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PubMed2006

Detection of tripeptidyl peptidase I activity in living cells by fluorogenic substrates.

Tripeptidyl peptidase I (TPP-I) is a lysosomal peptidase with unclear physiological function. TPP-I deficiency is associated with late-infantile neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease of childhood that is characterized by loss of neurons and photoreceptor cells. We have developed two novel fluorogenic substrates, [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110, that are cleaved by TPP-I in living cells. Fluorescence of liberated rhodamine 110 was detected by flow cytometry and was dependent on the level of TPP-I expression. Rhodamine-related fluorescence could be suppressed by preincubation with a specific inhibitor of TPP-I. When investigated by fluorescent confocal microscopy, rhodamine signals colocalized with lysosomal markers. Thus, cleavage of these rhodamide-derived substrates is a marker for mature enzymatically active TPP-I. In addition, TPP-I-induced cleavage of [Ala-Ala-Phe]2-rhodamine 110 could be visualized in primary neurons. We conclude that [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110 are specific substrates for determining TPP-I activity and intracellular localization in living cells. Further, these substrates could be a valuable tool for studying the neuronal pathology underlying classical late-infantile NCL. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Aminopeptidases↗

PubMed2001

Rat tripeptidyl peptidase I: molecular cloning, functional expression, tissue localization and enzymatic characterization.

We purified tripeptidyl peptidase I (TPP I) to homogeneity from a rat kidney lysosomal fraction and determined its physicochemical properties, including its molecular weight, substrate specificity and partial amino acid sequence. The molecular weight of the enzyme was calculated to be 280,000 and 290,000 by non-denaturing PAGE and gel filtration, respectively, and to be 43 000 and 46 000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of six identical subunits. The Km, Vmax, kcat and kcat/Km values of TPP I at optimal pH (pH 4.0) were 680 microM, 3.7 micromol x mg(-1) x min(-1), 33.1 s(-1) and 4.87 x 10(4) s(-1) x M(-1) for Ala-Ala-Phe-MCA, respectively. TPP I was significantly inhibited by PCMBS and HgCl2, and moderately by DFP. These findings also suggest that TPP I is an exotype serine peptidase that is regulated by SH reagent. TPP I released the tripeptide Arg-Val-Tyr from angiotensin III more rapidly than from Ala-Ala-Phe-MCA, and also released Gly-Asn-Leu from neuromedin B with the same velocity as from Ala-Ala-Phe-MCA. Angiotensin III and neuromedin B have recently been found to be good natural substrates for lysosomal TPP I. Furthermore, we determined the rat liver cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRTI-1, is composed of 2485 bp and encodes 563 amino acids in the coding region. By Northern blot analysis, the order for TPP I mRNA expression was kidney > or = liver > heart > brain > lung > spleen >> skeletal muscle and testis. In parallel experiments, the TPP I antigen was detected in various rat tissues by immunohistochemical staining.

Amino Acid Sequence↗

PubMed1992

Purification and immunochemical studies of dipeptidyl peptidase IV from bovine kidney.

DPP IV from bovine kidney has been purified and characterized by molecular mass, pI, pH optimum and sensitivity to inhibitors. Polyclonal antibodies to the enzyme protein were used to show the location of dipeptidyl peptidase IV in microvillar membranes of bovine kidney using immunogold labelling by secondary antibody. The same antibodies reacted to DPP IV in Western blots of rat, pig and bovine kidney tissue, but failed to recognise the enzyme in bovine brain. This suggests that dipeptidyl peptidase IV may exist as different organ-specific rather than species-specific proteins.

Animals↗

PubMed2004

New fluorescent method for the histochemical detection of tripeptidyl peptidase I using glycyl-l-prolyl-l-met-2-anthraquinonyl hydrazide as substrate.

