The cloning and characterization of a second brain enzyme with NAAG peptidase activity

The peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non-overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar V(max) and K(m) values (1.4 micro m and 54 pmol/min/mg; 3.5 micro m and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG.     

Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44-750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.     

Poster sessions AP11: Neurotransmitters,Transporter and Enzymes. Structure and activity of recombinant human glutamate carboxypeptidase II

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as N‐acetylated‐α‐linked‐acidic dipeptidase, NAALADase) cleaves N‐acetyl‐aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. We report the mapping of the entire coding region of GCPII and identification of the region sufficient and necessary for the production of active recombinant protein. Extracellular portion of human glutamate carboxypeptidase II (amino acids 44–750) was expressed in Drosophila Schneider's cells and purified to homogeneity. A novel assay for hydrolytic activity of GCPII, based on fluorimetric detection of released alpha‐amino groups was established, and used for enzymological characterization of GCPII. The potential of this assay for high‐throughput inhibitor testing was evaluated and pH dependence for the enzymatic activity have been analysed. Using a complete set of protected dipeptides, substrate specificity of recombinant GCPII was elucidated. Ac‐Glu‐Met, Ac‐Asp‐Met and surprisingly Ac‐Ala‐Met were identified as novel substrates for GCPII. The glycosylation has been found indispensable for the activity of the enzyme. A series of point mutants of the enzyme has been expressed and purified and the glycosylation sites critical for the proteolytic activity have been identified.     

Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis

Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1' site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N-acetyl-l-aspartyl-l-glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1' pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the 'fine-tuning' of GCPII substrate specificity.     

Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding.

Human glutamate carboxypeptidase II (GCPII) is a co-catalytic metallopeptidase and its putative catalytic domain is homologous to the aminopeptidases from Vibrio proteolyticus and Streptomyces griseus. In humans, the enzyme is expressed predominantly in the nervous system and the prostate. The prostate form, termed prostate-specific membrane antigen, is overexpressed in prostate cancer and is used as a diagnostic marker of the disease. Inhibition of the form of GCPII expressed in the central nervous system has been shown to protect against ischemic injury in experimental animal models. Human GCPII consists of 750 amino acids, and six individual domains were predicted to constitute the protein structure. Here, we report the analysis of the contribution of these putative domains to the structure/function of recombinant human GCPII. We cloned 13 mutants of human GCPII that are truncated or extended at one or both the N- and C-termini of the GCPII sequence. The clones were used to generate stably transfected Drosophila Schneider's cells, and the expression and carboxypeptidase activities of the individual protein products were determined. The extreme C-terminal region of human GCPII was found to be critical for the hydrolytic activity of the enzyme. The deletion of as few as 15 amino acids from the C-terminus was shown to completely abolish the enzymatic activity of GCPII. Furthermore, the GCPII carboxypeptidase activity was abrogated upon removal of more than 60 amino acid residues from the N-terminus of the protein. Overall, these results clearly show that amino acid segments at the N- and C-termini of the ectodomain of GCPII are essential for its carboxypeptidase activity and/or proper folding.     

Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity

Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor-associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate-specific membrane antigen (PSMA), is up-regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N-glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N-glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N-glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG-hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question.     

Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer

Membrane-bound glutamate carboxypeptidase II (GCPII) is a zinc metalloenzyme that catalyzes the hydrolysis of the neurotransmitter N-acetyl-L-aspartyl-L-glutamate (NAAG) to N-acetyl-L-aspartate and L-glutamate (which is itself a neurotransmitter). Potent and selective GCPII inhibitors have been shown to decrease brain glutamate and provide neuroprotection in preclinical models of stroke, amyotrophic lateral sclerosis, and neuropathic pain. Here, we report crystal structures of the extracellular part of GCPII in complex with both potent and weak inhibitors and with glutamate, the product of the enzyme's hydrolysis reaction, at 2.0, 2.4, and 2.2 A resolution, respectively. GCPII folds into three domains: protease-like, apical, and C-terminal. All three participate in substrate binding, with two of them directly involved in C-terminal glutamate recognition. One of the carbohydrate moieties of the enzyme is essential for homodimer formation of GCPII. The three-dimensional structures presented here reveal an induced-fit substrate-binding mode of this key enzyme and provide essential information for the design of GCPII inhibitors useful in the treatment of neuronal diseases and prostate cancer.     

