out for microorganisms able to produce N-(l-alpha-l-aspartyl)-l-phenylalanine methyl ester [APM] from l-isoasparagine and l-phenylalanine methyl ester hydrochloride. Of the 422 strains examined, 44 strains belonging to the family Enterobacteriaceae were found to produce APM. The enzyme catalyzing APM production was purified and identified as dipeptidase E.

Brain type carnosinase in dementia: a pilot study.

BACKGROUND: The pathological processes underlying dementia are poorly understood and so are the markers which identify them. Carnosinase is a dipeptidase found almost exclusively in brain and serum. Carnosinase and its substrate carnosine have been linked to neuropathophysiological processes. METHODS: Carnosinase activity was measured by a flourometric method in 37 patients attending a Geriatric Outpatient Clinic. There were 17 patients without dementia, 13 had Alzheimer's disease (AD) and 7 had mixed dementia (MD). RESULTS: The range of serum carnosinase activity for patients without dementia was 14.5 - 78.5 micromol/ml/h. There was no difference in carnosinase activity between patients without dementia (40.3 +/- 15.2 micromol/ml/h) and patients with AD (44.4 +/- 12.4 micromol/ml/h) or MD (26.6 +/- 15 micromol/ml/h). However, levels in the MD group were significantly lower than the AD group (p = 0.01). This difference remained significant after adjusting for gender, MMSE score, exercise, but not age, one at a time and all combined. The effect of other medical conditions did not remove the significance between the AD and MD groups. The MD group, but not the AD group, demonstrated a significant trend with carnosinase activity decreasing with duration of disease (from first recorded date of diagnosis to date of blood collection) (r = -0.76, p = 0.049). There was no association with carnosinase activity and MMSE score in the AD or MD group. Both AD and MD patients on any dementia medication (donepezil, galantamine, memantine) had higher carnosinase activity compared to those not taking a dementia medication. Carnosinase activity was higher in patients who regularly exercised (n = 20) compared to those who did not exercise regularly (n = 17)(p = 0.006). CONCLUSION: This exploratory study has shown altered activities of the enzyme carnosinase in patients with dementia.

Dipeptidase-inactivated tACE action in vivo: selective inhibition of sperm-zona pellucida binding in the mouse.

The angiotensin-converting enzyme (ACE) plays a crucial role in male fertilization and is a key regulator of blood pressure. Testicular ACE (tACE), the germinal specific isozyme expressed on different promoters, exclusively carries out the role of ACE in fertility, although the site and mode of action are not well known. To investigate the contribution of tACE in fertilization, we produced transgenic mouse lines carrying a dipeptidase-inactivated mutant. Although the transgenic mice showed normal blood pressure, kidney morphology, and fertility, reduced fertilization was observed after in vitro fertilization (IVF). The sperm-zona pellucida (ZP) binding was exclusively impaired in these lines in a manner similar to that observed in an Ace knockout mouse. The dipeptidase activity was reduced in epididymal ingredients but not in the testis. Furthermore, direct application of mutant protein did not suppress sperm-ZP binding of intact sperm during IVF, implying that the dipeptidase-inactivated mutant affects sperm modification in the epididymis for ZP binding. Our results indicate that the dipeptidase-inactivated tACE acts in vivo, suggesting that tACE contributes to fertilization as a dipeptidase at least in the epididymis.

Modulation of vaginal immune response among pregnant women with bacterial vaginosis by Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, and yeast.

