Katsuhiko Fukasawa

Successive cleavage of N-terminal Arg1--Pro2 and Lys3-Pro4 from substance P but no release of Arg1-Pro2 from bradykinin, by X-Pro dipeptidyl-aminopeptidase.

X-Pro dipeptidyl-aminopeptidase (EC 3.4.14.1) purified homogeneously from the human submaxillary gland was proved to hydrolyze N-terminal dipeptide Arg1-Pro2 and subsequent dipeptide Lys3-Pro4 from substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-gly-Leu-Met-NH2). Km and V values of hydrolysis of substance P were 2.0 mM and 3.6 mumol/min per mg protein, respectively. In contrast, the N-terminal Arg-Pro of bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) was not cleaved by the enzyme.

Dipeptidyl aminopeptidase IV, a glycoprotein from pig kidney

Dipeptidyl aminopeptidase IV was purified 350 fold from pig kidney by chromatographic procedures including affinity chromatography with conjugates of Gly-Pro linked to Sepharose 4.B. Purified enzyme existed in a dimeric form as determined by sodium dodecyl sulfate polyacrylamide-gel electrophoresis using dimethyl suberimidate (a cross-linking reagent). The molecular weight of the subunit was estimated to be 100 000 by gel filtration with 6 M guanidine hydrochloride and to be 94 000 based on analysis of N-terminal residue (dinitrophenyl-serine). The amino acid composition of the purified enzyme was also determined. The enzyme contained 18.3% of carbohydrate consisting of mannose, galactose, fucose, glucosamine and sialic acid. The enzyme desialized with sialidase was found to still possess full enzyme activity.

Comparison of dipeptidyl peptidase IV prepared from pig liver and kidney

Dipeptidyl peptidase IV (dipeptidylpeptide hydrolase, EC 3.4.14.-) has been purified from the microsomal fraction of pig liver, using an immunoaffinity chromatography, and its properties compared with those of the enzyme purified from pig kidney. The amino acid compositions of both enzymes were similar. The same kinds of carbohydrates were found in both enzymes, but there were differences in the molar concentrations of individual sugars. The liver enzyme had greater concentrations of mannose, fucose and sialic acid than the kidney enzyme, while the concentrations of galactose and glucosamine were greater in the kidney enzyme. The carbohydrates accounted for approx. 18.3 and 22.7% of the weight of the kidney and liver enzymes, respectively. The pH optima, molecular weights, substrate specificities and Km values of the two enzymes and the effects of diisopropylfluorophosphate on their activities were nearly identical. The liver enzyme was heat- and pH-sensitive, but not attacked by proteinases.

A sensitive and specific assay for dipeptidyl-aminopeptidase II in serum and tissues by liquid chromatography-fluorometry

A highly sensitive and specific method for the assay of dipeptidyl-aminopeptidase II (DAP II) in crude enzyme preparations such as serum and tissue homogenates has been established by using a newly synthesized fluorogenic substrate, 7-Lys-Ala-4-methylcoumarinamide. The enzymatically formed 7-amino-4-methylcoumarin was determined by high-performance liquid chromatography with fluorescence detection. The activities of other aminopeptidases in human serum and rat brain homogenates were completely inhibited by o-phenanthroline without any effect on DAP II activity to permit specific determination of DAP II. The limit of sensitivity for DAP II activity was about 300 fmol/30 min. DAP II activity was found to be increased in sera from cancer patients, in contrast to the decrease in serum DAP IV activity. DAP II activity was found to be unequally distributed in rat brain regions, and the highest activity was found in the hypothalamus.

Molecular cloning and expression of rat liver aminopeptidase B.

We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-β-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl−-dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.

Aminopeptidase B is structurally related to leukotriene-A4 hydrolase but is not a bifunctional enzyme with epoxide hydrolase activity.

Aminopeptidase B (Ap B; EC 3.4.11.6) is a zinc-binding protein that contains the consensus sequence HEXXHX18E (324-347), conserved among the M1 family of metallopeptidases. To determine if these putative zinc-binding residues (His324, His328 and Glu347) and the active-site Glu325 are essential for the enzyme activity, we replaced the histidines with tyrosines and the glutamic acid residues with alanines using site-directed mutagenesis. The cDNAs were expressed in Escherichia coli, and the resulting recombinant proteins, named H324Y, E325A, H328Y and E347A, were purified to apparent homogeneity. None of the expressed mutated proteins showed aminopeptidase activity. The E325A enzyme contained 1 mol of zinc per mol of protein, and the other three mutants, H324Y, H328Y and E347A, did not contain significant amounts of zinc, as determined by atomic absorption spectrometry. From sequence-homology searches, Ap B is known to be closely related to leukotriene (LT)-A4 hydrolase (EC 3.3.2.6). We examined human placental Ap B and recombinant rat Ap B, both of which had been purified previously [Fukasawa, Fukasawa, Kanai, Fujii and Harada (1996) J. Biol. Chem. 271, 30731-30735], to determine whether or not they had epoxide hydrolase activities. However, neither enzyme hydrolysed LTA4 into LTB4. We then replaced some amino acids in the domain of the rat enzyme similar to the LTA4-binding site of LTA4 hydrolase. However, these mutants, Y408F, N409S and NE409-410SS also did not possess any epoxide hydrolase activity. We concluded that Ap B is an M1-family zinc metallopeptidase without epoxide hydrolase activity.