A new substrate for tripeptidyl peptidase I (TPP I; E.C.3.4.14.9)-Gly-L-Pro-L-Met-2-anthraquinonyl hydrazide (Gly-Pro-Met-2-AH) is synthesized and used for the fluorescent histochemical detection of the enzyme. The enzyme liberates low soluble 2-anthraquinonylhydrazine, which-couples quickly with 3-nitrobenzaldehyde (3-NBA) yielding a highly fluorescent water-insoluble hydrazone--3-nitrobenzylidene-2-anthraquinonylhydrazone. The latter compound is localized precisely at sites of enzymatic activity and marks them with a very bright and stable orange-red fluorescence after excitation with conventional monochromatic andlaser green light (lambda(exc)=520-580 nm). The new technique is used successfully for the visualization of the enzyme in tissue sections of different rat organs - and represent the first fluorescent histochemical method for that peptidase.

Aminopeptidases↗

PubMed2006

[Tripeptidyl-peptidase I--distribution, biogenesis, and mechanisms of activation].

Tripeptidyl-peptidase I (TPPI) is an acidic lysosomal peptidase that removes tripeptides from an unmodified N-terminus of small proteins and polypeptides. In humans, TPP I constitutes an integral part of the lysosomal proteolytic apparatus, which, includes numerous hydrolytic enzymes, mostly cysteine proteases (cathepsin B, C, H, K, L, and others), but also serine (cathepsin A) and aspartic (cathepsin D) proteases. The combination of endo- and exopeptidase activities of these enzymes allows for efficient digestion of the diverse proteins transported to the lysosomes, releasing free amino acids and dipeptides that are transported back to the cytoplasm and reused according to the metabolic needs of the cell. The role of TPP I in normal lysosome functioning is underscored by the genetic association of the enzyme with one form of a group of the developmental neurodegenerative disorders of childhood--the neuronal ceroid lipofuscinoses (NCLs). The scope of this article is to review the most recent data, mostly from author's laboratory, on the biology and pathology of TPP I. NCLs are also shortly reviewed with the special emphasis on CLN2 form resulting from mutations in TPP I gene.

Aminopeptidases↗

PubMed1991

Membrane-bound peptidases of lymphocytes: functional implications.

The paper is aimed towards the role of the membrane ecto-enzymes aminopeptidase N (CD13, AP-N) and dipeptidyl peptidase IV (CD26, DP IV) in the immune system. Both peptidases have been identified on T lymphocytes, AP-N also on monocytes and non-T cells. Using enzyme inhibitors and antibodies it has been shown by different groups that DP IV plays a key role in T cell activation and growth. Inhibition studies of our laboratory, using bestatin, actinonin and probestin, also demonstrated an essential role of AP-N in regulation of T cell growth. Moreover, the action of cytokines/lymphokines having DP IV susceptible bonds, as IL-1, IL-2 and IL-6, on lymphocyte proliferation, was found to be suppressed by specific inhibitors of DP IV as well as AP-N. These results are in favour of our hypothesis that DP IV and AP-N, possibly both in a concerted action, are involved in cytokine/lymphokine mediated signalling between immune cells.

Amino Acid Sequence↗

PubMed1990

[Histochemistry of selected peptidases in small intestine mucosa in piglets experimentally infected with Isospora suis].

In the small intestine mucosa of 24 gnotobiotical piglets experimentally infected the first day post partum with oocysts of the coccidium Isospora suis, the activities of dipeptidylpeptidase IV (EC.3.4.14.5.; DAP IV) and gamma-glutamyl transferase (EC.2.3.2.2.; GGT) in the microvillous zone of enterocytes were evaluated by scanning densitometry. The tissue of the small intestine in piglets infected with a dose of 100,000 oocysts of the coccidia of I. suis was examined in the period from the first till the eleventh day post infection (DPI). In the control piglets at the age of 2-5 days it was found that most of the DAP IV activity was located in the microvillous zone of the enterocytes of the middle jejunum, rear jejunum and ileum. The DAP IV activity of duodenum mucosa was lower; as compared with the activity in the mucosa of the jejunum and ileum it reached 53-57%. In the case of GGT activity, the highest density values of the reaction product were recorded in the microvillous zone of enterocytes of the duodenum and the whole jejunum, the lowest in the ileum mucosa (86-89%) of the activity found in the duodenum and jejunum). During the experimental infection the infected piglets had a significant deficit of both peptidases, especially DAP IV (the whole studied period). The development of GGT activity was slightly different with the onset of the marked decline of the enzyme activity only on the fifth DPI. The lower GGT activity persisted till the eighth DPI. The density of the GGT reaction product began to return to the normal on the ninth to eleventh DPI. No predisposition in the location of the deficit was observed in the peptidases studied during the infection. The decline of the activity of both enzymes influenced also the mucosa of all studied parts of the small intenstine. The difference lay in the relevance of lowering of the density of reaction product of DAP IV and GGT on other DPI and in the different intensities of the return of the activity to the physiological normal.

