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During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dual-target anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei. We found that although the Plasmodium M1 aminopeptidases share a largely similar, broad specificity at the P1 position, the P. falciparum M1 displays the greatest diversity in specificity and P. berghei M1 showing a preference for charged P1 residues. In contrast, the Plasmodium M17 aminopeptidases share a highly conserved preference for hydrophobic residues at the P1 position. The aminopeptidases also demonstrated intra-peptide sequence specificity, particularly the M1 aminopeptidases, which showed a definitive preference for peptides with fewer negatively charged intrapeptide residues. Overall, the P. vivax and P. berghei enzymes had a faster substrate turnover rate than the P. falciparum enzymes, which we postulate is due to subtle differences in structural dynamicity. Together, these results build a kinetic profile that allows us to better understand the catalytic nuances of the M1 and M17 aminopeptidases from different Plasmodium species.

Toxoplasmosis is a parasitic disease caused by infection with Toxoplasma gondii that currently has few therapeutic options. The M1 aminopeptidase enzymes have been shown to be attractive targets for anti-parasitic agents and/or vaccine candidates, suggesting potential to re-purpose inhibitors between parasite M1 aminopeptidase targets. The M1 aminopeptidase TgAPN2 has been suggested to be a potential new drug target for toxoplasmosis. Here we investigate the structure and function of TgAPN2, a homologue of the antimalarial drug target PfA-M1, and evaluate the capacity to use inhibitors that target PfA-M1 against TgAPN2. The results show that despite a similar overall fold, the TgAPN2 has a unique substrate specificity and inhibition profile. Sequence and structure differences are investigated and show how comparative structure-activity relationships may provide a route to obtaining potent inhibitors of TgAPN2.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease tripeptidyl-peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6°C to 64.4°C and increased its enzymatic half-life at 60°C from 5.4 min to 21.9 min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL patient-derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.

Malaria is one of the world's most prevalent parasitic diseases, with over 200 million cases annually. Alarmingly, the spread of drug-resistant parasites threatens the effectiveness of current antimalarials and has made the development of novel therapeutic strategies a global health priority. Malaria parasites have a complicated lifecycle, involving an asymptomatic ‘liver stage’ and a symptomatic ‘blood stage’. During the blood stage, the parasites utilise a proteolytic cascade to digest host hemoglobin, which produces free amino acids absolutely necessary for parasite growth and reproduction. The enzymes required for hemoglobin digestion are therefore attractive therapeutic targets. The final step of the cascade is catalyzed by several metalloaminopeptidases, including aminopeptidase P (APP). We developed a novel platform to examine the substrate fingerprint of APP from Plasmodium falciparum (PfAPP) and to show that it can catalyze the removal of any residue immediately prior to a proline. Further, we have determined the crystal structure of PfAPP and present the first examination of the 3D structure of this essential malarial enzyme. Together, these analyses provide insights into potential mechanisms of inhibition that could be used to develop novel antimalarial therapeutics.

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln181 as important for enzymatic activity and substrate specificity. Replacement of Gln181 by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln181 is critical for the enzymatic activity and substrate specificity of ERAP-1.

Several investigators have independently identified membrane-associated aminopeptidases in the midgut of insect larvae as the initial interacting ligand to the insecticidal crystal proteins of Bacillus thuringiensis. Though several isoenzymes of aminopeptidases have been identified from the midgut of an insect and their corresponding cDNA cloned, only one of the isoform has been expressed heterologously and studied for its binding to Cry toxins. Here we report the cloning and expression of two aminopeptidases N from Helicoverpa armigera (American cotton bollworm) (HaAPNs). The full-length cDNA of H. armigera APN1 (haapn1) is 3205bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116bp in size and corresponds to a 1012-amino-acid protein. Structurally these proteins show sequence similarity to other insect aminopeptidases and possess characteristic aminopeptidase motifs. Both the genes have been expressed in Trichoplusia ni (cabbage looper) cells using a baculovirus expression vector. The expressed aminopeptidases are membrane-associated, catalytically active and glycosylated. Ligand-blot analysis of both these aminopeptidases with bioactive Cry1Aa, Cry1Ab and Cry1Ac proteins displayed differential interaction. All the three toxins bound to HaAPN1, whereas only Cry1Ac interacted with HaAPN2. This is the first report demonstrating differential Cry-toxin-binding abilities of two different aminopeptidases from a susceptible insect.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal recessive childhood disease caused by mutations in the CLN2 gene, which encodes the lysosomal enzyme tripeptidyl peptidase I. As a step towards understanding the protein and developing therapeutics for the disease, we have produced and characterized recombinant human CLN2 (ceroid lipofuscinosis, neuronal 2) protein from Chinese-hamster ovary cells engineered to secrete high levels of the enzyme. The protein was secreted as an inactive soluble proenzyme of approximately 65 kDa that appears as a monomer by gel filtration. Upon acidification, the protein is processed to mature form and acquires activity. The enzyme is efficiently delivered to the lysosomes of LINCL fibroblasts by mannose 6-phosphate-receptor-mediated endocytosis (EC(50) approximately 2 nM), where it remains active for long periods of time (t(1/2) approximately 12 days). In addition, the enzyme is taken up by rat cerebellar granule neurons by mannose 6-phosphate-dependent and -independent mechanisms. Treatment of LINCL fibroblasts with recombinant CLN2 protein restores normal enzyme activity and ameliorates accumulation of the major storage protein, mitochondrial ATP synthase subunit c.

