Crystal structure of the human carboxypeptidase N (kininase I) catalytic domain.

Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 A crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal beta-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1' pocket of CPN1 might better accommodate P1'-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain-TT interface, exposing the two active centers.

The purification and characterization of an 88-kDa Porphyromonas endodontalis 35406 protease.

A Porphyromonas endodontalis ATCC 35406 protease was purified from Triton X-114 cell extracts by preparative SDS-PAGE followed by electroelution. The purified enzyme exhibits a molecular size of 88 kDa and was dissociated into two polypeptides of 43 and 41 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The protease (pH optimum 7.5-8.0) degraded the extracellular matrix proteins fibrinogen and fibronectin. Collagen IV was also degraded at 37 degrees C but not at 28 degrees C. The protease also cleaved the bioactive peptide angiotensin at amino acid residue phenylalanine-8 and tyrosine-4 but failed to hydrolyze bradykinin, vasopressin and synthetic chromogenic substrates with phenylalanine or tyrosine at the P1 position. In addition, two peptidases were detected in P. endodontalis cells: a proline aminopeptidase that remained associated with the cell pellet after detergent extraction and peptidase/s that partitioned into the Triton X-114 phase after phase separation and degraded the bioactive peptides bradykinin and vasopressin. These P. endodontalis peptidases and proteases may play an important role in both the nutrition and pathogenicity of these assacharolytic microorganisms. The inactivation of bioactive peptides and degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of host tissues accompanied with endodontic infections.

Characterization of mouse carboxypeptidase N small active subunit gene structure.

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease comprised of two small subunits that have enzymatic activity, and two large subunits, which protect the enzyme from degradation. CPN cleaves the carboxyl-terminal amino acids arginine and lysine from biologically active peptides such as complement anaphylatoxins, kinins, and fibrinopeptides. To delineate the murine CPN small subunit coding region, gene structure, and chromosome location, cDNA and genomic clones were isolated, characterized, and used in Northern and fluorescence in situ hybridization analyses. The results from this study demonstrate that the murine CPN small subunit gene is a single copy gene of approximately 29 kb that is transcribed in the liver into a 1793-bp mRNA with an open reading frame of 1371 nucleotides encoding 457 aa. The gene contains nine exons ranging in size from 455 bp (exon 1) to 100 bp (exon 7), and eight introns ranging in size from 6.2 kb (intron 2) to 1.4 kb (intron 4). All intron/exon junctions follow the normal consensus rule. The mouse CPN small subunit gene localized to chromosomal band 19D2, which is syntenic to human chromosome 10q23-25. Primer extension experiments using mouse liver mRNA indicate one major transcriptional initiation site and three minor sites. Sequence analysis of the 5'-flanking region indicated a TATA-less promoter and numerous transcription factor binding sites, which may confer liver-specific expression of the CPN small subunit gene.

Carboxypeptidase U, a plasma carboxypeptidase with high affinity for plasminogen.

A novel basic carboxypeptidase clearly different from carboxypeptidase N has been isolated from human plasma. It circulates as an enzymatically inactive precursor enzyme bound to plasminogen. During fibrinolysis, it can be converted to its active form, carboxypeptidase U, through the action of plasmin. The active enzyme has an apparent molecular weight of 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzes the synthetic peptides hippuryl-L-arginine and hippuryl-L-lysine but, in contrast to other human basic carboxypeptidases, has only a limited esterase activity. After its activation, carboxypeptidase U tends to be very unstable.

On the specificity of carboxypeptidase N, a comparative study.

The structure of the enzymatically active subunit of human plasma carboxypeptidase N was modeled based on the homology with bovine carboxypeptidase A. The active site of carboxypeptidase N is well conserved in comparison with carboxypeptidase A. From a comparison of energetically favorable binding sites for different atomic probe groups a hypothesis for the differences in substrate specificity between carboxypeptidases A and N was derived. Small synthetic peptide substrates were synthesized to confirm this hypothesis. This study shows that even with very low homology model building by homology can be employed to build models of sufficient quality to aid in drug design.