OBJECTIVE: This study was undertaken to examine the influence of coinfections on vaginal innate and adaptive immunity, and microbial enzyme activities of pregnant women with bacterial vaginosis (BV). STUDY DESIGN: The population consisted of 265 singleton pregnant women in early gestation (<20 weeks) with BV (Nugent 7-10) who had vaginal fluid collected for measurement of interleukin-1beta (IL-1beta) and IL-8 concentrations, number of neutrophils, immunoglobulin A against Gardnerella vaginalis (anti-Gvh IgA), and activities of microbial sialidase and prolidase. RESULTS: Among women with BV, median levels of vaginal IL-1beta (4-fold, P = .005), IL-8 (4-fold, P < .001), and neutrophils (6-fold, P = .013) were greatly increased in women with T vaginalis with respect to women without any coinfection. Yeast increased the level of IL-8 (5-fold, P < .001), but not IL-1beta (P = .239) and neutrophils (P = .060). Chlamydia trachomatis and Neisseria gonorrhoeae had no effect on vaginal cytokines. None of the coinfections influenced vaginal anti-Gvh IgA, sialidase and prolidase activities. CONCLUSION: The strong proinflammatory cytokine induction by T. vaginalis may contribute to the observed increase in preterm birth among BV positive women coinfected with T. vaginalis treated with metronidazole.

Enhanced prolidase activity and decreased collagen content in breast cancer tissue.

Adherent interactions between integrins and extracellular matrix (ECM) proteins play an important role in tumorigenicity and invasiveness. The major component of ECM is collagen that plays a central role in the interaction with integrins. The expression of certain collagenases (gelatinases) by tumour cells is one of the characteristic features of the so-called metastatic phenotype, presumably by breaking down ECM barriers as well by altering the ECM-cell interaction. Although extracellular collagenases initiate the breakdown of collagen, the final step of collagen degradation is catalysed by intracellular prolidase. Collagen deposition, gelatinolytic and prolidase activities, expression of beta(1)-integrin receptor and their possible relationships were studied in seven operable breast cancer cases. In breast cancer tissue, we have found significant decrease in the amount of collagen. The decrease in collagen deposition in breast cancer tissue was accompanied by increase in the tissue gelatinolytic and prolidase activities. Simultaneously, a slight decrease in the expression of beta(1)-integrin receptor in breast cancer tissue was observed. These results suggest that alteration in collagen metabolism in breast cancer tissue may reflect tissue remodelling, characteristic for invasive phenotype of cancer cells. Increased gelatinolytic and prolidase activities in breast cancer tissue may enhance stromal matrix degradation and thus may promote metastatic dissemination. On the basis of the data, it seems that compounds endowed with gelatinolytic and prolidase inhibitory activities may be considered as a potential drug candidates for breast cancer therapy.

Identification of a new chromophoric substrate in the library of amino acid p-nitroanilides for continuous assay of VanX, a D,D-dipeptidase essential for vancomycin resistance.

As one of key bacterial proteins involved in vancomycin resistance, VanX is a D,D-dipeptidase that impedes bacterial cell wall biosynthesis by hydrolyzing the essential D-Ala-D-Ala dipeptide. Based on a report by Crowder and co-workers that L-alanine-p-nitroanilide (L-Ala-pNA) was a useful substrate for continuous assay of VanX, we constructed a library of 35 L- and D-amino acid p-nitroanilides to provide the needed diversity to discover new substrates that are more specific than L-Ala-pNA. We report here that, among all compounds tested, D-leucine-p-nitroanilide (D-Leu-pNA) was found to be the best substrate for VanX enzyme (KM=8.9+/-1.2 mM, kcat=0.0102+/-0.0016 s(-1), kcat/KM=0.0012 mM(-1)s(-1)). Although it is catalytically inefficient, this new VanX substrate needs essentially no sophisticated synthetic chemistry for preparation and therefore offers a convenient means for routine analysis of enzyme catalysis and the screening of potential inhibitors. Moreover, because it is the uncommon leucine in its D form in D-Leu-pNA, enzymatic activities due to other contaminated species in Escherichia coli used for VanX overproduction should be greatly reduced.

Variation in vaginal immune parameters and microbial hydrolytic enzymes in bacterial vaginosis positive pregnant women with and without Mobiluncus species.