An aminopeptidase from Vibrio proteolyticus was translated as a preproprotein consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. Protein expression and analysis of the activity results demonstrated that the N-terminal propeptide was essential to the formation of the active enzyme in Escherichia coli. Urea dissolution of inclusion bodies and dialysis indicated that the N-terminal propeptide could facilitate the correct folding of the enzyme in vitro. Using l-Leu-p-nitroanilide as the substrate, the kinetic parameters (kcat and Km) of the pro-aminopeptidase and processed aminopeptidases were analysed. The results suggested that the N-terminal propeptide inhibited enzyme activity of the mature region. In contrast, the C-terminal propeptide did not show evidence of forming an active enzyme, of correctly folding in vitro or of inhibiting the active region.

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5´-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca2+-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the metal chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.

An aminopeptidase gene named XAP has been isolated from Xanthomonas campestris pv. citri, a plant pathogenic bacterium. The bacterium is one of the rare micro-organisms susceptible to ascamycin, an aminoacyl nucleoside antibiotic that inhibits protein synthesis. Sequence analysis reveals that the gene encodes a 311 amino acid protein with a calculated molecular mass of 35134 Da and approx. 50% identity for amino acids to the proline iminopeptidase from Neisseria gonorrhoeae. The XAP gene product, Xap, expressed in Escherichia coli has proline iminopeptidase activity as well as ascamycin dealanylating activity in vitro.

Desensitization of recombinant human thrombin receptors expressed in Sf9 insect cells was compared with native thrombin receptors in megakaryoblast erythroleukaemia (HEL) cells. Addition of thrombin (2 units/ml) or agonist peptide SFLLRN (10 microM) to HEL cells, or to Sf9 cells infected with recombinant baculovirus containing the thrombin receptor cDNA, produced an increase in the free cytosolic Ca2+ concentration ([Ca2+]i) as measured by fura-2. The response in HEL cells was transient, reflecting a rapid homologous desensitization. In contrast, [Ca2+]i in Sf9 cells expressing the thrombin receptor increased rapidly to a peak value that slowly declined, but remained elevated for at least 12 min following stimulation by thrombin. The sustained [Ca2+]i response to thrombin was not reversed by washout of thrombin or by any subsequent addition of hirudin. Pretreatment of Sf9 cells with either thrombin (2 units/ml) or SFLLRN (10 or 50 microM) for 5 min produced a shift in the ED50 for SFLLRN (added 10 min after washout) from 0.4 microM to 20 and 7 microM, respectively. Thus, desensitization of thrombin receptors expressed in Sf9 cells occurs slowly and reflects a decrease in receptor affinity. The sustained [Ca2+]i response in Sf9 cells stimulated by thrombin may reflect continuous activation by the tethered ligand. To test this hypothesis, the effect of protease treatment during the sustained phase of the response was examined. Addition of either aminopeptidase M or thermolysin reversed the sustained response to SFLLRN, but only thermolysin reversed the sustained response to thrombin. Thermolysin had no effect on the change in [Ca2+]i observed following carbachol stimulation of Sf9 cells expressing the M5 muscarinic receptor. Furthermore, following thermolysin treatment, the cells remained responsive to a subsequent application of SFLLRN. These results demonstrate that the tethered ligand remains active for extended periods of time after thrombin stimulation and suggests that further hydrolysis by extracellular proteases may represent an important mechanism of rapid receptor deactivation.

Tripeptidyl-peptidase II (TPP II) is a cytosolic high-M(r) exopeptidase with an active site of the subtilisin type. This paper describes cloning of cDNA encoding murine TPP II. Four clones were isolated from a murine mastocytoma cDNA library and the 5′-end was isolated by use of 5′-RACE (rapid amplification of cDNA ends). A total of 4611 bp were isolated, including the complete coding region. The deduced amino acid sequence shows a 96% overall identity when compared with the previously cloned human TPP II. The remarkably high identity indicates that not only the catalytic domain, but almost the entire subunit, must be of functional importance. Alignment with subtilisin-like serine peptidases identified Asp44, His264 and Ser449 as the catalytic triad, thus defining an extra domain of approximately 200 amino acids between the catalytic Asp and His in TPP II as compared with other subtilases. In addition, it was demonstrated that different polyadenylation signals can be utilized, since two different clones with untranslated 3′-ends of 155 bp and 781 bp respectively have been isolated. Finally, one of the isolated clones contains an extra 39 bp insert encoding 13 amino acids, which implies alternative splicing of the mRNA.

Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5′-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.

Although lymphocytes are CD-13-negative and therefore should not express the ectoenzyme aminopeptidase N (AP-N), there have been a number of reports suggesting the presence of a cell-surface aminopeptidase with many similarities to AP-N. We have determined aminopeptidase activity with 4-methyl-7-coumarylamide (NMec) derivatives of alanine, leucine, lysine and arginine in Jurkat cells (a human T-cell lymphoma line) and in HL60 cells (a CD-13-positive myeloid leukaemia line) and compared the activities with those of purified pig AP-N and human renal microvillar membranes. Jurkat cell aminopeptidase activity doubled on disrupting the cells and the sensitivity to amastatin increased. When the cells were fractionated only 4% of the activity was recovered in the membrane fraction, compared with 87% recovery for alkaline phosphatase. The profile of activities for intact Jurkat cells was Leu > Ala > Lys > Arg, changing in the cytosolic fraction to Lys > or = Arg > Leu = Ala; the profiles for intact HL60 cells and AP-N were identical, namely Ala > Leu > Arg > Lys. The Km values for the hydrolysis of Ala-NMec and Leu-NMec by Jurkat cells were 65 microM and 11 microM, in each case some 6-fold lower than those for AP-N. The pH-activity curves for the hydrolysis of Ala-NMec by Jurkat cells and human renal microvillar membranes were displaced by almost 1 pH unit and the activity was not sensitive to the anionic composition of the buffers. However, a 3-fold activation of the cytosolic activity by 0.1 M NaCl was observed with Arg-NMec as substrate. With Ala-NMec as substrate, the sensitivity of the aminopeptidase activity to inhibitors increased markedly after disrupting the cells, but still differed from that observed with purified pig AP-N; the concentrations giving 50% inhibition were as follows (values for AP-N in parentheses): amastatin. 28 nM (150 nM); bestatin, 12 microM (43 microM), probestin, 100 nM (< 10 nM), puromycin, 30 microM (> 1 mM). Anion exchange chromatography on Mono Q revealed two activities: that of peak I preferentially hydrolysed Arg-NMec, was activated by NaCl and was insensitive to amastatin; while that of peak II was strongly inhibited by amastatin and had a broad specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

To investigate the role of post-translational events in intestinal cell differentiation we have studied the effects of heat shock on processing and cell surface delivery of sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV) and aminopeptidase N (APN) in Caco-2 cells. In cells cultured at 42.5 degrees C there was a rapid decline in sucrase activity, while DPPIV and APN were unaffected over a 3-day period. Immunofluorescence staining confirmed the selective disappearance of SI from the surface membrane after only 1 day of culture at 42.5 degrees C. Cell-surface biotinylation of cells metabolically labelled with [35S]methionine 4 h after a switch from 37 degrees C to 42.5 degrees C demonstrated that newly synthesized APN and DPPIV were associated with the surface membrane, while SI was almost completely retained intracellularly. Pulse-chase experiments confirmed that, in these cells, DPPIV and APN were normally processed and vectorially delivered to the cell surface; in contrast, conversion between the two conformationally distinct high-mannose precursor forms of SI (hmP1 and hmP2) was markedly inhibited, a significant fraction of newly synthesized enzyme was degraded, probably in the ER, and an immature form of complex-glycosylated SI precursor (cP) was produced and mostly retained intracellularly. Double labelling of Caco-2 cells for SI and cathepsin D excluded an accumulation of SI in the lysosomes, suggesting that this organelle was not involved in the degradation of SI. These results indicate that the ER may play an important role in intestinal cell differentiation by regulating the conformational maturation, degradation and eventual cellular localization of some digestive enzymes.