Structural characterization of P1′-diversified urea-based inhibitors of glutamate carboxypeptidase II

Urea-based inhibitors of human glutamate carboxypeptidase II (GCPII) have advanced into clinical trials for imaging metastatic prostate cancer. In parallel efforts, agents with increased lipophilicity have been designed and evaluated for targeting GCPII residing within the neuraxis. Here we report the structural and computational characterization of six complexes between GCPII and P1′-diversified urea-based inhibitors that have the C-terminal glutamate replaced by more hydrophobic moieties. The X-ray structures are complemented by quantum mechanics calculations that provide a quantitative insight into the GCPII/inhibitor interactions. These data can be used for the rational design of novel glutamate-free GCPII inhibitors with tailored physicochemical properties.     

The discovery and structure-activity relationships of indole-based inhibitors of glutamate carboxypeptidase II

A series of N-substituted 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acids were synthesized as inhibitors of glutamate carboxypeptidase II (GCPII). Those containing carboxybenzyl or carboxyphenyl groups at the N-position exhibited potent inhibitory activity against GCPII. These indole-based compounds represent the first example of achiral GCPII inhibitors and demonstrate greater tolerance of the GCPII active site for ligands with significant structural difference from the endogenous substrate, N-acetyl-aspartylglutamate.     

Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.     

Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs

Human glutamate carboxypeptidase II (GCPII) is involved in neuronal signal transduction and intestinal folate absorption by means of the hydrolysis of its two natural substrates, N-acetyl-aspartyl-glutamate and folyl-poly-γ-glutamates, respectively. During the past years, tremendous efforts have been made toward the structural analysis of GCPII. Crystal structures of GCPII in complex with various ligands have provided insight into the binding of these ligands, particularly to the S1′ site of the enzyme. In this article, we have extended structural characterization of GCPII to its S1 site by using dipeptide-based inhibitors that interact with both S1 and S1′ sites of the enzyme. To this end, we have determined crystal structures of human GCPII in complex with phosphapeptide analogs of folyl-γ-glutamate, aspartyl-glutamate, and γ-glutamyl-glutamate, refined at 1.50, 1.60, and 1.67 Å resolution, respectively. The S1 pocket of GCPII could be accurately defined and analyzed for the first time, and the data indicate the importance of Asn519, Arg463, Arg534, and Arg536 for recognition of the penultimate (i.e., P1) substrate residues. Direct interactions between the positively charged guanidinium groups of Arg534 and Arg536 and a P1 moiety of a substrate/inhibitor provide mechanistic explanation of GCPII preference for acidic dipeptides. Additionally, observed conformational flexibility of the Arg463 and Arg536 side chains likely regulates GCPII affinity toward different inhibitors and modulates GCPII substrate specificity. The biochemical experiments assessing the hydrolysis of several GCPII substrate derivatives modified at the P1 position, also included in this report, further complement and extend conclusions derived from the structural analysis. The data described here form an a solid foundation for the structurally aided design of novel low-molecular-weight GCPII inhibitors and imaging agents.     

Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery

Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure–activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in K_i value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.     

Acetylation regulates the stability of glutamate carboxypeptidase II protein in human astrocytes

Glutamate carboxypeptidase II (GCPII) is known to be implicated in brain diseases such as schizophrenia and bipolar disorder, and dramatically increases in prostate cancer. Here, we investigated the regulation of GCPII expression in astrocytes and examined whether GCPII is epigenetically regulated through histone modification. In this study, valproic acid (VPA), a drug used for bipolar disorder and epilepsy and a known histone deacetylase (HDAC) inhibitor was used. We found that acute exposure of VPA for 4–6 h increased the GCPII protein level in human astrocyte U87MG cells but did not have a similar effect after 12–24 h exposure. Real-time polymerase chain reaction analysis revealed that VPA did not affect the GCPII mRNA expression. In contrast, decrease in GCPII protein level by cycloheximide treatment was blocked by VPA, indicating that VPA increases GCPII protein stability. Treatment with MG132, a proteasome inhibitor, suggested that the VPA-induced increase of GCPII protein level is dependent on the ubiquitin/proteasome pathway. In addition, immunoprecipitation analysis revealed that VPA increased the acetylation of GCPII protein at the lysine residues and facilitated a decrease of the poly-ubiquitinated GCPII level. Similarly, M344, a specific HDAC 1/6 inhibitor, also increased the GCPII protein level. In contrast, treatment with C646, a histone acetyltransferase inhibitor of p300/CBP, significantly reduced the level of GCPII protein. Taken together, this study demonstrated that the increase in GCPII induced by VPA is not due to the classical epigenetic mechanism, but via enhanced acetylation of lysine residues in GCPII.     

