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PubMed1997

Use of site-directed chemical modification to study an essential lysine in Escherichia coli leader peptidase.

Escherichia coli leader peptidase, which catalyzes the cleavage of signal peptides from pre-proteins, is an essential, integral membrane serine peptidase that has its active site residing in the periplasmic space. It contains a conserved lysine residue that has been proposed to act as the general base, abstracting the proton from the side chain hydroxyl group of the nucleophilic serine 90. To help elucidate the role of the essential lysine 145 in the activity of E. coli leader peptidase, we have combined site-directed mutagenesis and chemical modification methods to introduce unnatural amino acid side chains at the 145-position. We show that partial activity can be restored to an inactive K145C leader peptidase mutant by reacting it with 2-bromoethylamine.HBr to produce a lysine analog (gamma-thia-lysine) at the 145-position. Modification with the reagents 3-bromopropylamine.HBr and 2-mercaptoethylamine also allowed for partial restoration of activity showing that there is some flexibility in the length requirements of this essential residue. Modification with (2-bromoethyl)trimethylammonium.Br to form a positively charged, nontitratable side chain at the 145-position failed to restore activity to the inactive K145C leader peptidase mutant. This result, along with an inactive K145R mutant result, supports the claim that the lysine side chain at the 145-position is essential due to its ability to form a hydrogen bond(s) or to act as a general base rather than because of an ability to form a critical salt bridge. We find that leader peptidase processes the pre-protein substrate, pro-OmpA nuclease A, with maximum efficiency at pH 9.0, and apparent pKa values for titratable groups at approximately 8.7 and 9.3 are revealed. We show that the lysine modifier maleic anhydride inhibits leader peptidase by reacting with lysine 145. The results of this study are consistent with the hypothesis that the lysine at the 145-position of leader peptidase functions as the active site general base. A model of the active site region of leader peptidase is presented based on the structure of the E. coli UmuD', and a mechanism for bacterial leader peptidase is proposed.

Amino Acid Sequence↗
PubMed1976

Rat intestinal brush border membrane peptidases. I. Solubilization, purification, and physicochemical properties of two different forms of the enzyme.

Two brush border peptidases have been isolated from the particulate fraction of the rat intestinal mucosa and purified to homogeneity as judged by polyacrylamide gel electrophoresis, starch gel electrophoresis, isoelectric focusing, and double immunodiffusion. For convenience, the peptidases have been designated peptidase F (fast) and S (slow) on the basis of their anodic mobilities. The isoelectric point of peptidase F was 4.76 and of peptidase S, 5.10. Both enzymes are glycoproteins. The amino acid compositions of the two peptidases are similar. The same carbohydrates are found in both enzymes, but there are differences in the molar concentrations of individual sugars. Peptidase S has greater concentrations of mannose and galactose and of hexosamines than peptidase F, while sialic acid is slightly greater in peptidase F. Carbohydrate accounted for approximately 19% and 23% of the weight of peptidases F and S, respectively. Estimates of the molecular weights of both enzymes by gel filtration gave values of 280,000. Electrophoresis of the enzymes under denaturing conditions on sodium dodecyl sulfate polyacrylamide gels indicated that each enzyme is a dimer consisting of two subunits of equal molecular weight, 140,000.

Amino Acids↗
PubMed1986

Is the peptidase activity of highly purified human plasma cholinesterase due to a specific cholinesterase isoenzyme or a contaminating dipeptidylaminopeptidase?