[Purification and physico-chemical properties of soluble carboxypeptidase N from cat brain gray matter]. / Ochistka i fiziko-khimicheskie svoistva rastvorimoi karboksipeptidazyN iz serogo veshchestva golovnogo mozga koshki.

Using affinity chromatography on diasorb-L-arginine and polyacrylamide gel electrophoresis, soluble carboxypeptidase H (E. C. 3.4.17.10) has been isolated from cat brain cortex and purified 598-fold with a 16% yield. The enzyme has a molecular mass of 50 kDa, consists of one polypeptide chain, and displays the maximum activity at pH 5.6. Carboxypeptidase H is a thiol-dependent metalloenzyme and contains a Zn2+ ion in its active center. The Km and V values for dansyl-Phe-Leu-Arg are 100 +/- 5 microM and 12.5 +/- 1.4 microM/min/mg of protein, respectively. The existence of two forms of soluble carboxypeptidase differing in isoelectric points and pH optima has been demonstrated. The enzyme with a pI of 4.8 has a pH optimum at 5.5-5.6, while that with a pI of 5.25-at 6.0.

Inhibition of plasma kallikrein. Kininase and kinin-like activities of preparations from the medicinal leeches.

The medicinal leech salivary gland secretion deprived of hirudin antithrombin activity inhibits amidolytic (substrate S-2302) and kininogenase (substrate kininogen) activities of plasma kallikrein, the main component of the intrinsic mechanism of blood coagulation. It therefore possesses high anticoagulant properties. Kininase (substrate bradykinin) activity of leech saliva and extracts from the medicinal leeches, as well as kinin-like effects of extracts heated at 100 degrees C have been detected. The last one is correlated with the hyperalgetic property of the heated extract. An analgetic effect was observed with the unheated extract but not with leech saliva after intranasal administration to rats.

Aminopeptidase P: purification of a membrane-bound bradykininase from rat lung.

Aminopeptidase P that hydrolyzes the Arg1-Pro2-bond of bradykinin was solubilized from rat lung microsomes using phosphatidylinositol-specific phospholipase C. The enzyme was purified 420-fold by chromatography on decylagarose (two steps), omega-aminodecyl-agarose and DEAE-Sephacel. A single stained band was observed following native gradient (4-15%) polyacrylamide gel electrophoresis. Dipeptidylaminopeptidase IV-like activity was also present in the final preparation and co-migrated with aminopeptidase P in the above gel system.

Purification and characterization of new arginine esteropeptidase from the soluble fraction of human submaxillary glands.

An arginine esteropeptidase was completely purified from the soluble fraction of human submaxillary glands. The molecular weight was calculated to be 12,000, having 2 species of subunits. The study of the effect of inhibitors confirmed the enzyme's serine protease-like characteristics. The best ester and amide substrates were Tos-Arg-OMe and D-Ile-Pro-Arg-pNA, respectively.

A new latent arginine esteropeptidase from human submaxillary gland.

One of the arginine esteropeptidases in human submaxillary gland was purified from microsomal membranes. The enzyme is inactive in membranes and requires trypsin treatment for its full activation. The trypsin-activated enzyme was purified to homogeneity. Its molecular weight was determined to be 94,000 by SDS-polyacrylamide gel electrophoresis. Among various substrates examined, the obtained enzyme exhibited high specific activities toward Tos-Arg-OMe (esterolysis) and D-Ile-Pro-Arg-pNA (amidolysis). The enzyme was inhibited by some serine proteinase inhibitors, whereas inhibitors of other types of proteinases did not affect or only scarcely affected it. The enzyme appears to be distinct from other arginine esteropeptidases previously described.

[Kininase from the Latrodectus tredecimguttatus venom. Characteristics of the enzyme as a thiol endopeptidase hydrolyzing the -Pro7-Phe8- bond of bradykinin]. / Kininaza iada pauka Latrodectus tredecimguttatus. Kharakteristika fermenta kak tiolovoi éndopeptidazy, rasshchepliaiushchei sviaz' -Pro7-Phe8- v bradikinine.