A method to determine the mode of binding for GCPII inhibitors using bio-layer interferometry

The rapid dilution of the enzyme-inhibitor complex assay to monitor the recovery of enzyme activity is a well-established assay to determine the reversibility of inhibition. Our laboratory has previously employed this method to ascertain the reversibility of known glutamate carboxypeptidase II (GCPII)-targeting agents. Due to the tedious and time-consuming nature of the assay, we sought to develop a facile method to determine the reversibility of well-characterized GCPII inhibitors using bio-layer interferometry (BLI). The results from the BLI assay are in agreement with the rapid dilution method. Herein, we report for the first time, a rapid, novel real-time BLI method to determine reversibility of inhibition.     

Purification of carboxypeptidase B from human pancreas.

Carboxypeptidase B of the human pancreas was purified by chromatography on DEAE-cellulose and CM-cellulose columns. Two forms of the enzyme, named carboxypeptidase B1 and B2, were separated. They have similar mol.wts. (34250 +/- 590) as established by polyacrylamide-gel disc electrophoresis and by gel filtration. Carboxypeptidase B2 migrates further towards the anode in disc electrophoresis. When the amino acid content of the enzymes was analysed, carboxypeptidase B2 had four more glycine and three more aspartic acid residues than had form B1. The amino acid sequence of the human carboxypeptidase B1 differs from that of the bovine enzyme only in two places in the N-terminal 20-amino-acid sequence. The N-terminal amino acid in carboxypeptidase B1 and B2 is alanine. The peptide 'map' of the tryptic digest of carboxypeptidase B1 contained more peptides than did that of form B2. The Km, the Vmax. and the pH optimum of the cleavage of the peptide substrate hippurylarginine and the ester substrate hippurylargininic acid were similar for both enzymes. CoCl2 accelerated the peptidase activity, and cadmium acetate enhanced the esterase activity, of human carboxypeptidases B1 and B2. Urea and sodium dodecyl sulphate inhibited the enzymes.     

Mice lacking glutamate carboxypeptidase II develop normally,but are less susceptible to traumatic brain injury

Glutamate carboxypeptidase II (GCPII) is a transmembrane zinc metallopeptidase found mainly in the nervous system, prostate and small intestine. In the nervous system, glia‐bound GCPII mediates the hydrolysis of the neurotransmitter N‐acetylaspartylglutamate (NAAG) into glutamate and N‐acetylaspartate. Inhibition of GCPII has been shown to attenuate excitotoxicity associated with enhanced glutamate transmission under pathological conditions. However, different strains of mice lacking the GCPII gene are reported to exhibit striking phenotypic differences. In this study, a GCPII gene knockout (KO) strategy involved removing exons 3–5 of GCPII. This generated a new GCPII KO mice line with no overt differences in standard neurological behavior compared to their wild‐type (WT) littermates. However, GCPII KO mice were significantly less susceptible to moderate traumatic brain injury (TBI). GCPII gene KO significantly lessened neuronal degeneration and astrocyte damage in the CA2 and CA3 regions of the hippocampus 24 h after moderate TBI. In addition, GCPII gene KO reduced TBI‐induced deficits in long‐term spatial learning/memory tested in the Morris water maze and motor balance tested via beam walking. Knockout of the GCPII gene is not embryonic lethal and affords histopathological protection with improved long‐term behavioral outcomes after TBI, a result that further validates GCPII as a target for drug development consistent with results from studies using GCPII peptidase inhibitors.