The expression of cell-surface peptidases was examined in two human colon carcinoma cell lines, Caco-2 and HT-29. Enzymic assays revealed the presence of eight cell-surface peptidases on a Caco-2 cell line (passage number 82-88), namely aminopeptidase N, dipeptidyl peptidase IV, peptidyl dipeptidase A (angiotension-converting enzyme), aminopeptidase P, aminopeptidase W, endopeptidase-24.11, gamma-glutamyl transpeptidase and membrane dipeptidase. The presence of dipeptidyl peptidase IV and endopeptidase-24.11 was also confirmed immunochemically. After 15 days culture, the activities of aminopeptidase P, peptidyl dipeptidase A and alkaline phosphatase activities on Caco-2 cells reached a plateau, and that of membrane dipeptidase began to decline. In contrast, aminopeptidase N, dipeptidyl peptidase IV and endopeptidase-24.11 activities were still rising after 26 days in culture. Caco-2 cells of passage number 181-183 were found to lack endopeptidase-24.11, but maintained dipeptidyl peptidase IV expression. Two populations of HT-29 cells were surveyed. Both the standard, undifferentiated population and a differentiated population expressed only three peptidases: dipeptidyl peptidase IV, aminopeptidase W and carboxypeptidase M. In the differentiated HT-29 cells the activity of dipeptidyl peptidase IV after 14-21 days was beginning to plateau whereas aminopeptidase W activity was still rising and that of carboxypeptidase M had begun to decline. These differences in activity profiles observed among this group of cell-surface peptidases indicate that these cell lines, especially Caco-2, are useful models to study the regulation of their expression.

A 130 kDa glycoprotein (GP 130) was purified from porcine renal brush-border membranes by affinity chromatography using immobilized 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate (SITS)- and concanavalin A-Sepharose. GP 130 was the major concanavalin A-binding protein in porcine renal brush-border membranes and also bound Ricinus communis (castor-bean) and wheat-germ agglutinins. Endo-beta-N-acetylglucosaminidase F reduced the molecular mass of GP 130 by 20 kDa as determined by SDS/PAGE, whereas endo-beta-N-acetylglucosaminidase H reduced the molecular mass by 5 kDa, showing that GP 130 contained both complex and high-mannose carbohydrate structures. Western-blot analyses using an antibody raised against GP 130 showed that it was localized to the brush-border membrane fraction and was present in a membrane fraction of the pig kidney cell line LLC-PK1. The N-terminal sequence and amino acid composition of GP 130 showed that GP 130 is similar to rat kidney zinc peptidase and human intestinal aminopeptidase N. GP 130 had aminopeptidase N enzymic activity and was inhibited by bestatin (Ki = 36 microM), 1,10-phenanthroline (Ki 30 microM), Zn2+ (Ki 26 microM), Cu2+ (Ki 260 microM), pre-incubation with EDTA and by a polyclonal antibody against GP 130. Bicarbonate and iodide blocked the binding of GP 130 to the SITS-affinity resin, showing that GP 130 has an anion-binding site. Neither these anions nor stilbene disulphonates affected the aminopeptidase N activity of GP 130.

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)–MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.

By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour.

Aminopeptidase W is a newly discovered enzyme of the renal and intestinal brush borders, having been first isolated as a 130 kDa glycoprotein recognized by a monoclonal antibody [Gee & Kenny (1985) Biochem. J. 230, 753-764]. It is particularly effective in the hydrolysis of dipeptides, Glu-Trp (Km 0.57 mM; kcat. 6770 min-1) being a favoured substrate. Dipeptides with tryptophan, phenylalanine or tyrosine in the P1 position were rapidly hydrolysed, but the requirements in respect of the P1 residue were not stringent. The activity of aminopeptidase W is markedly influenced by ionic conditions. The highest activity was observed in 100 mM-Tris/HCl, pH 8; phosphate ions were strongly inhibitory. Activity was also greatly affected by bivalent metal ions, and the magnitude and direction of the effects depended on the nature of the buffer anions and on pH. The most effective inhibitors were amastatin and bestatin. Some thiols also inhibited, but other chelating agents, EDTA and 1,10-phenanthroline, had no effect over the concentration range 1-10 mM. Other group-specific inhibitors, for cysteine, serine or aspartic peptidases, were also ineffective. Some molecular properties were studied. Deglycosylation by treatment with N-glycanase diminished the apparent subunit Mr from 130,000 to 90,000. The enzyme contained zinc, 1.2 atoms/subunit, and in spite of the atypical properties of this enzyme in respect of chelating agents, a zinc-catalysed mechanism is the most probable. Its roles in digestion and in renal function are not yet clear.

Orientation of rat renal and intestinal brush-border membrane vesicles was studied with two independent methods: electron-microscopic freeze-fracture technique and immunological methods. With the freeze-fracture technique a distinct asymmetric distribution of particles on the two membrane fracture faces was demonstrated; this was used as a criterion for orientation of the isolated membrane vesicles. For the immunological approach the accessibility or inaccessibility of aminopeptidase M localized on the outer surface of the cell membrane to antibodies was used. With both methods we showed that the brush-border membrane vesicles isolated from rat kidney cortex and from rat small intestine for transport studies are predominantly orientated right-side out.

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a ‘high-mannose’ state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.