Purification and properties of dipeptidyl peptidase II from rat kidney.

Dipeptidyl peptidase II (EC 3.4.14.2) from rat kidney was purified to a specific activity of 66.2 mumol/min per mg protein by a series of column chromatographic techniques. The purified enzyme was apparently homogeneous as judged by disc gel electrophoresis. Gel filtration on a calibrated column indicated an apparent molecular weight of 130 000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate in a constant acrylamide concentration resulted in the appearance of a single component for which a molecular weight of 64 000 was calculated. The pH optima for dipeptidyl arylamides and for tripeptides were pH 5.5 and 4.5, respectively, and the isoelectric point was 4.8. Substrate specificity studies indicated that the purified enzyme hydrolyzes specifically N-terminal X-alanine or X-proline from their respective arylamides and from tri- or tetrapeptides, but not from pentapeptides.

Serum dipeptidyl peptidase activities as a possible marker of oral cancer

Serum glycyl‐1‐prolyl 4‐methyl‐coumaryl‐7‐amide (gly‐pro‐MCA) hydrolase (DPP IV) and L‐lysyl‐L‐alanyl β‐naphthylamide (lys‐ala‐β NA) hydrolase (assumed to be DPP II) activities were measured in patients with oral squamous cell carcinoma and healthy subjects. The mean serum DPP IV activity of all cancer patients was significantly (P < 0.001) decreased, compared with that of healthy subjects. Although there was no significant difference between the stages by International Union Against Cancer (UICC) classification (1978), DPP IV levels tended to change dynamically, reflecting the clinical status during therapies. The serum DPP IV activity of patients with a fair prognosis was significantly elevated toward the normal range, whereas the activity of patients with a poor prognosis was significantly decreased (P < 0.05). In contrast, the mean serum lys‐ala‐β NA hydrolytic activity of cancer patients was significantly (P < 0.001) increased, compared with that of healthy subjects, and was changed reciprocally to DPP IV activity. The correlation of these two serum enzyme activities with tumor weights also was observed in animal models using nude mice transplanted with human KB carcinoma cells and hamsters transplanted with BHK21 cells. These results indicate that these serum enzyme levels may become an aid for the diagnosis of malignant tumors and for estimating the prognosis of the patients.

Cloning and functional expression of rat kidney dipeptidyl peptidase II.

Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4 micromol/min per mg of protein for Lys-Ala-beta-naphthylamide. The N-terminal and partial amino acid sequences of the enzyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST clones as a probe, we isolated a cDNA clone with 1710 bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open reading frame of 1500 bp, coding for a protein of 500 amino acids. The first 10 residues of the purified enzyme matched the deduced protein sequence starting with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51400 Da, which was lower than the value (about 60000 Da) determined by SDS/PAGE; and the deduced amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quiescent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (38% identity) and bore the putative catalytic triad (Ser, Asp, His) conserved in serine peptidase families. We transiently transfected COS-7 cells with pcDNA3.1 containing the cloned cDNA and obtained the overexpression of an immunoreactive protein (of about 60000 Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.

Inhibitory action of proline-containing peptides on Xaa-Pro-dipeptidylaminopeptidase

Abstract The inhibitory action of proline-containing peptides such as Gly-Pro, Gly-Pro-Hyp, Pro-Gly, Pro-Gly-Gly, Pro-Pro and Pro-Pro-Pro-Pro ((PrO)4) on Xaa-Pro-dipeptidylaminopeptidase from pig kidney was investigated. The enzyme activity towards Gly-Pro-pNA was competitively inhibited by Gly-Pro, Gly-Pro-Hyp, Pro-Pro and (Pro)4 but was not inhibited by Pro-Gly and Pro-Gly-Gly. The 1Ki value of Pro-Pro or (Pro)4 was much smaller than that of Gly-Pro or Gly-Pro-Hyp, indicating that rite binding affinity of Pro-Pro and (Pro)4 to the enzyme active site is much stronger than that of Gly-Pro or Gly-Pro-Hyp.