Animals↗

PubMed1985

Purification and properties of human placental dipeptidyl peptidase IV.

Dipeptidyl peptidase IV (EC 3.4.14.5; Gly-Pro-p-nitroanilidase) was purified 1840-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100 and subjected to zinc acetate fractionation, hydroxylapatite chromatography, Sephacryl S-300 chromatography and then affinity chromatographies with Lentil Lectin-Sepharose 4B and Gly-Pro-NH-(CH2)6-NH-Sepharose 4B. Dipeptidyl peptidase IV was completely separated from leucine aminopeptidase by the final affinity chromatography. The apparent molecular weight of the enzyme was estimated to be 350,000 by gel filtration. The purified enzyme gave a single band with a weight of 124,000 with sodium dodecyl sulfate (SDS) gel-electrophoresis, suggesting that the enzyme consists of three subunits. The isoelectric point of the enzyme was 4.4. The purified enzyme was most active at pH 8.0 with Gly-Pro-p-nitroanilide as substrate and the Km value for this substrate was 2.27mM.

Chromatography, Affinity↗

PubMed1987

Stepwise degradation of the hexapeptide Met-Ala-Ser-Pro-Phe-Ala by dipeptidyl peptidase IV.

Dipeptidyl peptidase IV (dipeptidyl-peptide hydrolase, EC 3.4.14.-) purified from pig kidney was proved to split off the N-terminal dipeptide Met1-Ala2 and, subsequently the dipeptide Ser3-Pro4 from the synthetic model peptide Met-Ala-Ser-Pro-Phe-Ala representing the N-terminal part of a signal sequence of leucocyte interferon. The kinetic parameters for the release of Met1-Ala2 (Km 3.2.10(-5) mol.l-1 and Ser3-Pro4 (Km 1.65.10(-4) mol.l-1, kcat57.9 s-1) were determined. The dipeptides Ser-Pro and Phe-Ala were found to be competitive inhibitors of the hydrolysis of Gly-Pro-NHNp by dipeptidyl peptidase IV.

Animals↗

PubMed1988

Activity of dipeptidyl peptidase IV and post-proline cleaving enzyme in sera from osteoporotic patients.

The activity of dipeptidyl peptidase IV (EC 3.4.14.5) in human sera from normal controls and osteoporotic patients was assayed with Gly-Pro-4-methylcoumaryl-7-amide (Gly-Pro-MCA) as substrate, at pH 8.7. The mean normal value for the activity in serum of 120 healthy subjects (ages 20-86 y) was 54.0 (SE 0.9, range 34.3-81.6) U/L at 37 degrees C and differed slightly but significantly between the younger group (less than 50 y old) and those older than 50 y. Values for the younger men were slightly but significantly higher than for the older men. Overall, however, the enzyme activities in serum were above normal in patients with osteoporosis. In contrast, the activity of post-proline cleaving enzyme (PPCE) was not increased in serum from patients with osteoporosis, as determined with succinyl-Gly-Pro-4-methylcoumaryl-7-amide (Suc-Gly-Pro-MCA) as substrate. These results suggest that high activities of dipeptidyl peptidase IV in serum of patients with osteoporosis are probably related to its severity.

Adult↗

PubMed1988

Dipeptidyl peptidases of human lymphocytes.