The peptidase site of human plasma cholinesterase (butyrylcholinesterase) is distinct from its esteratic site. We found that the number of peptidase sites on an enzyme highly purified from pooled plasma is less than 0.1, as compared with 4 esteratic sites, per tetramer. However, the subunits which carry the peptidase sites are electrophoretically indistinguishable from esteratic subunits. The atypical-silent enzyme (Ea1Es1) had a much higher absolute peptidase activity when substance P was used as the substrate, and we found that the number of peptidase and esteratic sites of this enzyme was roughly the same. This suggests that the mutated esteratic site of the silent possesses a peptidase activity. The esteratic site of the usual allozyme (Eu1Eu1) has no peptidase activity towards substance P. However, a small proportion of peptidase subunits are present in all preparations of enzymes purified from the plasmas of homozygote individuals. The peptidase activity of butyrylcholinesterase might therefore correspond to a specific isoenzyme produced by an epigenetic mechanism or produced by a gene distinct from genes E1 and E2 encoding for cholinesterase subunits. However, the possibility that highly purified cholinesterase contains traces of a dipeptidylaminopeptidase cannot be completely ruled out.

Binding Sites↗
PubMed1994

Expression and localization of aminopeptidase N, neutral endopeptidase, and dipeptidyl peptidase IV in the human placenta and fetal membranes.

OBJECTIVE: Our purpose was to determine the distribution of membrane-bound cell surface peptidases, namely aminopeptidase N, neutral endopeptidase, and dipeptidyl peptidase IV in the human placenta and fetal membranes. STUDY DESIGN: Frozen tissue sections of the first-trimester chorionic villi, term placentas, and term fetal membranes were stained by indirect immunofluorescence with specific monoclonal antibodies. RESULTS: In the first trimester chorionic villi cytotrophoblasts expressed both neutral endopeptidase and dipeptidyl peptidase IV, but syncytiotrophoblasts expressed only neutral endopeptidase. Stromal cells in the chorionic villi expressed the three peptidases at various intensities. In the term placentas villous syncytiotrophoblasts expressed neutral endopeptidase weakly, and the villous stromal cells expressed large amounts of both aminopeptidase N and dipeptidyl peptidase IV but neutral endopeptidase weakly or faintly. In the term fetal membranes amniotic epithelial cells and chorion laeve expressed both neutral endopeptidase and dipeptidyl peptidase IV. Decidual cells in the decidua parietalis moderately or highly expressed aminopeptidase N. CONCLUSION: Three peptidases, aminopeptidase N, neutral endopeptidase, and dipeptidyl peptidase IV, are expressed by different cell populations in the human placenta and fetal membranes, suggesting their respective and important roles at the maternofetal interface.

Aminopeptidases↗
PubMed2004

Mutations in classical late infantile neuronal ceroid lipofuscinosis disrupt transport of tripeptidyl-peptidase I to lysosomes.

Classical late infantile neuronal ceroid lipofuscinosis is an autosomal recessive disease caused by mutations in the CLN2 gene resulting in functional defects of the gene product tripeptidyl-peptidase I. This disease is associated with a progressive neurodegenerative course beginning at the age of two years with developmental stagnation, finally leading to a complete loss of motor function, vision and speech by the age of 10 years. We analyzed the functional consequences of the mutations R127Q, R208X, N286S, I287N, T353P and Q422H, which were previously identified in patients with late infantile ceroid lipofuscinosis, with regard to enzymatic activity, stability, post-translational processing and intracellular localization of tripeptidyl-peptidase I. We could not detect any translational product for the mutant R208X. We found that four missense mutations, N286S, I287N, T353P and Q422H, which are located in conserved protein regions of tripeptidyl-peptidase I, decreased the enzymatic activity dramatically, blocked processing to mature size peptidase and led to protein retention in the endoplasmatic reticulum and rapid degradation in non-lysosomal compartments. We conclude that these amino-acid substitutions induce major misfolding of the precursor peptidase and hence prevent post-translational processing and lysosomal targeting of tripeptidyl-peptidase I. In contrast, the amino-acid substitution R127Q within a non-conserved protein region did not significantly affect enzymatic activity, stability, processing and lysosomal targeting of tripetidyl-peptidase I. Thus, our functional analyses of CLN2 mutations reveal novel insight into the molecular defect underlying dysfunction of tripeptidyl-peptidase I.

Amino Acid Sequence↗
PubMed1993

Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum.

