A bioluminescent probe for in vivo imaging of pyroglutamate aminopeptidase in a mouse model of inflammation.

Pyroglutamate aminopeptidase (PGP) specifically cleaves the peptide bond of pyroglutamic acid linked to the N-terminal end of a polypeptide or protein. Previous studies showed that PGP was associated with several physiological processes and diseases especially those involving inflammation. Utilizing a 'caging' strategy, we designed and synthesized a bioluminescence probe (PBL) with a limit-of-detection of 3.7 * 10-4 mU/mL. In vivo imaging in a mouse model of inflammatory liver disease revealed that the probe has excellent sensitivity and selectivity and provides a powerful tool for studying the physiological and pathological processes involving PGP.

Activity of soluble aminopeptidase A and dipeptidyl peptidase IV and membrane-bound aminopeptidase B and pyroglutamyl peptidase I in adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis.

OBJECTIVE: To analyze soluble and membrane-bound peptidase activities in the tonsils and adenoids removed from patients with adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis. METHODS: A total of 48 tissue samples from patients undergoing adenoidectomy and tonsillectomy for adenoid hyperplasia, tonsillar hyperplasia or chronic tonsillitis were analyzed. The catalytic activity of a pool of peptidases in the soluble (dipeptidyl peptidase IV, aminopeptidase A, aminopeptidase N and cystinyl aminopeptidase) and membrane-bound (prolyl endopeptidase, aspartyl aminopeptidase, aminopeptidase B and pyroglutamyl peptidase I) fractions was measured fluorometrically. RESULTS: The activity of membrane-bound aminopeptidase B was higher in cases of chronic tonsillitis and adenoid hyperplasia than in tonsillar hyperplasia, p=0.004. Soluble dipeptidyl peptidase IV and membrane-bound pyroglutamyl peptidase I were found to be more active in tissues from male chronic tonsillitis tissues, p<0.05, while membrane-bound aminopeptidase B activity was higher in tissues of females with tonsillar hyperplasia, p<0.001. In the case of chronic tonsillitis, soluble aminopeptidase A was found to have a higher level of activity in tissues from children than those from adults, p=0.005. CONCLUSIONS: Our results suggest a potential role of soluble aminopeptidase A, soluble dipeptidyl peptidase IV, membrane-bound aminopeptidase B and membrane-bound pyroglutamyl peptidase I in the pathobiology of adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis that is differently regulated as a function of gender. These finfings may modify in the future the clinical approach to these diseases.

Continuous spectrometric assays for glutaminyl cyclase activity.

The enzymatic conversion of one chromogenic substrate, l-glutamine-p-nitroanilide, and two fluorogenic substrates, l-glutaminyl-2-naphthylamide and l-glutaminyl-4-methylcoumarinylamide, into their respective pyroglutamic acid derivatives by glutaminyl cyclase (QC) was estimated by introducing a new coupled assay using pyroglutamyl aminopeptidase as the auxiliary enzyme. For the purified papaya QC, the kinetic parameters were found to be in the range of those previously reported for other glutaminyl peptides, such as Gln-Gln, Gln-Ala, or Gln-tert-butyl ester. The assay can be performed in the presence of ammonia up to a concentration of 50 mM. Increasing ionic strength, e.g., potassium chloride up to 300 mM, resulted in an increase in enzymatic activity of about 20%. This is the first report of a fast, continuous, and reliable determination of QC activity, even in the presence of ammonium ions, during the course of protein purification and enzymatic analysis.

Use of pyrrolidonyl peptidase to distinguish Citrobacter from Salmonella.

In the routine testing of foods for Salmonella, Citrobacter and other members of the Enterobacteriaceae often produce colonies which are almost indistinguishable from Salmonella on commonly used selective agars. Biochemical confirmation of such colonies can be expensive and time-consuming. It has been suggested that the enzyme pyrrolidonyl peptidase (PYRase) could be used as a rapid test to distinguish Citrobacter colonies (PYRase-positive) from Salmonella (PYRase-negative). Pure cultures of Salmonella, Citrobacter and other Enterobacteriaceae were tested for PYRase activity; all strains of Salmonella tested were PYRase-negative, and all Citrobacter tested were PYRase-positive. Inoculated and naturally contaminated food samples were tested for the presence of Salmonella by a standard cultural method. A PYR test was used to test Salmonella-like colonies isolated on selective agar and potentially, eliminate PYR-positive isolates from further biochemical testing. The test was able to screen out 6% of colonies selected from samples inoculated with Salmonella, and 43% of colonies selected from uninoculated samples.

Effect of acute lithium administration on pyroglutamyl-aminopeptidase I activity in several brain areas of the rat.