In human lymphocytes three dipeptidyl peptidases were discovered in our laboratory. For a correct demonstration of activities of these enzymes discriminating substrates must be used. Dipeptidyl peptidase IV (DPP IV) is revealed with Gly-Pro-4-methoxy-2-naphthylamide (Gly-Pro-MNA) and Fast Blue B (FBB). It is present in the surface membrane of about 40% lymphocytes of the peripheral blood. Only T-lymphocytes bear the reaction. Reacting lymphocytes belong predominantly to OKT4+ subset. Some OKT8+ lymphocytes also react. With more sensitive substrates (Lys-Pro-MNA, Phe-Pro-MNA and Ala-Pro-MNA) a co-reaction of DPP II was demonstrated "in situ" and in zymograms. In haemoblastoses a positive reaction in cells indicates their derivation from the T-lineage of lymphocytes. A negative reaction does not exclude a T-cell malignancy, however. A decreased number of DPP IV positive lymphocytes in the peripheral blood indicates a diminished immunocompetent potential of T-cells, e.g. immunodeficiency in patients with malignant lymphoma, gastric and colocrectal carcinoma, AIDS, etc. DPP II demonstrated with Lys-Ala-MNA occurs in about 60% of lymphocytes belonging to T and B subsets. It is localized in lysosomes. Although Lys-Pro-MNA is a more sensitive substrate a co-reaction of DPP IV must always be considered. Patients with chronic B-lymphocytic leukaemia displaying a high number of DPP II+ cells usually have a worse prognosis. DPP I assessed with Gly-Pro-MNA and nitrosalicylaldehyde occurs in about 20% of T and B lymphocytes. The number of positively reacting cells increases after corticosteroid therapy. The influence of the treatment on the activity can be shown very well in histograms of DPP I activity measured by computer-assisted microfluorometry.

Cathepsin C↗

PubMed1985

Proteinase-like peptidase activities in malignant and non-malignant gastric tissue.

Activities of several proteinase-like peptidases have been determined in homogenates of malignant tissue, non-malignant tissue adjacent to the tumour (A-NM) and non-malignant tissue distant to the tumour (D-NM) from 17 patients undergoing surgery for histologically confirmed gastric malignancies. In homogenates of malignant tissues the activities of collagenase, cathepsin B, cathepsin (B+L), cathepsin H and cathepsin D were significantly higher than in D-NM tissues. By contrast, the levels of plasminogen activator were significantly lower in malignant tissues than in the D-NM tissues. Furthermore, the activities of collagenase-like and the cysteine-proteinase-like peptidases in the A-NM tissues were lower than in malignant tissues but higher than in the D-NM tissues. Separation of full-thickness non-malignant tissues into mucosal and seromuscular layers revealed significantly higher activities in the former. The elevated levels of these proteinase-like peptidases in homogenates of gastric cancer tissue suggests an important role for these enzymes in tumour invasion.

Aged↗

PubMed1986

The protein inhibitors of amylases and peptidases isolated from cereal grains.

The peptidase and amylase inhibitors were isolated from rye and preliminarily purified to investigate some of their properties. It was shown, that both are of albumin character despite the acetic acid solubility of the former. They are not active against the native cereal grain peptidases or amylases (rye, wheat, triticale) but the latter inhibited the animal (hog pancreas, man salivary) and bacterial (Thermamyl, NOVO) alpha-amylases. In the long-term experiments on various rye genotypes there was shown, that the peptidase inhibitor level was rather differentiated between them, but more stable as concerns particular ones. On the other hand the level differentiation of alpha-amylase inhibitor between the genotypes was rather small, but that of climate much more significant.

Amylases↗

PubMed1998

Peptidase activities of the multicatalytic protease in rat liver after voluntary and intragastric ethanol administration.

