Continuous assays for meprin alpha and beta using prolyl tripeptidyl aminopeptidase (PtP) from Porphyromonas gingivalis.

Common assays for endoprotease activity of meprin α and ß are based on cleavage of internally quenched substrates. Although direct and convenient, for meprins these assays bear disadvantages such as, e.g., significant substrate inhibition or potential fluorescence quenching by compounds applied in inhibitor analysis. Here, we present a novel continuous assay by introducing an auxiliary enzyme, prolyl tripeptidyl aminopeptidase (PtP) and the chromogenic substrate KKGYVADAP-p-nitroanilide. We provide a quick strategy for expression and one-step-purification of the auxiliary enzyme. The enzyme kinetic data for meprin α and ß suggest hyperbolic v/S-characteristics, the kinetic parameters of substrate conversion by meprin ß were Km = 184 ±â€¯32 µM and kcat = 20 ±â€¯4 s-1. We also present conditions for the use of the fluorogenic substrate KKGYVADAP-AMC to assess meprin ß activity. The assays were applied for determination of inhibitory parameters of the natural inhibitor actinonin and two recently published hydroxamates. Hence, we present two novel methods, which can be applied to assess inhibitory mechanism and potency with the attractive current drug targets meprin α and ß. Furthermore, the assay might also provide implications for analysis of other endoproteases as well as their inhibitors.

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells.

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G0/G1 cell cycle arrest and increased levels of the CDK inhibitor p27(kip1) and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G0/G1 cell cycle phase arrest and increased levels of p27(kip1) in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G0 state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.

An Arabidopsis FAD pyrophosphohydrolase, AtNUDX23, is involved in flavin homeostasis.

Although flavins, riboflavin (RF), FMN and FAD, are essential for primary and secondary metabolism in plants, the metabolic regulation of flavins is still largely unknown. Recently, we found that an Arabidopsis Nudix hydrolase, AtNUDX23, has FAD pyrophosphohydrolase activity and is distributed in plastids. Levels of RF and FAD but not FMN in Arabidopsis leaves significantly increased under continuous light and decreased in the dark. The transcript levels of AtNUDX23 as well as genes involved in flavin metabolism (AtFADS, AtRibF1, AtRibF2, AtFMN/FHy, LS and AtRibA) significantly increased under continuous light. The pyrophosphohydrolase activity toward FAD was enhanced in AtNUDX23-overexpressing (OX-NUDX23) plants and reduced in AtNUDX23-suppressed (KD-nudx23) plants, compared with the control plants. Interestingly intracellular levels of RF, FMN and FAD significantly decreased in not only OX-NUDX23 but also KD-nudx23 plants. The transcript levels of the flavin metabolic genes also decreased in both plants. Similarly, the increase in intracellular levels on treatment with flavins caused a reduction in the transcript levels of genes involved in flavin metabolism. These results suggest that negative feedback regulation of the metabolism of flavins through the hydrolysis of FAD by AtNUDX23 in plastids is involved in flavin homeostasis in plant cells.

Isoquinoline alkaloid biosynthesis is regulated by a unique bHLH-type transcription factor in Coptis japonica.

Specific plant species produce unique isoquinoline alkaloids (IQAs); however, the mechanism of their evolution and the regulation of their biosynthesis are largely unknown. We report here the isolation of a novel basic helix-loop-helix protein, CjbHLH1, from IQA-producing Coptis japonica. A BLAST search indicated that CjbHLH1 homologs were only found in plant species that produce IQAs. Transient RNA interference (RNAi) and overexpression of CjbHLH1 in C. japonica protoplasts revealed the activity of CjbHLH1 in transcription of IQA biosynthetic genes, and little activity in the transcription of genes involved in primary metabolism or the stress response. A chromatin immunoprecipitation experiment using CjbHLH1-specific antibodies revealed the direct interaction of CjbHLH1 with promoter sequences of IQA biosynthetic genes in vivo. We discuss the unique role of CjbHLH1 in IQA biosynthesis.

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line.

Prolyl oligopeptidase (POP) is a post-proline cleaving enzyme, which is widely distributed in various organs, with high levels in the brain. In this study, we investigated the effects of a selective POP inhibitor, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746), on the growth of NB-1 human neuroblastoma cells. SUAM-14746 treatment for 24-72 h suppresses the growth of NB-1 cells without cell death in a dose-dependent manner (10-60 µM). Similar suppressive effects were observed with another POP inhibitor benzyloxycarbonyl-thioprolyl-thioprolinal. The SUAM-14746-induced growth inhibition in NB-1 cells was associated with pronounced G(0)/G(1) arrest and reduced levels of phosphorylated retinoblastoma protein (pRb), cyclin E, and cyclin dependent kinase (CDK) 2, and increased levels of the CDK inhibitor p27(kip1) and the tumor suppressor p53. SUAM-14746 also induced transient inhibition of S and G(2)/M phase progression, which was correlated with retardation of the decrease in the levels of cyclins A and B. Moreover, RNAi-mediated knockdown of POP also led to inhibition of NB-1 cell growth and the effect was accompanied by G(0)/G(1) arrest. These results indicate that POP is a part of the machinery that controls the cell cycle.