The nature of the bradykinin (BK)-hydrolyzing (kininase) activity of peptidhydrolase isolated from spider (Latr. tredecimguttatus) venom has been studied. It was found that the BKase activity of the enzyme is fully inhibited by organic mercurials (10(-5)-10(-6) M) as well as by 5,5'-dithiobis(2-nitrobenzoic acid) (10(-7) M); the latter blocks three SH-groups within the enzyme molecule. Serine and metalloproteinase inhibitors have no effect on the kininase activity. Thin-layer chromatography on silicagel revealed that the highly purified enzyme hydrolyzes the -Pro7-Phe8- bond of BK liberating the C-terminal dipeptide, HPhe-ArgOH. Besides, the kininase splits off the C-terminal tripeptide from angiotensin I by hydrolyzing its -Pro7-Phe8-bond. The enzyme does not exhibit any exopeptidase activity with free and N-substituted tri- and pentapeptides. The data obtained suggest that the Latr. tredecimguttatus kininase can be related to thiol endopeptidases hydrolyzing the peptide bonds formed by proline carboxyl.

Purification and characterization of a new arginine carboxypeptidase in human serum.

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37 degrees C, we named it unstable carboxypeptidase (carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl-L-arginine and hippuryl-L-lysine, but at a different relative rate than carboxypeptidase N, and has no esterase activity on hippuryl-L-argininic acid. Its activity was inhibited by o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate carboxypeptidase U from carboxypeptidase N and other known carboxypeptidases.

Purification and properties of microsomal carboxypeptidase N (kininase I) in human placenta.

Carboxypeptidase N (kininase I, EC 3.4.17.3) was found in human placenta and purified 600-fold. The enzyme was solubilized from membrane fractions with Triton X-100 and was purified by affinity chromatography with histargin, a potent inhibitor of this enzyme. The pH optimum of the enzyme was 7.8. The Km values for L-hippuryl-L-lysine and bradykinin were 1.25 and 0.43 mmol/l, respectively. The apparent molecular mass (Mr) of the enzyme determined by gel filtration was estimated to be 280,000, which is identical to that of the human serum enzyme. We propose that the placenta is a major source of carboxypeptidase N and thus may be involved in the physiological control of fetal circulation by regulating the kallikrein-kinin and renin-angiotensin systems.

Characterization of the carboxypeptidase N secreted by Hep G2 cells.

Human hepatoma (Hep G2) cells secrete nanogram quantities of carboxypeptidase enzymes which are capable of hydrolyzing COOH-terminal lysine and arginine residues. A carboxypeptidase with a neutral pH optimum (greater than pH 7.0) was partially purified from the conditioned medium and compared with pure plasma carboxypeptidase N. The two enzymes behaved in a similar manner on gel filtration (apparent Mr = 280,000), DE52 ion exchange chromatography, and concanavalin A-affinity chromatography and were indistinguishable enzymatically and immunologically. Immunoblots of the Hep G2 and plasma carboxypeptidase N before and following deglycosylation with peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase F revealed a similar, if not identical, multimeric structure. A second carboxypeptidase with a lower molecular weight and a pH optimum of 5.0 was also detected in the Hep G2 medium.

Amino acid sequence of the N-terminus and selected tryptic peptides of the active subunit of human plasma carboxypeptidase N: comparison with other carboxypeptidases.

Human plasma carboxypeptidase N was purified to homogeneity and its active and inactive subunits were separated. By introducing a novel technique, both forms of the active subunit (Mr = 55,000 and Mr = 48,000) were isolated. N-terminal sequencing of the active subunit of human carboxypeptidase N revealed significant homology with the N-terminal sequence of bovine carboxypeptidase H (43% identity) and to a lesser extent with carboxypeptidase A (29% identity) or carboxypeptidase B (18% identity). The active subunit of carboxypeptidase N was hydrolyzed with trypsin and 4 of the tryptic peptides were isolated by HPLC and sequenced. The sequences of the four peptides were homologous (39-64% identity) with regions of carboxypeptidase H corresponding to the middle (residues 148-175) and C-terminal portion (residues 321-408). These regions had essentially no homology with carboxypeptidase A or B. These data indicate that carboxypeptidase H and the active subunit of carboxypeptidase N may have diverged from a common ancestral gene.