The activity of a purified cytosolic aminopeptidase (Mr 79,000) from monkey brain was stimulated about 4-fold by ATP-Mg2+. The stimulation was seen with either synthetic aminopeptidase substrates or natural peptides such as enkephalins. Both ATP and Mg2+ were required for stimulation, and ADP did not inhibit the stimulation. Non-hydrolysable analogues of ATP, deoxy-ATP and other nucleoside triphosphates stimulated to a lesser extent compared with ATP, whereas nucleoside mono- or di-phosphates were ineffective. The enzyme did not exhibit any ATPase activity. An ATPase inhibitor, orthovanadate, had no inhibitory effect on the ATP-Mg2+ stimulation. The aminopeptidase was not autophosphorylated by [gamma-32P]ATP and Mg2+, but in the presence of cyclic AMP-dependent protein kinase underwent phosphorylation on serine residue(s). Phosphorylation resulted in inactivation of the aminopeptidase activity, and also resulted in a decreased stimulation of the enzyme by ATP-Mg2+.

The biogenesis of two microvillar enzymes, aminopeptidase N (EC 3.4.11.2) and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. Both enzymes became inserted into the membrane during or immediately after polypeptide synthesis, indicating that translation takes place on ribosomes attached to the rough endoplasmic reticulum. The earliest detectable forms of aminopeptidase and sucrase-isomaltase were polypeptides of Mr 140 000 and 240 000 respectively. These polypeptides were susceptible to treatment with endo-β-N-acetylglucosaminidiase H (EC 3.2.1.96), suggesting that the microvillar enzymes during or immediately after completion of protein synthesis become glycosylated with a ‘high-mannose’ oligosaccharide structure similarly to other plasma-membrane and secretory proteins. After 20-40 min or 60-90 min of chase, respectively, aminopeptidase N and sucrase-isomaltase were reglycosylated to give the polypeptides of Mr 166 000 (aminopeptidase N) and 265 000 (sucrase-isomaltase). These were expressed at the microvillar membrane after 60-90 min. During the entire process of synthesis and transport to the microvillar membrane the enzymes were bound to membranes, indicating that the biogenesis of aminopeptidase N and sucrase-isomaltase occurs in accordance with the membrane flow hypothesis.

A novel two-step enzyme-linked assay for aminopeptidase W is described and validated by comparison with other assays. L-alpha-Glutamyl-L-tryptophan (Glu-Trp) is a favoured substrate for this enzyme. With the use of glutamate dehydrogenase (EC 1.4.1.2) in a second step, the assay measured the release of free glutamate from L-alpha-glutamyl-L-tryptophan by the increase in NADH fluorescence. In the presence of 5 mM-1,10-phenanthroline and 50 microM-cilastatin the contribution of other membrane peptidases, in particular aminopeptidases N and A and microsomal dipeptidase in kidney, was very small. Residual cytosolic activities hydrolysing Glu-Trp were sensitive to inhibition by 2.5 mM-N-ethylmaleimide. The activity of aminopeptidase W was unaffected by these inhibitors. There was good correlation between the fluorimetric assay and those in which the free tryptophan released by kidney membrane fractions was determined by h.p.l.c. or the aminopeptidase W was measured immunoradiometrically with a monoclonal antibody.

Pig kidney microvillar proteins were extracted with octyl β-glucoside and reconstituted in liposomes prepared from microvillar lipids of known composition. Four peptidases, namely endopeptidase (EC 3.4.24.11), aminopeptidases N (EC 3.4.11.2) and A (EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), were shown to be reconstituted. At lipid/protein ratios greater than 4:1, about half the detergent-solubilized protein and nearly all of the activity of the four peptidases were reconstituted. Dissolution of the liposomes with Triton X-100 did not increase the activity of any of these peptidases, a result consistent with an asymmetric, ‘right-side-out’, orientation of these enzymes. When purified, endopeptidase was subjected to the same procedure; the two amphipathic forms of the enzyme (the detergent form and the trypsin-treated detergent form) were fully reconstituted. The amphiphilic form, purified after toluene/trypsin treatment, failed to reconstitute. Electron microscopy of microvilli showed that the appearance of the surface particles was profoundly altered by treatment with papain. Before treatment, the microvilli were coated with particles of stalk lengths ranging from 2.5 to 9 nm. After papain treatment nearly all the particles had stalks of 2-3 nm. Reconstituted microvillar proteins in liposomes showed the same heterogeneity of stalk length. In contrast, liposomes containing reconstituted endopeptidase revealed a very homogeneous population of particles of stalk length 2 nm. Since the smallest dimension of a papain molecule is 3.7 nm, the ability of papain, and other proteinases of similar molecular size, to release microvillar enzymes is crucially affected by the length of the junctional peptide that constitutes the stalk of this type of membrane protein.