Peptides of the glucagon/vasoactive-intestinal-peptide (VIP) peptide family share a considerable sequence similarity at their N-terminus. They either start with Tyr-Ala, His-Ala or His-Ser which might be in part potential targets for dipeptidyl-peptidase IV, a highly specialized aminopeptidase removing dipeptides only from peptides with N-terminal penultimate proline or alanine. Growth-hormone-releasing factor (1-29)amide and gastric inhibitory peptide/glucose-dependent insulinotropic peptide (GIP) with terminal Tyr-Ala as well as glucagon-like peptide-1(7-36)amide/insulinotropin [GLP-1(7-36)amide] and peptide histidine methionine (PHM) with terminal His-Ala were hydrolysed to their des-Xaa-Ala derivatives by dipeptidyl-peptidase IV purified from human placenta. VIP with terminal His-Ser was not significantly degraded by the peptidase. The kinetics of the hydrolysis of GIP, GLP-1(7-36)amide and PHM were analyzed in detail. For these peptides Km values of 4-34 microM and Vmax values of 0.6-3.8 mumol.min-1.mg protein-1 were determined for the purified peptidase which should allow their enzymic degradation also at physiological, nanomolar concentrations. When human serum was incubated with GIP or GLP-1(7-36)amide the same fragments as with the purified dipeptidyl-peptidase IV, namely the des-Xaa-Ala peptides and Tyr-Ala in the case of GIP or His-Ala in the case of GLP-1(7-36)amide, were identified as the main degradation products of these peptide hormones. Incorporation of inhibitors specific for dipeptidyl-peptidase IV, 1 mM Lys-pyrrolidide or 0.1 mM diprotin A (Ile-Pro-Ile), completely abolished the production of these fragments by serum. It is concluded that dipeptidyl-peptidase IV initiates the metabolism of GIP and GLP-1(7-36)amide in human serum. Since an intact N-terminus is obligate for the biological activity of the members of the glucagon/VIP peptide family [e. g. GIP(3-42) is known to be inactive to release insulin in the presence of glucose as does intact GIP], dipeptidyl-peptidase-IV action inactivates these peptide hormones. The relevance of this finding for their inactivation and their determination by immunoassays is discussed.

Amino Acid Sequence↗
PubMed1984

A distinct signal peptidase for prolipoprotein in Escherichia coli.

We have previously demonstrated the modification and processing of Escherichia coli prolipoprotein (Braun's) in vitro ( Tokunaga M, Tokunaga H, Wu HC: Proc Natl Acad Sci USA 79:2255, 1982). Using this in vitro assay of prolipoprotein signal peptidase and globomycin selection, we have isolated and partially characterized an E coli mutant which contained a higher level of prolipoprotein signal peptidase activity. In contrast, the procoat protein signal peptidase activity was not increased in this mutant as compared to the wild-type strain. Furthermore, E coli strains containing cloned procoat protein signal peptidase gene were found to contain elevated levels of procoat protein signal peptidase, but normal levels of prolipoprotein signal peptidase. These two signal peptidase activities were also found to exhibit different stabilities during storage at 4 degrees C. Thus biochemical, immunological, and genetic evidence clearly indicate that prolipoprotein signal peptidase is distinct from procoat protein signal peptidase in E coli.

Anti-Bacterial Agents↗
PubMed2002

Identification and characterization of a dense cluster of placenta-specific cysteine peptidase genes and related genes on mouse chromosome 13.

Genes encoding novel murine cysteine peptidases of the papain family C1A and related genes were cloned and mapped to mouse chromosome 13, colocalizing with the previously assigned cathepsin J gene. We constructed a <460-kb phage artificial chromosome (PAC) contig and characterized a dense cluster comprising eight C1A cysteine peptidase genes, cathepsins J, M, Q, R, -1, -2, -3, and -6; three pseudogenes of cathepsins M, -1, and -2; and four genes encoding putative cysteine peptidase inhibitors related to the proregion of C1A peptidases (trophoblast-specific proteins alpha and beta and cytotoxic T-lymphocyte-associated proteins 2alpha and -beta). Because of sequence homologies of 61.9-72.0% between cathepsin J and the other seven putative cysteine peptidases of the cluster, these peptidases are classified as "cathepsin J-like". The absence of cathepsin J-like peptidases and related genes from the human genome suggests that the cathepsin J cluster arose by partial and complete gene duplication events after the divergence of primate and rodent lineages. The expression of cathepsin J-like peptidases and related genes in the cluster is restricted to the placenta only. Clustered genes are induced at specific time points, and their expression increases toward the end of gestation. The specific expression pattern and high expression level suggest an essential role of cathepsin J-like peptidases and related genes in formation and development of the murine placenta.