Lithium (CAS 7439-93-2) is widely accepted as an effective treatment in acute mania. A considerable body of literature exists on the effects of lithium on the central nervous system. However, the neurochemical mechanism of the ion action remains still relatively obscure. In the present work, the effect of acute lithium administration on pyroglutamyl-aminopeptidase I activity in discrete areas of the rat brain, including the hypothalamic-pituitary axis, is described. Increases of the enzyme activity in the striatum and the hypothalamus after lithium treatment were observed. It is suggested that this enzyme activity plays a part in the lithium action mechanism, possibly through the regulation of the activity of TRH (Thyrotropin Releasing Hormone) and/or histidyl-proline diketopiperazine.

Pyroglutamyl [correction of Pyroglutamil] -peptidase I activity in the cortex of the cat brain during development.

Thyrotropin Releasing Hormone (TRH) is a principal regulator of thyroid system function. However, significant concentrations of TRH were found throughout the central nervous system, the cortex being one of the areas most richly endowed with thyroliberin. Research concerning the functional role of this brain peptide is performed, in part, by studying peptidase enzymes which may be involved in the inactivation of the peptide. The pGlu-His bond is cleaved by two pyroGlu-peptidases: I (soluble) and II (membrane-bound). In the present investigation, developmental activity of the soluble form is described in the cortices of the cat brain. The selected maturation stages were 15 and 30 days postnatal. The cortices were the frontal, parietal, area 17 and areas 18 and 19 as a whole, distinguishing brain hemispheres in all cases. PyroGlu-aminopeptidase I activity increased significantly with age in all the brain regions except area 17. It is suggested that this enzyme activity plays a part in the neurochemical changes that take place during brain maturation.

Regional distribution of pyroglutamyl peptidase II in rabbit brain, spinal cord, and organs.

Pyroglutamyl peptidase II (PPII) is a narrow specificity ectoenzyme that degrades thyrotropin-releasing hormone (TRH). We detected the enzyme in the brain of various mammals, with highest specific activity in rabbit brain. In this species, activity was heterogeneously distributed in the central nervous system. There was a 28-fold difference between regions of highest and lowest PPII activity. Enzyme activity was highest in the olfactory bulb and posterior cortex. In the spinal cord, activity was low but unevenly distributed, with highest values detected in the thoracic (T) region. Segments T1 and T2 activities were particularly high. Other organs contained low or undetectable levels of activity. The levels of TRH-like immunoreactivity (TRH-LI) in spinal cord segments were greatest in T3-T4 and lumbar L2-L6. Low concentrations were found in T1 and T9-T12. There was a partial correlation between the distribution of PPII activity and TRH receptors but not with TRH-LI levels. These results demonstrate that PPII is predominantly a central nervous system enzyme, and they support the hypothesis that PPII is responsible for degrading TRH released into the synaptic cleft.

Bilateral distribution of aminopeptidase activities in selected structures of a photoneuroendocrine circuit and other rat brain areas.

Provided that soluble aminopeptidases, the most abundant proteolytic enzymes found in brain, are involved in the metabolism of several neuropeptides, their activity could be a reflect of neuropeptide function. Therefore, in order to analyze their rate of participation, we have measured 4 soluble aminopeptidase activities: leucine aminopeptidase, arginine aminopeptidase, aspartate aminopeptidase, and pyroglutamate aminopeptidase, using arylamide derivatives as substrates, in selected structures integrating the photoneuroendocrine circuit related to the melatonin rhythm generating system and other rat brain areas. The regional distribution of all the activities was heterogenous: a 3-fold (leucine-, arginine- and aspartate-aminopeptidase) and 5-fold (pyroglutamate aminopeptidase) difference was observed between the regions with the highest and lowest activity. Significant differences were displayed between the left and right retina for pyroglutamate aminopeptidase and arginine aminopeptidase activities. High levels of pyroglutamate aminopeptidase were evident in the retina and adenohypophysis, which is consistent with a role for thyrotropin releasing hormone in photoreceptive mechanisms, and support its well established role in controlling thyrotropin releasing hormone in anterior pituitary. The presence of a high activity rate of aspartate aminopeptidase in adenohypophysis implies an active participation of angiotensin peptides at this level.

Few-minutes tests for the identification of group A streptococci and enterococci with chromogenic substrates.

The usefulness of paper strip tests for rapid identification of Streptococcus pyogenes and enterococci cultured on blood agar plates was investigated. The paper strips used contain dried chromogenic substrates for pyrrolidonyl peptidase (PYRase) and beta-glucosidase (beta-Gluc). Material from only a few colonies needed to be applied to the test strips. The reactions could be read after two min (PYRase) and 5-7 min (beta-Gluc), respectively. Results from testing 503 streptococcal strains were evaluated. The reactions proved very useful for rapid differentiation of S. pyogenes and enterococci from human sources. Nearly all strains of these streptococcal species showed positive reactions in the PYRase test whereas only the enterococci (E. faecalis and E. faecium) were positive for both enzymes.

Rapid identification of Streptococcus bovis by using combination constitutive enzyme substrate hydrolyses.

