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Intermediate cleavage peptidase (Icp55) processes a subset of mitochondrial matrix proteins by removing a bulky residue at their N termini, leaving behind smaller N-terminal residues (icp activity). This contributes towards the stability of the mitochondrial proteome. We report crystal structures of yeast Icp55 including one bound to the apstatin inhibitor. Apart from icp activity, the enzyme was found to exhibit Xaa-Pro aminopeptidase activity in vitro. Structural and biochemical data suggest that the enzyme exists in a rapid equilibrium between monomer and dimer. Furthermore, the dimer, and not the monomer, was found to be the active species with loop dynamics at the dimer interface playing an important role in activity. Based on the new evidence, we propose a model for binding and processing of cellular targets by Icp55. DATABASE: The atomic coordinates and structure factors for the structures of Icp55 (code 6A9T, 6A9U, 6A9V) have been deposited in the Protein Data Bank (PDB) (http://www.pdb.org/).

Peptidase-E, a nonclassical serine peptidase, is specific for dipeptides with an N-terminal aspartate. This stringent substrate specificity remains largely unexplained. We report an aspartate-bound structure of peptidase-E at 1.83 A resolution. In contrast to previous reports, the enzyme forms a dimer, and the active site is located at the dimer interface, well shielded from the solvent. Our findings further suggest that the stringent aspartate specificity of the enzyme is due to electrostatics and molecular complementarity in the active site. The new structural information presented herein may provide insights into the role of functionally important residues in peptidase-E.

The accumulation of beta amyloid (Aβ) has been a primary target for Alzheimer disease therapeutic strategies. Previously, we discovered an activity from Streptomyces sp. KK565 growth media that inhibits Aβ aggregation. The active component was an aminopeptidase and named Streptomyces sp. KK565 aminopeptidase (SKAP). SKAP cleaved N-terminal amino-acids of Aβ1–42 monomer, inhibited formation of fibrils and protected Aβ1–42-induced neurotoxicity. Over-expression of a human homolog of SKAP, glutamate carboxypeptidase II (hGCPII) in Aβ-oversynthesizing cells dramatically reduced the Aβ levels. These findings suggest a possible role of M28 family peptidases in preventing Aβ deposits in mammalian brain. Structured summary MINT- 7992796 : SKAP (uniprotkb: Q306T3 ) physically interacts (MI: 0915 ) with Abeta (uniprotkb: P05067 ) by protease assay (MI: 0435 ) MINT- 7992752 , MINT- 7992778 : SKAP (uniprotkb: Q306T3 ) binds (MI: 0407 ) to Abeta (uniprotkb: P05067 ) by protease assay (MI: 0435 )

In the yeast, Saccharomyces cerevisiae , two ubiquitin-like modifications, Apg12 conjugation with Apg5 and Apg8 lipidation with phosphatidylethanolamine, are essential for autophagy and the cytoplasm-to-vacuole transport of aminopeptidase I (Cvt pathway). As a unique E1-like enzyme, Apg7 activates two modifiers (Apg12 and Apg8) in an ATP-dependent manner and, for this activity, the carboxyl terminal 40 amino acids are essential. For a better understanding of the function of the carboxyl terminus of Apg7, we performed a sequential deletion of the region. A mutant expressing Apg7ΔC17 protein, which lacks the carboxyl 17 amino acids of Apg7, showed defects in both the Cvt pathway and autophagy. Apg8 lipidation is inhibited in the mutant, while Apg12 conjugation occurs normally. A mutant expressing Apg7ΔC13 protein showed a defect in the Cvt pathway, but not autophagy, suggesting that the activity of Apg7 for Apg8 lipidation is more essential for the Cvt pathway than for autophagy. Mutant Apg7ΔC17 protein is still able to interact with Apg8, Apg12 and Apg3, and forms a homodimer, indicating that the deletion of the carboxyl terminal 17 amino acids has little effect on these interactions and Apg7 dimerization. These results suggest that the carboxyl terminal 17 amino acids of Apg7 play a specific role in Apg8 lipidation indispensable for the Cvt pathway and autophagy.

Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin-binding region of each APN isoform. The results suggest that APN isoforms have a common toxin-binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS–PAGE is determined by factors such as expression level in conjunction with differences in binding affinity.