Amino Acid Sequence↗
PubMed1980

Oxytocin biotransformation in the rat limbic brain: characterization of peptidase activities and significance in the formation of oxytocin fragments.

The enzymatic conversion of oxytocin by brain peptidases has been studied. Oxytocin was incubated with synaptosomal plasma membranes (SPM) isolated from the rat brain. Qualitative studies using a microdansylation technique revealed two types of oxytocin converting peptidases, e.g. aminopeptidase and C-terminal cleaving peptidase activities. Both enzyme activities were quantitated using [14C]oxytocin labeled at either the tyrosine-2 or the glycinamide-9 residue. Radiolabeled products were separated by high-voltage paper electrophoresis or high-pressure liquid chromatography. The aminopeptidase activity was optimally active at pH 6.9 with a Michaelis constant (Km) of 6.1 x 10(-5) M. The pH optimum of the C-terminal cleaving peptidase activity was pH 6.0 with Km = 1.3 x 10(-5) M. Subcellularly, highest amino-peptidase activities were associated with SPM, synaptosomal and microsomal preparations, while the C-terminal cleaving peptidase prevailed in the cytosol and mitochondrial fractions. The regional distribution of both peptidases showed differences between several brain areas and indicated the medial basal hypothalamus as a locus of high oxytocin biotransformation. In the course of this investigation an oxytocin fragment of unknown structure was detected in the digests and its accumulation was studied together with the determination of peptidase activities. It is suggested that the SPM-associated peptidases may have a role in the modulation of oxytocin action in the brain.

Aminopeptidases↗
PubMed1990

Electrophoretic detection of Trypanosoma cruzi peptidases.

Peptidases of Trypanosoma cruzi epimastigotes were examined by polyacrylamide gel electrophoresis in gels containing gelatin as peptidase substrate. Mini-gels were far superior to large gels in their sensitivity of peptidase detection. Patterns of peptidases were similar between different strains of T. cruzi, although some inter-strain heterogeneity was found. In strain Y, at least five peptidases were detected: four of these enzymes were shown to be cysteine-type peptidases with acidic pH optima. The other peptidase was a 60-kDa membrane-associated peptidase that was sensitive to o-phenanthroline; it was tentatively characterised as a metallopeptidase, and was optimally active at alkaline pH. This membrane-associated peptidase was conserved between strains of T. cruzi.

Animals↗
PubMed1998

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation.

Cholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae. Although the eps gene locus encodes several type four prepilin-like proteins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepilin peptidase from the V. cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa. Plasmid disruption or deletion of this peptidase gene in either EI Tor or classical V. cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion. In the case of the EI Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose-sensitive haemagglutination is abolished. The cloned V. cholerae peptidase processes either EpsI or MshA preproteins when co-expressed in E. coli. Mutation of the V. cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU. The V. cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepilin peptidase required for pilus assembly and cytolysin secretion in V. vulnificus. Accordingly, the V. cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.

Adhesins, Bacterial↗
PubMed1992

Genetic and environmental effects on the expression of peptidases and larval viability in Drosophila melanogaster.