Studies of crude extracts of pea seeds (Pisum sativum, var. Green feast) revealed the presence of three enzymes that hydrolyse the amide bond of aminoacyl β-naphthylamides. They differ in their specificity towards the aminoacyl moiety; one is proline-specific, whereas the other two hydrolyse the β-naphthylamides of primary amino acids. Of the latter, one is highly specific for hydrophobic aminoacyl residues whereas the other has a broader, somewhat complementary specificity, showing preferential hydrolysis of non-hydrophobic aminoacyl residues. These latter two aminoacyl-β-naphthylamidases have been separated and partly characterized with regard to substrate specificity and antagonism by inhibitors. Both are true aminopeptidases, requiring the presence of a free amino group and hydrolysing the amide bonds of amino acid amides, dipeptides and oligopeptides consecutively from the N-terminal end.

Human peripheral lymphocytes were found to contain at least two distinct aminopeptidases, designated cytosol aminopeptidase and microsomal aminopeptidase, which differed from one another with respect to intracellular localization, substrate specificity, metal-ion activation, Km value and electrophoretic mobility. No change in these aminopeptidase activities was observed in cultured lymphocytes in the absence of mitogen throughout the cultivation period. The addition of phytohaemagglutinin or concanavalin A to the culture medium caused, in dose-dependent manner, a significant increase in cytosol aminopeptidase activity in lymphocytes. On the other hand, no increase in microsomal aminopeptidase activity was observed under the same conditions. The biochemical properties of aminopeptidases in stimulated cultured lymphocytes were identical with those of the enzymes in peripheral lymphocytes and unstimulated cultured lymphocyte. The phytohaemagglutinin dose-response curves for lymphocyte activation as measured by the DNA synthesis rate and for cytosol aminopeptidase activity were observed to be similar. However, when DNA synthesis was temporarily blocked by hydroxyurea, the rate of increase of aminopeptidase activity was unaffected. Pokeweed mitogen only slightly increased the cytosol aminopeptidase activity in cultured lymphocytes, although the lymphocytes were highly activated.

Hydrolysis of histones by proteinases from rat liver, skin and other sources was studied by using a rat thymus histone preparation as the substrate and polyacrylamide-gel electrophoresis and densitometric analysis as the methods to detect histone subtypes and their hydrolysis. The rat mast-cell proteinase I effectively hydrolysed histones except type H4. Thrombin hydrolysed effectively histones H1 and H2A, whereas plasmin hydrolysed all types of histones. Cathepsin D hydrolysed especially histone H2A. Cathepsins B and L hydrolysed all histones more slowly, and cathepsin H hydrolysed them extremely slowly. Epidermal aminoendopeptidase did not hydrolyse histones. Trypsin and chymotrypsin were used as reference enzymes, which hydrolysed all types of histones in very low concentrations. This study suggests that a variety of proteinases could play a role in histone hydrolysis. Hydrolysis of a specific subtype of histones, such as histone H2A at pH 6 by cathepsin D, may be directly involved in regulation of epidermal-cell differentiation.

Aminopeptidase N (EC 3.4.11.2), when isolated from pig intestine in either the proteinase- or detergent-released form, frequently appears to contain three polypeptide chains, here termed alpha, beta and gamma. We have established by an immunological technique that the beta- and gamma-polypeptides are derived from the alpha-chain and that the intact enzyme is a dimer, alpha 2. Each alpha-chain of the detergent form was shown to contain a hydrophobic anchor peptide about 35 amino acid residues in length, which included the N-terminal sequences. A peptide bond in the alpha-chain was very sensitive to proteolysis. Its cleavage generated the commonly observed forms: alpha beta gamma and beta 2 gamma 2. The gamma-fragment, which lacked the anchor peptide, was derived from the C-terminal part of the alpha-chain.

Tripeptidyl peptidase II is an extralysosomal serine peptidase of an unusually large size, i.e. Mr greater than 10(6) for the native enzyme and Mr 135000 for the subunit. The enzyme from human erythrocytes was studied by electron microscopy on samples negatively stained by ammonium molybdate. Two different structural representations of the purified enzyme were obtained, both with a length of about 50 nm, and consisting of repetitive substructures. Upon dialysis of the enzyme against a Tris/HCl buffer, the activity was gradually decreased. This decrease was shown to parallel the dissociation of the large enzyme structures into smaller ones, the smallest measuring 3 nm by 10 nm and apparently corresponding to the repetitive substructures. The results indicate that a large polymeric form of the enzyme is a prerequisite for full activity.