Ethanol consumption slows down the rate of hepatic protein catabolism. The present study was conducted to determine whether ethanol consumption, given by voluntary (pair) feeding or by intragastric administration, affected the peptidase activities of the proteasome in rat liver. Rats were pair-fed liquid diets containing either ethanol or isocaloric maltose-dextrin. A separate group of animals was intragastrically infused continuously with similar liquid diets containing either ethanol or isocaloric dextrose. Crude liver homogenates and their cytosolic fractions were assayed for their chymotrypsin-like (Cht-L), trypsin-like (T-L), and peptidyl-glutamyl-peptide hydrolase (PGPH) activities, using specific fluorogenic peptides as substrates. Voluntary ethanol feeding did not affect the three peptidase activities of the proteasome. However, intragastric ethanol administration caused a 35% to 40% decline in the Cht-L and the T-L activities, but did not significantly change the PGPH activity. The lower peptidase activities in cytosol samples from intragastrically ethanol-fed rats were not restored to control levels by overnight dialysis, nor by the inclusion of low levels of sodium dodecyl sulfate (SDS) or of 0.5 mmol/L adenosine triphosphate (ATP) in the proteasome assay mixture. Immunoblot analyses using anti-rat liver proteaseome exhibited equal levels of immunoreactive proteasome subunits in livers of control and ethanol-fed rats. Similar results were obtained when blots were probed with antibody made specifically against the proteasome subunit, LMP-7. The results indicate that intragastric, but not voluntary, ethanol consumption differentially affects the separate catalytic activities of the proteasome without affecting its steady-state levels. Such changes may be related to the degree of ethanol-induced oxidative stress.

Animals↗

PubMed1979

Peptidase activity in Tetrahymena.

This report strongly suggests that two compartments in Tetrahymena thermophila contain peptidase activity: the cytoplasm and the outer cell surface. Determinations of amino acid concentrations in the extracellular medium upon incubation of cells with peptides suggest that the surface-bound peptidase activity hydrolyses di- and tri-phenylalanine equally fast on a molar basis. Growth experiments designed to characterize the in vivo peptidase specificities showed that both T. thermophila and T. pyriformis can use L-leucyl-L-leucine, but not L-leucyl-D-leucine as a leucine donor. These results are independent of whether the cells form food vacuoles or not.

Animals↗

PubMed1982

Peptidase isozymes of the leopard frog Rana pipiens: properties and genetics.

The substrate specificity of five peptidases in tissues of the leopard frog, Rana pipiens, corresponds to mammalian peptidases (Pep) A, B, C, D, and S. The inheritance of electrophoretic variants of Pep A, B, C, and D verifies the genetic basis of the variants and the control of the enzymes by separate genetic loci. Electrophoretic patterns of heterozygotes suggest that Pep A, B, and D are dimers, whereas Pep C has a monomer structure. The tissue distribution and developmental patterns suggest that frog peptidases are subject to cellular regulation. Tests of genetic linkage show independent assortment for Pep A and Pep C and for Pep C and Pep D. Pep C is closely linked to superoxide dismutase-1 (6.87% recombination), and Pep B is linked to mannose phosphate isomerase (30.4% recombination).

Aminopeptidases↗

PubMed1995

Crystallization of a soluble, catalytically active form of Escherichia coli leader peptidase.

Leader peptidase, a novel serine protease in Escherichia coli, catalyzes the cleavage of the amino-terminal leader sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Here, we report a procedure for the purification and the crystallization of a soluble non-membrane-bound form of leader peptidase (delta 2-75). Crystals were obtained by the sitting-drop vapor diffusion technique using ammonium dihydrogen phosphate as the precipitant. Interestingly, we have found that the presence of the detergent Triton X-100 is required to obtain crystals sufficiently large for X-ray analysis. The crystals belong to the tetragonal space group P4(2)2(1)2, with unit cell dimensions of a = b = 115 A and c = 100 A, and contain 2 molecules per asymmetric unit. This is the first report of the crystallization of a leader (or signal) peptidase.

Crystallization↗

PubMed1995

Families and clans of serine peptidases.

It has become clear that the proteolytic enzymes that depend upon a serine residue for their catalytic activity belong to many different families of proteins. We have attempted to group these families in "clans." A clan is defined as a group of families the members of which have a common ancestor. That is to say, they are homologous, although some of the similarities are in the "twilight zone" and the relationships cannot be proven by rigorous statistical methods. We believe we can recognize five, or perhaps six, clans of serine peptidases. In view of the separate evolutionary origins of the serine peptidases in different clans, it is not surprising to find that they may differ greatly and that indeed there are few generalizations that can be applied to all serine peptidases. In contrast, the members of a clan commonly have marked similarities.

Amino Acid Sequence↗