Several studies have documented the association of blood and rectal-culture positivity for Streptococcus bovis with gastrointestinal neoplasia, especially colonic carcinoma. Conventional methods using bile-esculin hydrolysis, salt tolerance, and sugar fermentations to differentiate S. bovis from other streptococci are laborious, slow, and relatively expensive. Commercially available systems are costly and require at least 24 to 48 h of incubation. A rapid identification procedure for S. bovis and related bacteria was developed. The method uses a reagent containing two hydrolyzable substrates, p-nitrophenyl-alpha-D-galactopyranoside and 4-methylumbilliferyl-beta-D-glucoside, in the presence of 2.5% sodium deoxycholate. This combination test, performed with a rapid assay for L-pyrrolidonyl-aminopeptidase, could distinguish S. bovis, Streptococcus equinus, Enterococcus spp., Streptococcus pneumoniae, and the viridans group streptococci in culture within 30 min. Twelve species of the genera Streptococcus and Enterococcus were tested. The rapid method correlated well with conventional techniques. The reagents are readily available, inexpensive, and easy to make and can be stored in the refrigerator for at least 6 months.

Occurrence of pyroglutamyl peptidase II, a specific TRH degrading enzyme in rabbit retinal membranes and in human retinoblastoma cells.

Pyroglutamyl peptidase II, a highly specific thyrotropin releasing hormone (TRH)-degrading enzyme is found in highest concentration in brain where it is localized to synaptic membranes. Retina contains relatively high concentrations of both immunoreactive TRH and TRH receptors. We report that the specific activity of pyroglutamyl peptidase II in rabbit retinal membranes exceeds that of all non-CNS tissues thus far studied. Nine clonal cell lines were screened for this enzymatic activity. The specific activity of pyroglutamyl peptidase II in Y79 retinoblastoma cells was greater than the highest activity found in other cell lines by approximately one order of magnitude. These studies further support a functional relationship between pyroglutamyl peptidase II and TRH and identify a cell line suitable for studies on the regulation of this enzyme.

Measurement of active constitutive L-pyrrolidonyl-peptidase from the genera Streptococcus and Enterococcus.

In the family Streptococceae the ability to measure L-pyrrolidonyl-peptidase is limited to Lancefield group D Enterococcus and group A Streptococcus pyogenes. A number of methods exist to assay this enzyme. All measure pyrrolidonyl-peptidase by the ability of the bacterium to cleave L-pyrrolidonyl-beta-naphthylamide to form free beta-naphthylamine and L-pyrrolidone-carboxylic acid. Free beta-naphthylamine is then reacted with N,N-dimethylamino-cinnamaldehyde to form a red color complex. These methods are generally expensive and require a 2-4 h incubation period before results are available. A method, which employs the substrate dried on paper discs and can be easily made, is described herein. It is simple to perform, inexpensive, rapid, and has a long storage life. The results of this constitutive method are equivalent to those obtained using a commercial system.

Regional distribution of the membrane-bound pyroglutamate amino peptidase-degrading thyrotropin-releasing hormone in rat brain.

The brain regional distribution of membrane-bound pyroglutamate aminopeptidase-degrading thyrotropin-releasing hormone (TRH) in rat was studied using a specific radiometric assay. The distribution was not homogeneous: a 10-fold difference was observed between regions. The highest activity was detected in olfactory bulb while the lowest was in the cervical part of spinal cord. There was no correlation with the regional distribution of enzyme activity vs TRH levels, previously reported TRH receptors or in vitro TRH release. The differential distribution of this enzyme is consistent with the hypothesis that it is responsible for extracellular degradation of neuroactive peptides.

Assay of pyroglutamyl aminopeptidase by high-performance liquid chromatography and its use in peptide sequencing.

Assay of pyroglutamyl aminopeptidase by HPLC methodology allows determination of the kinetic parameters of the enzyme for a variety of natural peptide substrates. Moreover, by this method it is possible both to evaluate the extent of the enzymatic reaction by determining the amount of pyrrolidone carboxylic acid released and to obtain the unblocked peptide for structural and functional characterization.

New chromogenic and fluorogenic substrates for pyrrolidonyl peptidase.

L-Pyroglutamyl derivatives of p-nitroaniline and 7-amino-4-methylcoumarin were synthesized as new sensitive substrates for pyrrolidonyl peptidase (pyrrolidonecarboxylyl peptidase) from Bacillus amyloliquefaciens. Their hydrolyses could be followed by conventional colorimetric and fluorometric procedures; i.e., in terms of the increase in absorbance at 410 nm caused by the liberation of p-nitroaniline and the emission at 440 nm after excitation at 370 nm depending on the liberation of 7-amino-4-methylcoumarin. Values of Km were estimated to be 0.69 mM for anilide substrate and 0.33 mM for methylcoumarin substrate in the pyrrolidonyl peptidase reaction at pH 8.0. The methylcoumarin compound was about one thousand fold more sensitive than the anilide substrate.