OBJECTIVE: This study was undertaken to assess if levels of interleukin-1beta (IL-1beta), IL-8, sialidase, prolidase and immunoglobulin A against Gardenerella vaginalis hemolysin (anti-Gvh IgA) in vaginal secretions differ between BV+ women with (M+) and without (M-) Mobiluncus spp. STUDY DESIGN: Vaginal secretions were obtained from 265 women at their first prenatal care visit and assessed for all study parameters. Gram stain evaluation using Nugent criteria was performed and coinfection with sexually transmitted infections determined. Differences between BV+/M+ and BV+/M- women were evaluated using the chi2 statistic or Mann-Whitney test. RESULTS: Of the 265 BV+ women, 43% (n = 113) were M+ of which 97% (n = 110) had Nugent scores of 9 or 10 . BV+/M+ women had elevated levels of sialidase (median value: 4.11 nmol vs 1.91 nmol of converted substrate; P = .003) but no difference in prolidase, anti-Gvh IgA, IL-1beta, IL-8, levels were found between the two groups. BV+/M- women had significantly higher rates of coinfection with Trichomonas vaginalis. CONCLUSION: BV+/M+ women have higher vaginal concentrations of sialidase and lower rates of T. vaginalis compared with BV+/M- women. Further research is needed to assess the association of this, and other, microbiologic profiles to risk of adverse pregnancy outcome.

Characterization of prolidase activity using capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection and application to evaluate collagen degradation in diabetes mellitus.

A new method for prolidase (PLD, EC 3.4.13.9) activity assay was developed based on the determination of proline produced from enzymatic reaction through capillary electrophoresis (CE) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)3(2+)] electrochemiluminescence detection (ECL). A detection limit of 12.2 fmol (S/N = 3) for proline, corresponding to 1.22 x 10(-8) units of prolidase catalyzing for 1 min was achieved. PLD activity determined by CE-ECL method was in agreement with that obtained from the classical Chinard's one. CE-ECL showed its powerful resolving ability and selectivity as no sample pretreatment was needed and no interference existed. The clinical utility of this method was successfully demonstrated by its application to assay PLD activity in the serum of diabetic patients in order to evaluate collagen degradation in diabetes mellitus (DM). The results indicated that enhanced collagen degradation occurred in DM.

Phenotypic diversity of the lung vasculature in experimental models of metastases.

In vivo phage display is a screening method in which peptides homing to specific vascular beds are selected after IV administration of a random peptide library. This strategy has revealed a vascular address system that allows tissue-specific targeting of normal blood vessels and angiogenesis-related targeting of tumor blood vessels by selected peptides. Many vascular receptors or "addresses" targeted by homing peptides have been identified. One such vascular receptor of normal lung endothelium is membrane dipeptidase (MDP), which was found by in vivo phage display to bind the tripeptide motif gly-phe-glu (GFE). Our studies with GFE peptide and lung vasculature suggest that MDP mediates cancer cell adhesion to lung vasculature and the development of lung metastases, but that MDP is not present in the vasculature of lung metastases. MDP appears to occupy a vascular distribution that is similar to the pulmonary artery circulation. These results demonstrate the promise of defining critical functional and anatomic characteristics of endothelial cells in lung and other organs by in vivo phage display.

Identification and characterization of a mouse dipeptidase that hydrolyzes L-carnosine.

L-Carnosine is a bioactive dipeptide present in mammalian tissues including the central nervous system. We have recently shown that L-carnosine is involved in the regulation of energy homeostasis through the autonomic nervous system, but the mechanisms for its biosynthesis and degradation have not yet been fully elucidated. Here we report the biochemical and immunohistochemical characterization of a mammalian protein that has a 17% overall amino acid sequence homology with a Lactobacilus carnosinase, PepV. A recombinant protein expressed in E. coli has the enzymatic ability to digest L-carnosine and various other dipeptides, and this activity is inhibited by bestatin. It requires Mn2+ for enzymatic activity and its effect is reversible. Immunohistochemical analysis showed that a few neuronal populations express this protein at very high levels. It is highly expressed in the parafascicular nucleus of the thalamus, tuberomammillary nucleus of the hypothalamus and the mitral cell layer of the olfactory bulb. In addition, neuronal processes, but not cell bodies, are stained in the striatum. In all these areas, the protein did not colocalize with the glial fibrilary acidic protein. These results suggest that a peptidase that digests L-carnosine is enriched in several specific neuronal populations in the central nervous system.