Tripeptidyl peptidase-I (TPP-I) is a lysosomal peptidase which cleaves tripeptides from the N-terminus of peptides. The function of the enzyme is unclear but its importance is demonstrated by the fact that mutations in TPP-I are responsible for late infantile neuronal ceroid lipofuscinosis, a lethal lysosomal storage disease. As a step towards identifying its natural substrates, we have used a series of synthetic peptides, based on angiotensin-II, to explore the effects of peptide chain length and the effects of amino acid substitutions at the P1 and P1' positions on the rate of catalysis. With the exception of angiotensin-(1-8) (angiotensin-II), which is a relatively poor substrate for TPP-I, the rate of catalysis increases with increasing chain length. K(cat)/K(m) values increase 50-fold between angiotensin-(1-5) and angiotensin-(1-14). TPP-I shows little specificity for the nature of the amino acids in the P1 and P1' positions, K(cat)/K(m) values varying only 5-fold for a range of substitutions. However, Pro or Lys in the P1 position and Pro in the P1' positions are incompatible with TPP-I activity. These observations suggest that TPP-I is a non-specific, but essential, peptidase involved in the latter stages of lysosomal protein degradation.

Phosphate shows a non-competitive inhibition toward a Streptomyces aminopeptidase (sAP) between pH 5.85 (Ki = 0.48 mM) and 9.0 (110 mM), with a pKa of 7.1 likely due to ionization of H2PO 3 . This non-competitive inhibition pattern indicates that phosphate binding to sAP in solution is different from that in the crystal structure, where phosphate is bound to the active site Zn(II) ions. Fluoride uncompetitively inhibits sAP from pH 5.5 (Ki = 3.72 mM) to 9.0 (43.6 mM), with ap Ka of V6.2 likely due to a coordinated water. The different inhibition natures and pKa values indicate that the two inhibitors bind at different locations. z 1999 Federation of European Biochemical Societies.

Leukotriene A4 hydrolase is a cytosolic metalloenzyme of the arachidonic acid biosynthetic pathway responsible for leukotriene A4 conversion into leukotriene B4. In addition to its epoxide hydrolase properties, this enzyme exhibits an aminopeptidase activity which was used as an assay to monitor the purification of a novel form of leukotriene A4 hydrolase from Xenopus laevis skin exudate. This 70 kDa, secreted, form of leukotriene A4 hydrolase was identified by immunochemical cross-reactivity with anti-human leukotriene A4 hydrolase antibodies and by its capacity to convert leukotriene A4 into leukotriene B4. Moreover this enzyme produced a second metabolite which could be the leukotriene B4 isomer 5S,12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, previously shown by Strömberg et al. (Eur. J. Biochem. 238 (1996) 599–605) to be formed by incubation of the leukotriene A4 with amphibian tissue extracts. Partial amino acid sequencing of peptides generated by endolysin C fragmentation of the purified enzyme confirmed the presence, in X. laevis skin secretions, of a related but distinct form of leukotriene A4 hydrolase which is likely to be responsible for the production of these eicosanoid metabolites of leukotriene A4.

Transcription of the vacuolar aminopeptidase yscI-encoding gene (APE1) is regulated by the carbon source used for yeast growth, responding to carbon catabolite repression. By Northern blot analyses, we determined the kinetics of glucose repression in growth-shift experiments. When added to induced cells, glucose leads to the disappearance of hybridizable aminopeptidase yscI RNA sequences within 30 min. However, the amount of inmunoreactive protein, once induced, is not affected by the addition of glucose. By deletion analysis of the fusion gene APE1-lacZ we have identified a number of strong regulatory regions in the APE1 promoter. Consensus sequences for the binding of yAP1 and the HAP2/HAP3/HAP4 complex are contained in those regions. Control of the APE1 gene expression is not mediated by the HXK2 regulatory gene, but a strain bearing a deletion in the CAT1 gene can not derepress APE1 transcription to wild-type levels.

The importance of HNF1 binding proteins for intestinal aminopeptidase N expression was investigated using the Caco-2 cell-line. Aminopeptidase N promoter activity in Caco-2 cells depends on the HNF1 element (positions -85 to -58) and co-transfection with an HNF1 alpha expression vector demonstrates a direct activation of the promoter by HNF1 alpha through this element. Electrophoretic mobility shift assays using nuclear extracts from Caco-2 cells show the presence of high amounts of HNF1 binding proteins irrespective of their state of differentiation.