The peptidase system in Drosophila melanogaster, consisting of dipeptidase-A, dipeptidase-B, dipeptidase-C and the leucine aminopeptidases, was used as a model to study the adaptive significance of enzyme activity variation. The involvement of the peptidases in osmoregulation has been suggested from the ubiquitous distribution of peptidase activities in nearly all tissues and the high concentration of amino acids and oligopeptides in the hemolymph. Under this hypothesis, larvae counteract increases in environmental osmotic stress by hydrolyzing peptides into amino acids both intra- and extracellularly to increase physiological osmotic concentration. The expression of the peptidases was studied by assaying for peptidase activities in third instar larvae of isogenic lines, which were reared under increasing levels of environmental osmotic stress using either D-mannitol or NaCl. Second and third chromosome substitution isogenic lines were used to assess the relative contribution of regulatory and structural genes in enzyme activity variation. Results indicate that: (1) genetic variation exists for peptidase activities, (2) the effect of osmotic stress is highly variable among peptidases, (3) changes in peptidase activities in response to osmotic stress depend on both genetic background and osmotic effector and (4) peptidase activities are correlated with each other, but these phenotypic correlations depend on genetic background, osmotic effector, and level of osmotic stress. Osmotic concentration in the larval hemolymph is correlated with leucine aminopeptidase activity, but changes in hemolymph osmotic concentration in response to environmental osmotic stress depend on the osmotic effector in the environment. Although these findings suggest that genetic and environmental factors contribute significantly toward the expression of enzymes with similar functions, a relative larval viability study of genotypes that differed significantly in dipeptidase-B (DIP-B) activity revealed that low DIP-B activity did not confer any measurable reduction in larval viability under increasing levels of environmental osmotic stress. These negative results suggest that, either DIP-B does not play a major role in osmoregulation or differential osmoregulation is not related to egg to adult viability in these tests.

Animals↗
PubMed2000

Endothelin-1 inactivating peptidase in the human kidney and urine.

OBJECTIVE: Recently, an apparently novel, specific endothelin-1 inactivating metalloendopeptidase (ET-1 peptidase) has been isolated from the rat kidney. In this study we attempted to determine whether the same or a similar peptidase is present in the human kidney, and whether the enzyme is excreted into the urine. The urinary ET-1 peptidase could serve as an indirect index of the renal endothelin system, both in physiology and pathophysiology. METHODS: Kidney specimens were obtained from part of nephrectomized kidneys unaffected by any neoplastic process from six adult patients. The enzyme was purified using differential centrifugation, detergent solubilization of the membrane proteins, ultrafiltration and nondenaturing gel electrophoresis. The enzyme activity assays were performed at pH 5.5 and 37 degrees C in the presence of increasing concentrations of unlabelled peptides and inhibitors using a fixed amount of [125I]ET-1 as substrate. The degradation extent was quantified with trichloroacetic acid precipitation and high performance liquid chromatography. The degrading activity of ET-1 was determined in urine samples from adult patients with hypertension, children with chronic renal failure and those with stable renal allograft RESULTS: ET-1 peptidase from the human kidney displays characteristics close to that of the rat ET-1 peptidase we have recently described (J. Hypertens 1994; 12:1155-1162). The enzyme, a membrane-bound metalloendopeptidase, exhibits low electro- phoretical mobility on nondenaturing gel (Rf 0.08); it is an apparently heterologous structure comprising three enzymatically inactive subunits, it has a pH optimum at 5.5, a nanomolar range affinity to the ET-1 (KM 180 nmol/l) that is hydrolysed to two main degradation products, and a 10-100-fold lower affinity to big ET-1 (KM 11.5 micromol/l), endothelin 11 21 fragment (KM 15.3 micromol/l), endothelin antagonist Trp-Leu-Asp-Ile-Ile-Trp (KM 3.1 micromol/I), gastrin (KM 2.2 micromol/l) and cholecystokinin (KM 4.0 micromol/l). Substance P, neuropeptide Y, atrial natriuretic peptide, bradykinin, angiotensin II and enkephalin were poor substrates for the enzyme. The most powerful inhibitors of the ET-1 peptidase included thiorphan (IC50 0.28 nmol/l), phosphoramidon (IC50 0.55 nmol/l), phenanthroline (IC50 11.5 micromol/l), cyclosporin (IC50 400 micromol/l), phosphate (IC50 1.2 mmol/l), citrate (IC50 0.6 mmol/l) and aniline naphthalene sulphonic acid (IC50 0.25 mmol/l). Our data suggest that three ET-1 degrading peptidases with optimal activity at pH 4.5, 5.5 and 7.0, respectively, are excreted into the urine. The enzyme with a pH optimum 4.5 is of lysosomal origin whereas the two other enzymes correspond by their pH optima to the renal ET-1 peptidase and neutral endopeptidase. We have found statistically significant increases (P < 0.001) in the activity of both lysosomal and ET-1 peptidase in the urine in patients with hypertension and in children with chronic renal failure compared with healthy subjects or children with stable renal allograft CONCLUSIONS: Human kidney contains an acidic, highly specific endothelin-1 inactivating metalloendopeptidase that may have a key role in the regulation of concentrations of renal and circulating endothelins. The enzyme is excreted into the urine where its activity seems to be increased in patients with hypertension and chronic renal failure; it may potentially serve as an indirect index of the renal endothelin system.