The amounts of lactase (beta-D-galactosidase, EC 3.2.1.23), sucrase (sucrose alpha-D-glucohydrolase, EC 3.2.1.48), maltase (alpha-D-glucosidase, EC 3.2.1.20) microvillus aminopeptidase (EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.-) in tangentially sectioned biopsies from jejunum were studied by quantitative immunoelectrophoresis and enzymic assays. All enzymes had their maximum activities near the mid-region of the villi and their lowest activities at the bases of the crypts. The ratio between enzyme activity and immunoreactive protein was constant along the villus-crypt axis. This result is consistent with a continuous brush-border-enzyme synthesis as the enterocytes migrate up the villi.

A nucleoside antibiotic, ascamycin (9-beta-[5′-0-(N-L-alanyl) sulphamoyl-D-ribofuranosyl]-2-chloroadenine), has a selective antibacterial activity against Xanthomonas species. When ascamycin was dealanylated, dealanylascamycin showed a broad antibacterial activity against various Gram-negative and Gram-positive bacteria. Xanthomonas citri is susceptible to ascamycin by virtue of the ascamycin-dealanylating enzyme on the cell surface [Osada & Isono (1985) Antimicrob. Agents Chemother. 27, 230-233]. The enzyme (Xc aminopeptidase) was purified from X. citri cells by successive DEAE-cellulose, chromatofocusing and Sephadex G-100 column chromatography to a homogeneous state. The purified enzyme exhibited a single band with an Mr of 38 000 in SDS/polyacrylamide-gel electrophoresis. Gel filtration on a calibrated column indicated a similar Mr value. The isoelectric point of the enzyme was 5.7. The enzyme catalysed the hydrolysis of the alanyl group of ascamycin and liberated alanine from the sulphamoyl nucleoside. The enzyme also catalysed the hydrolysis of L-proline beta-naphthylamide and L-alanine beta-naphthylamide. The optimal pH and temperature for enzyme activity were pH 7.5-8.0 and 35-40 degrees C respectively. The enzyme was inhibited by thiol-enzyme inhibitors (i.e. rho-chloromercuribenzoate and N-ethylmaleimide), but was not affected by various naturally occurring aminopeptidase inhibitors (i.e. amastatin, bestatin, pepstatin and leupeptin). Mn2+ and Mg2+ activated the enzyme, whereas Cu2+, Zn2+ and Cd2+ were inhibitory.

1. The peptides obtained by tryptic digestion of S-[14C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.

An organ culture employing slices of renal-cortex tissue from piglets of the Yucatan strain was used to study the biogenesis of four microvillar peptidases: endopeptidase-24.11 (EC 3.4.24.11), dipeptidyl peptidase IV (EC 3.4.14.5), aminopeptidase N (EC 3.4.11.2) and aminopeptidase A (EC 3.4.11.7). The viability of the culture system was confirmed by the preservation of ultrastructural integrity and by an unchanged uptake of [3H]alanine into cells during the period of the experiments. After labelling with [35S]methionine, treatment with Mg2+ yielded two fractions, one containing microvilli and another, the Mg2+ pellet, containing intracellular and basolateral membranes. The labelled forms of the peptidases, isolated by immunoprecipitation, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The Mg2+ pellet contained the earliest detectable forms of the enzymes. In each case, a polypeptide of lower Mr than the mature form and sensitive to treatment with endo-beta-N-acetylglucosaminidase H was the first form to be detected. These high-mannose forms were followed, about 30 min after the pulse, by a complex glycosylated form of higher Mr. Only the latter form was observed in microvilli and then only after 90 min of the chase period. A quantitative study of dipeptidyl peptidase IV showed that the forms observed in the Mg2+ pellet were precursors of those in the microvillar fraction. No labelled forms were observed in the cytosol. All four peptidases were thus synthesized within membrane compartments and glycosylated in two steps before assembly in microvilli.

A crude RNA fraction, prepared from pig small intestine, was found to be more efficient than a fraction enriched in polyadenylated RNA in directing translation of polypeptides with Mr greater than 100000 in a rabbit reticulocyte lysate system. Aminopeptidase N (EC 3.4.11.2) synthesized in vitro was immunopurified from the translation mixture and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It was found to have an apparent Mr of 115000 regardless of whether the translation was performed in the absence or presence of proteinase inhibitors. This result contradicts the possibility of aminopeptidase N being synthesized as a large single-chain precursor polypeptide.

Human muscle enzymes that hydrolyse dipeptide 2-naphthylamides were separated by chromatography on DEAE-Sephadex. Evidence was obtained that these substrates are hydrolysed by arylamidase as well as by dipeptidyl aminopeptidases.

The enzymes required to convert the prohormone angiotensin I into angiotensins II and III, secretagogues of aldosterone, are enriched in association with capillary endothelium isolated from rat adrenal cortex. Thus the secretion of aldosterone may be controlled, in part, by processing of peptides occurring within the adrenal gland itself.