Structure of aminopeptidase N from Escherichia coli complexed with the transition-state analogue aminophosphinic inhibitor PL250.

Aminopeptidase N (APN; EC 3.4.11.2) purified from Escherichia coli has been crystallized with the optically pure aminophosphinic inhibitor PL250, H(3)N(+)-CH(CH(3))-P(O)(OH)-CH(2)-CH(CH(2)Ph)-CONH-CH(CH(2)Ph)CO(2)(-), which mimics the transition state of the hydrolysis reaction. PL250 inhibits APN with a K(i) of 1.5-2.2 nM and its three-dimensional structure in complex with E. coli APN showed its interaction with the S(1), S'(1) and S'(2) subsites of the catalytic site. In this structure, the Zn ion was shown to be pentacoordinated by His297, His301 and Glu320 of APN and the two O atoms of the phosphinic moiety of PL250. One of these O atoms is also involved in a hydrogen bond to Tyr381, supporting the proposed role of this amino acid in the stabilization of the transition state of the enzymatic process. The strength of the phosphinic zinc binding and the occupancy of the S'(2) subsite account for the 100-fold increase in affinity of PL250 compared with the dipeptide-derived inhibitor bestatin (K(i) = 4.1 x 10(-6) M). Accordingly, the removal of the C-terminal phenylalanine of PL250 resulted in a large decrease in affinity (K(i) = 2.17 x 10(-7) M). Furthermore, it was observed that the C-terminal carboxyl group of the inhibitor makes no direct interactions with the amino acids of the APN active site. Interestingly, PL250 exhibits the same inhibitory potency for E. coli APN and for mammalian enzymes, suggesting that the structure of the complex could be used as a template for the rational design of various human APN inhibitors needed to study the role of this aminopeptidase in various pathologies.

Dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia exhibits activity against a substrate containing a 4-hydroxyproline residue.

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.

A cysteine residue near the propionate side chain of heme is the radical site in ascorbate peroxidase.

The radical scavenger 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used in conjunction with mass spectrometry to identify the protein-based radical sites of the H(2)O(2)-tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H(2)O(2)-sensitive stromal APX of tobacco. A cysteine residue in the vicinity of the propionate side chain of heme in both enzymes was labeled with TEMPO(*) and DMPO in an H(2)O(2)-dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H(2)O(2). TEMPO(*) bound to the cysteine thiyl radicals, and sulfinylated and sulfonylated them. Other oxidized cysteine residues were found in both APXs. Experiments with a cysteine-to-serine point mutation showed that formation of TEMPO adducts and subsequent oxidation of the cysteine residue located near the propionate group of heme leads to loss of enzyme activity, in particular in the Galdieria APX. When treated with glutathione and H(2)O(2), both cysteine residues in both enzymes were glutathionylated. These results suggest that, under oxidative stress in vivo, cysteine oxidation is involved in the inactivation of APXs in addition to the proposed H(2)O(2)-mediated crosslinking of heme to the distal tryptophan residue [Kitajima S, Shimaoka T, Kurioka M & Yokota A (2007) FEBS J274, 3013-3020], and that glutathione protects APX from irreversible oxidation of the cysteine thiol and loss of enzyme activity by binding to the cysteine thiol group.

D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme.

The gene coding for d-3-hydroxybutyrate dehydrogenase (HBDH) was cloned from Pseudomonas fragi. The nucleotide sequence contained a 780 bp open reading frame encoding a 260 amino acid residue protein. The recombinant enzyme was efficiently expressed in Escherichia coli cells harboring pHBDH11 and was purified to homogeneity as judged by SDS-PAGE. The enzyme showed a strict stereospecificity to the D-enantiomer (3R-configuration) of 3-hydroxybutyrate as a substrate. Crystals of the ligand-free HBDH and of the enzyme-NAD+ complex were obtained using the hanging-drop, vapor-diffusion method. The crystal structure of the HBDH was solved by the multiwavelength anomalous diffraction method using the SeMet-substituted enzyme and was refined to 2.0 A resolution. The overall structure of P.fragi HBDH, including the catalytic tetrad of Asn114, Ser142, Tyr155, and Lys159, shows obvious relationships with other members of the short-chain dehydrogenase/reductase (SDR) family. A cacodylate anion was observed in both the ligand-free enzyme and the enzyme-NAD+ complex, and was located near the catalytic tetrad. It was shown that the cacodylate inhibited the NAD+-dependent D-3-hydroxybutyrate dehydrogenation competitively, with a Ki value of 5.6 mM. From the interactions between cacodylate and the enzyme, it is predicted that substrate specificity is achieved through the recognition of the 3-methyl and carboxyl groups of the substrate.

Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis.

The crystal structure of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis was determined. Prolyl tripeptidyl aminopeptidase consists of beta-propeller and catalytic domains, and a large cavity between the domains; this structure is similar to dipeptidyl aminopeptidase IV. A catalytic triad (Ser603, His710, and Asp678) was located in the catalytic domain; this triad was virtually identical to that of the enzymes belonging to the prolyl oligopeptidase family. The structure of an inactive S603A mutant enzyme complexed with a substrate was also determined. The pyrrolidine ring of the proline residue appeared to fit into a hydrophobic pocket composed of Tyr604, Val629, Trp632, Tyr635, Tyr639, Val680, and Val681. There were characteristic differences in the residues of the beta-propeller domain, and these differences were related to the substrate specificity of tripeptidyl activity. The N-terminal amino group was recognized by salt bridges, with two carboxyl groups of Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the Glu205 (located in the loop) and Glu636 were found to carry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues (Xaa-Xaa-Pro) of substrates. This is the first report of the structure and substrate recognition mechanism of tripeptidyl peptidase.

[Biochemistry and structural biology of microbial enzymes and their medical applications].

Microbial enzymes were studied from two medicinal viewpoints. First, we examined proline-specific peptidases from pathogenic microorganisms. We found several proline-specific peptidases in pathogenic bacteria. Among them, prolyl tripeptidyl aminopeptidase from Porphylomonas gingivals and prolyl aminopeptidase from Serratia marcescens were crystallized. The complex structures of those enzymes and inhibitors were clarified in X-ray crystallography. Aminopeptidase N, which has wide specificity for amino acids, was distributed in the pathogens. The crystal structure of the aminopeptidase N elucidated the reasons for its wide substrate specificity but inertness to the X-Pro bond. It was also revealed that proline-specific peptidases and aminopeptidase N cooperatively degrade collagen for the uptake of amino acids as nutrition when these bacteria infect cells. Second, we applied enzymes from microorganisms to diagnostic analyses. We found a series of creatinine-metabolizing enzymes in Pseudomonas putida. Creatininase, creatinase, and sarcosine oxidase were coupled and have been developed for a diagnostic analysis kit that examines renal function. The structures of the native and the Mn2+-activated creatininases were determined in X-ray crystallography. Based on the structure, the activated enzyme was used for an improved assay kit. The structure of D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi was also clarified in crystallography. The enzyme is useful for diagnostic analysis of diabetes mellitus while monitoring ketone bodies.

Crystallization and preliminary X-ray characterization of aminopeptidase N from Escherichia coli.

A recombinant form of aminopeptidase N (molecular weight 99 kDa) from Escherichia coli was crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitating agent. The crystals belong to the hexagonal space group P3(1)21, with unit-cell parameters a = b = 120.5, c = 171.0 angstroms. The crystals are most likely to contain one molecule in the asymmetric unit, with a V(M) value of 3.62 angstroms3 Da(-1). Diffraction data were collected to 2.0 angstroms resolution using Cu Kalpha radiation from a rotating-anode X-ray generator.

Characterization of the Promoter Region of Biosynthetic Enzyme Genes Involved in Berberine Biosynthesis in Coptis japonica.

The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs), a plant-specific WRKY-type TF, CjWRKY1, and a basic helix-loop-helix TF, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4'OMT and CYP719A1) were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC) reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay and by a chromatin immunoprecipitation assay. In addition, CjbHLH1 also activated transcription from truncated 4'OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. japonica are discussed.

Oral Administration of Collagen Hydrolysates Improves Glucose Tolerance in Normal Mice Through GLP-1-Dependent and GLP-1-Independent Mechanisms.