Adolescent↗
PubMed1987

A peptidase activity exhibited by human serum pseudocholinesterase.

The identity of a peptidase activity with human serum pseudocholinesterase (PsChE) purified to apparent homogeneity was demonstrated by co-elution of both peptidase and PsChE activities from procainamide-Sepharose and concanavalin-A--Sepharose affinity chromatographic columns; comigration on polyacrylamide gel electrophoresis; co-elution on Sephadex G-200 gel filtration and coprecipitation at different dilutions of an antibody raised against purified PsChE. The purified enzyme showed a single protein band on gel electrophoresis under non-denaturing conditions. SDS gel electrophoresis under reducing conditions, followed by silver staining, also gave a single protein band (Mr approximately equal to 90,000). Peptidase activity using different peptides showed the release of C-terminal amino acids. Blocking the carboxy terminal by an amide or ester group did not prevent the hydrolysis of peptides. There was no evidence for release of N-terminal amino acids. Potent anionic or esterase site inhibitors of PsChE, such as eserine sulphate, neostigmine, procainamide, ethopropazine, imipramine, diisopropylfluorophosphate, tetra-isopropylpyrophosphoramide and phenyl boronic acid, did not inhibit the peptidase activity. An anionic site inhibitor (neostigmine or eserine) in combination with an esterase site inhibitor (diisopropylfluorophosphate) also did not inhibit the peptidase. However, the choline esters (acetylcholine, butyrylcholine, propionylcholine, benzoylcholine and succinylcholine) markedly inhibited the peptidase activity in parallel to PsChE. Choline alone or in combination with acetate, butyrate, propionate, benzoate or succinate did not significantly inhibit the peptidase activity. It appeared that inhibitor compounds which bind to both the anionic and esteratic sites simultaneously (like the substrate analogues choline esters) could inhibit the peptidase activity possibly through conformational changes affecting a peptidase domain.

Amino Acids↗
PubMed1978

Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.

Alleles↗
PubMed1998

Quantitation and regulation of pyroglutamyl peptidase II messenger RNA levels in rat tissues and GH3 cells.

The distribution of the mRNA of the specific thyrotropin-releasing-hormone (TRH)-degrading enzyme pyroglutamyl peptidase II (EC 3.4.19.6) in rat tissues and brain regions and its regulation in rat tissues and in GH3 cells was studied by a reliable and quantitative solution hybridization ribonuclease protection assay. The distribution of pyroglutamyl peptidase II mRNA levels was uneven with the highest level of mRNA found in brain. Within brain the distribution of pyroglutamyl peptidase II mRNA was heterogeneous. A single dose of T3 markedly increased the level of pyroglutamyl peptidase II mRNA in the pituitary (p < 0.01) and in the liver (p < 0.05). In GH3 cells, exposure to T3 at concentrations from 10(-10) to 10(-6) M for time periods of 2-24 h, did not change pyroglutamyl peptidase II mRNA levels. Acute administration of TRH to rats had no effect on pyroglutamyl peptidase II mRNA levels. By contrast, TRH down-regulated pyroglutamyl peptidase II mRNA in GH3 cells. A similar effect was produced in GH3 cells by activators of protein kinase C. These studies reveal fundamental differences in the mechanism of regulation of pyroglutamyl peptidase II mRNA in pituitary and in GH3 cells. Elevation of pyroglutamyl peptidase II mRNA in liver by T3 suggests that this organ is the source of the enzyme in serum.