1. A procedure was developed for the preparation of plasma membranes from experimental granulation tissue of the rat without the addition of enzymes. The yield is better than 20% and the purification at least tenfold. 2. Values are given for the activities of 5′-nucleotidase, Na-+, k-+-activated Mg-2+dependent adenosine triphosphatase and leucine β-naphthylamidase, for lipid composition, and for the gel-electrophoretic patterns of proteins and glycoporteins in the membrane preparations. 3. The plasma membranes from the mature granulation tissue contain proportionally more protein in the lipid phase, but the specific activities of 5′-nucleotidase and Na-+,K-+-activated Mg-2+-dependent adenosine triphosphatase are smaller than in the proliferating tissue. Certain differences were repeatedly observed in the gel-electrophoretic patterns of the developmental phases. 4. The plasma membranes from the granulation tissue were compared with those from rat peritoneal macrophages and from embryonic-chick tendon cells.

1. The preparation of gram quantities of isolated epithelial-cell ;ghosts' from mucosal scrapings of rat small intestine is described. The method involves dispersing the tissue by gentle homogenization in 6% dextran in Krebs-Ringer phosphate, pH7.4, followed by filtration through nylon cloth and sedimentation by low-speed centrifuging. 2. The isolated epithelial-cell ;ghosts' contained all of the DNA, but only 52% of the protein and 53-57% of the RNA of the original homogenate. They contained most of the activity of the following enzymes found in the homogenate: aminopeptidase (71%); alkaline beta-glycerophosphatase (82%); invertase (92%); adenosine triphosphatase (93-116%); acid beta-glycerophosphatase (83%); nonspecific esterase (76%); succinate dehydrogenase (96%). Only small proportions of the total lactate-dehydrogenase (10%) and phosphoglucose-isomerase (2%) activities found in the homogenate were recovered in the isolated cell ;ghosts'. 3. The epithelial-cell ;ghost' preparation did not respire unless cofactors and substrates were added, and did not consume glucose or produce lactic acid from glucose. 4. The effect of varying the composition of the homogenization medium was studied. Concentrations of dextran (mol.wt. 15x10(4)) from 1 to 12%, solutions of dextrans (all at 6%) with mol.wt. varying between 3.6x10(4) and 2x10(6), and a solution of 8% polyethylene glycol (mol.wt. 4000) served equally well for the production of epithelial-cell ;ghosts'. Two of these solutions, however, 12% dextran (mol.wt.15x10(4)) and 6% dextran (mol.wt. 2x10(6)), were too viscous to allow the complete sedimentation of the cell ;ghosts' at low relative centrifugal forces. Omission of either Krebs-Ringer phosphate or dextran from the medium resulted in almost complete cell breakage during the homogenization. 5. The isolated cell ;ghosts' were used as a starting material for subcellular fractionation of rat intestinal mucosa by differential centrifugation. The distributions of protein and succinate-dehydrogenase activity among the fractions were compared with corresponding values in fractions isolated by differential centrifugation of mucosa homogenized in 0.3m-sucrose-5mm-EDTA, pH7.4. The method in which cell ;ghosts' were used as starting material gave a better separation and cleaner fractions than the method in which untreated mucosal scrapings were used.

The degree of solubilization of pig lymphocyte plasma membrane by sodium deoxycholate was determined at a variety of temperatures and detergent concentrations. Approx. 95% of the membrane protein was soluble in 2% deoxycholate at 23 degrees C. Some of the biological activities of the membrane survived this treatment. The leucine beta-naphthylamidase activity was more readily soluble than the 5'-nucleotidase and these enzymes could be separated by extraction with 0.5% deoxycholate at 0 degrees C. Membrane solubilized in 2% deoxycholate at 23 degrees C was fractionated by sucrose-density-gradient centrifugation in 1% deoxycholate. The phospholipid was separated from the protein, which formed a fairly symmetrical peak that sedimented slightly slower than ovalbumin; the leucine naphthylamidase and 5'-nucleotidase activities were resolved from each other and from the main protein peak. Similar separations were achieved by elution from Sephadex G-200 and Sepharose 6B in 1% deoxycholate. The main proteins, however, appeared to possess much higher molecular weights than those indicated by sucrose-density-gradient centrifugation. This disparity suggests that many of the membrane proteins have a rod-like shape, especially since the results of experiments with [(14)C]deoxycholate revealed that the proteins did not bind significant amounts of deoxycholate. In contrast, 5'-nucleotidase and leucine naphthylamidase appeared to be globular. Polyacrylamide-gel electrophoresis of membrane solubilized in sodium dodecyl sulphate gave a similar distribution of protein to that achieved by sucrose-density-gradient centrifugation. Trace amounts only of polypeptides of molecular weight less than 10000 were detected.