     

Glutamate carboxypeptidase II (GCPII) inhibitor displays anti-glutamate and anti-cocaine effects in an invertebrate assay

Glutamate carboxypeptidase II (GCPII) inhibitors are promising anti-glutamatergic and anti-addictive agents. We hypothesized that a GCPII inhibitor 2 (phosphonomethyl) pentanedioic acid (2-PMPA) would display anti-stereotypical activity in planarians. Experiments revealed that 2-PMPA displayed no overt behavioral activity by itself but attenuated stereotypical counts (C-shape hyperkinesias) elicited by four compounds (2-PMPA rank order potency: glutamate>NMDA>pilocarpine>cocaine). These data suggest GCPII inhibitors display broad-spectrum efficacy against behavioral activity produced by glutamatergic and non-glutamatergic compounds in an invertebrate assay.     

Regulation of glutamate carboxypeptidase II hydrolysis of N-acetylaspartylglutamate (NAAG) in crayfish nervous tissue is mediated by glial glutamate and acetylcholine receptors

Glutamate carboxypeptidase II (GCPII), a glial ectoenzyme, is responsible for N-acetylaspartylglutamate (NAAG) hydrolysis. Its regulation in crayfish nervous tissue was investigated by examining uptake of [3H]glutamate derived from N-acetylaspartyl-[3H]glutamate ([3H]NAAG) to measure GCPII activity. Electrical stimulation (100 Hz, 10 min) during 30 min incubation with [3H]NAAG increased tissue [3H]glutamate tenfold. This was prevented by 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a GCPII inhibitor, suggesting that stimulation increased the hydrolysis of [3H]NAAG and metabolic recycling of [3H]glutamate. Antagonists of glial group II metabotropic glutamate receptors (mGLURII), NMDA receptors and acetylcholine (ACh) receptors that mediate axon-glia signaling in crayfish nerve fibers decreased the effect of stimulation by 58-83%, suggesting that glial receptor activation leads to stimulation of GCPII activity. In combination, they reduced [3H]NAAG hydrolysis during stimulation to unstimulated control levels. Agonist stimulation of mGLURII mimicked the effect of electrical stimulation, and was prevented by antagonists of GCPII or mGLURII. Raising extracellular K+ to three times the normal level stimulated [3H]NAAG release and GCPII activity. These effects were also blocked by antagonists of GCPII and mGLUR(II). No receptor antagonist or agonist tested or 2-PMPA affected uptake of [3H]glutamate. We conclude that NAAG released from stimulated nerve fibers activates its own hydrolysis via stimulation of GCPII activity mediated through glial mGLURII, NMDA and ACh receptors.     

Cathepsins X and B can be differentiated through their respective mono- and dipeptidyl carboxypeptidase activities

Several new cysteine proteases of the papain family have been discovered in the past few years. To help in the assignment of physiological roles and in the design of specific inhibitors, a clear picture of the specificities of these enzymes is needed. One of these novel enzymes, cathepsin X, displays a unique specificity, cleaving single amino acid residues at the C-terminus of substrates very efficiently. In this study, the carboxypeptidase activities and substrate specificity of cathepsins X and B have been investigated in detail and compared. Using quenched fluorogenic substrates and HPLC measurements, it was shown that cathepsin X preferentially cleaves substrates through a monopeptidyl carboxypeptidase pathway, while cathepsin B displays a preference for the dipeptidyl pathway. The preference for one or the other pathway is about the same for both enzymes, i.e., approximately 2 orders of magnitude, a result supported by molecular modeling of enzyme-substrate complexes. Cleavage of a C-terminal dipeptide of a substrate by cathepsin X can become more important under conditions that preclude efficient monopeptidyl carboxypeptidase activity, e.g., nonoptimal interactions in subsites S(2)-S(1). These results confirm that cathepsin X is designed to function as a monopeptidyl carboxypeptidase. Contrary to a recent report [Klemencic, I., et al. (2000) Eur. J. Biochem. 267, 5404-5412], it is shown that cathepsins X and B do not share similar activity profiles, and that reagents are available to clearly distinguish the two enzymes. In particular, CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X. The insights obtained from this and previous studies have been used to produce an inhibitor designed to exploit the unique structural features responsible for the carboxypeptidase activity of cathepsin X. Although of moderate potency, this E-64 derivative is the first reported example of a cathepsin X-specific inhibitor.     

Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.