The aim of this study was to evaluate the antidiabetic properties of collagen hydrolysates (CHs). CHs exhibited dipeptidyl peptidase-IV inhibitory activity and stimulated glucagon-like-peptide-1 (GLP-1) secretion in vitro. We also determined whether CHs improve glucose tolerance in normal mice. Oral administration of CHs suppressed the glycemic response during the oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT), but the effects were weaker in IPGTT than in OGTT. CHs had no effect on the gastric emptying rate. A pretreatment with the GLP-1 receptor antagonist, exendin 9-39 (Ex9), partially reversed the glucose-lowering effects of CHs, but only when coadministered with glucose. CHs administered 45 min before the glucose load potentiated the glucose-stimulated insulin secretion. This potentiating effect on insulin secretion was not reversed by the pretreatment with Ex9, it appeared to be enhanced. These results suggest that CHs improve glucose tolerance by inhibiting intestinal glucose uptake and enhancing insulin secretion, and also demonstrated that GLP-1 was partially involved in the inhibition of glucose uptake, but not essential for the enhancement of insulin secretion.

Crystal structure of formaldehyde dehydrogenase from Pseudomonas putida: the structural origin of the tightly bound cofactor in nicotinoprotein dehydrogenases.

Formaldehyde dehydrogenase from Pseudomonas putida (PFDH) is a member of the zinc-containing medium-chain alcohol dehydrogenase family. The pyridine nucleotide NAD(H) in PFDH, which is distinct from the coenzyme (as cosubstrate) in typical alcohol dehydrogenases (ADHs), is tightly but not covalently bound to the protein and acts as a cofactor. PFDH can catalyze aldehyde dismutations without an external addition of NAD(H). The structural basis of the tightly bound cofactor of PFDH is unknown. The crystal structure of PFDH has been solved by the multiwavelength anomalous diffraction method using intrinsic zinc ions and has been refined at a 1.65 A resolution. The 170-kDa homotetrameric PFDH molecule shows 222 point group symmetry. Although the secondary structure arrangement and the binding mode of catalytic and structural zinc ions in PFDH are similar to those of typical ADHs, a number of loop structures that differ between PFDH and ADHs in their lengths and conformations are observed. A comparison of the present structure of PFDH with that of horse liver ADH, a typical example of an ADH, reveals that a long insertion loop of PFDH shields the adenine part of the bound NAD(+) molecule from the solvent, and a tight hydrogen bond network exists between the insertion loop and the adenine part of the cofactor, which is unique to PFDH. This insertion loop is conserved completely among the aldehyde-dismutating formaldehyde dehydrogenases, whereas it is replaced by a short turn among typical ADHs. Thus, the insertion loop specifically found among the aldehyde-dismutating formaldehyde dehydrogenases is responsible for the tight cofactor binding of these enzymes and explains why PFDH can effectively catalyze alternate oxidation and reduction of aldehydes without the release of cofactor molecule from the enzyme.

Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism.

Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.

Crystallization and preliminary X-ray characterization of D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi.

A recombinant form of D-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) from Pseudomonas fragi has been crystallized by the hanging-drop method using PEG 3000 as a precipitating agent. The crystals belong to the orthorhombic group P2(1)2(1)2, with unit-cell parameters a = 64.3, b = 99.0, c = 110.2 A. The crystals are most likely to contain two tetrameric subunits in the asymmetric unit, with a VM value of 3.29 A3 Da(-1). Diffraction data were collected to a 2.0 A resolution using synchrotron radiation at the BL6A station of the Photon Factory.

Crystallization and preliminary X-ray characterization of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis.

A recombinant form of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis has been crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as a precipitating agent. The crystals belong to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 149.4, c = 159.7 A. The crystals are most likely to contain one subunit of a dimer in the asymmetric unit, with a VM value of 3.14 A3 Da(-1). Diffraction data were collected to 2.1 A resolution using synchrotron radiation at the BL5 station of the Photon Factory.

Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning.

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.

Localization of thymidine phosphorylase in advanced gastric and colorectal cancer.

Thymidine phosphorylase (TP) is known to be more concentrated in human cancer tissues than in adjacent normal tissue based on findings using enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. However, the ultrastructural localization of TP in cancer tissues has not previously been demonstrated. We investigated the localization of TP in gastric cancer and colorectal cancer tissue by ELISA, immunohistochemistry, and immunoelectron microscopy. Between April 1997 and May 2000, we obtained surgically resected specimens from 42, 46, and 36 cases of advanced gastric, colon, and rectal cancer, respectively. ELISA demonstrated that the TP level was higher in cancer tissues than in adjacent normal tissue. Immunohistochemically, cancer cells were positive for the enzyme in some cases. However, in a number of cases immunopositive inflammatory cells were also present in cancerous tissues. At the electron microscope level, TP was diffusely distributed in the cytoplasm of cancer cells and in the mitochondria of the neutrophil in gastric cancer tissue. In rectal cancer tissues, cytoplasmic granules in macrophages in cancer tissues were immunoreactive for the TP. These findings suggest that TP is produced by macrophages and exists in neutrophils and cancer cells.