Aminopeptidases↗
PubMed1990

Residues flanking the COOH-terminal C-region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase and the extent of coupling of its co-translational translocation and proteolytic processing in vitro.

The polar, COOH-terminal c-region of signal peptides has been considered to be most important for influencing the efficiency and fidelity of signal peptidase cleavage while the hydrophobic core or h-region appears indispensable for initiating translocation. To identify structural features of residues flanking the c-region that influence the fidelity and efficiency of signal peptidase cleavage as well as co-translational translocation, we introduced six amino acid substitutions into the COOH terminus of the hydrophobic core and seven substitutions at the NH2 terminus of the mature region (the +1 position) of a model eukaryotic preprotein-human pre(delta pro)apoA-II. This preprotein contains several potential sites for signal peptidase cleavage. The functional consequences of these mutations were assayed using an in vitro co-translational translocation/processing system and by post-translational cleavage with purified, detergent-solubilized, hen oviduct signal peptidase. The efficiency of translocation could be correlated with the hydrophobic character of the residue introduced at the COOH terminus of the h-region. Some h/c boundary mutants underwent co-translational translocation across the microsomal membrane with only minimal cleavage yet they were cleaved post-translationally by hen oviduct signal peptidase more efficiently than other mutants which exhibited a high degree of coupling of co-translational translocation and cleavage. These data suggest that features at the COOH terminus of the h-domain can influence "presentation" of the cleavage site to signal peptidase. The +1 residue substitutions had minor effects on the extent of co-translational translocation and processing. However, these +1, as well as h/c boundary mutations, had dramatic effects on the site of cleavage chosen by signal peptidase, indicating that residues flanking the c-region of this prototypic eukaryotic signal peptide can affect the fidelity of its proteolytic processing. The site(s) selected by canine microsomal and purified hen oviduct signal peptidase were very similar, suggesting that "intrinsic" structural features of this prepeptide can influence the selectivity of eukaryotic signal peptidase cleavage, independent of the microsomal membrane and associated translocation apparatus.

Amino Acid Sequence↗
PubMed1986

Intestinal brush border peptidases: activities in normal and abnormal peroral intestinal biopsy specimens.

Discriminating substrates were used to develop assays that accurately measure the activity of each of four brush border peptidases in peroral jejunal biopsy specimens. Seventy-five biopsy specimens, 43 normal and 32 abnormal, were assayed for aminopeptidase A (EC 3.4.11.7), aminopeptidase N (EC 3.4.11.2), and disaccharidases, and 37 of these were assayed for membrane glycylleucine peptidase and Zn2+-stable aspartyllysine peptidase. Peptidase levels in normal biopsy specimens from males and females did not differ. Only aminopeptidase A levels changed significantly with age. The mean +/- SD units per gram of protein for each enzyme in peroral biopsy specimens of normal intestine were aminopeptidase A 1.7 +/- 0.7 for children and 3.1 +/- 1.3 for adults, membrane glycylleucine peptidase 16.3 +/- 10.6, aminopeptidase N 35.5 +/- 13.2, and Zn2+-stable aspartyllysine peptidase 42.6 +/- 28.5. In abnormal biopsy specimens, levels of three of the peptidases and the disaccharidases were significantly decreased, 37% to 51%. In contrast, aminopeptidase N activity was decreased only 7% in the abnormal biopsy specimens. These results provide a basis for further studies investigating the role of brush border peptidases in normal digestion and various pathologic states, including the search for individuals with brush border peptidase deficiencies.

Adolescent↗