A number of natural cytokines are characterized as having dipeptidyl peptidase (DP) IV susceptible N-terminal peptide sequences. Here we demonstrate that oligopeptides with sequences analogous to the N-terminal part of human IL-1 beta, IL-2, TNF-beta and murine IL-6 were hydrolyzed by purified DP IV and aminopeptidase N (AP-N). The rate of DP IV-catalyzed hydrolysis of these peptides was negatively correlated with their chain length. In contrast to these results, no degradation was found under our conditions for the intact recombinant cytokines, IL-1 alpha, IL-1 beta, IL-2, G-CSF and for natural IL-2, independent of whether DP IV and AP-N were used separately or in combination.

A rat liver plasma membrane glycoprotein, gp110, was compared with dipeptidyl aminopeptidase IV (DAP IV) by using Wistar rats (DAP IV-positive rats) and Fischer 344 rats (DAP IV-negative rats). Fischer rats also lacked gp110 and gp110 of Wistar rats had DAP IV activity. Furthermore, we showed that the C-terminal sequence of gp110 was Ser-Leu-Arg, which was the same as the C-terminal amino acid sequence deduced from the nucleotide sequence of the cDNA of DAP IV. According to these results, we concluded that gp110 was identical with DAP IV.

Aminopeptidase B (AP-B) is a metallopeptidase that removes basic residues from the N-termini of neuropeptide substrates in secretory vesicles. This study assessed zinc regulation of AP-B activity, since secretory vesicles contain endogenous zinc. AP-B was inhibited by zinc at concentrations typically present in secretory vesicles. Zinc effects were dependent on concentration, incubation time, and the molar ratio of zinc to enzyme. AP-B activity was recovered upon removal of zinc. AP-B with zinc became susceptible to degradation by trypsin, suggesting that zinc alters enzyme conformation. Zinc regulation demonstrates the metallopeptidase property of AP-B.

HIV-1 viral protein R (Vpr) is one of the human immunodeficiency virus type 1 encoded proteins that have important roles in viral pathogenesis. However, no clinical drug for AIDS therapy that targets Vpr has been developed. Here, we have established a screening system to isolate Vpr inhibitors using budding yeast cells. We purified a Vpr inhibitory compound from fungal metabolites and identified it as fumagillin, a chemical already known to be a potent inhibitor of angiogenesis. Fumagillin not only reversed the growth inhibitory activity of Vpr in yeast and human cells, but also inhibited Vpr-dependent viral gene expression upon the infection of human macrophages.

We have investigated the physiological function of type 2 methionine aminopeptidases (MetAP2) using Caenorhabditis elegans as a model system. A homolog of human MetAP2 was found in the C. elegans genome, which we termed MAP-2. MAP-2 protein displayed methionine aminopeptidase activity and was sensitive to inhibition by fumagillin. Downregulation of map-2 expression by RNAi led to sterility, resulting from a defect in germ cell proliferation. These observations suggest that MAP-2 is essential for germ cell development in C. elegans and that this ubiquitous enzyme may play important roles in a tissue specific manner.

We here identify Mai1p, a homologue of the autophagy protein Aut10p, as a novel component essential for proaminopeptidase I (proAPI) maturation under non-starvation conditions. In mai1Delta cells mature vacuolar proteinases are detectable and vacuolar acidification is normal. In mai1Delta cells autophagy occurs, though at a somewhat reduced level. This is indicated by proAPI maturation during starvation and accumulation of autophagic bodies during starvation with phenylmethylsulfonyl fluoride. Homozygous diploid mai1Delta cells sporulate, but with a slightly reduced frequency. Biologically active Ha-tagged Mai1p, chromosomally expressed under its native promoter, is at least in part peripherally membrane-associated. In indirect immunofluorescence it localizes to the vacuolar membrane or structures nearby. In some cells Ha-tagged Mai1p appears concentrated at regions adjacent to the nucleus.

We here report the identification of AUT10 as a novel gene required for both the cytoplasm to vacuole targeting of proaminopeptidase I and starvation-induced autophagy. aut10Delta cells are impaired in maturation of proaminopeptidase I under starvation and non-starvation conditions. A lack of Aut10p causes a defect in autophagy prior to vacuolar uptake of autophagosomes. Homozygous aut10Delta diploids do not sporulate. Vacuolar acidification indicated by accumulation of quinacrine is normal in aut10Delta cells and mature vacuolar proteinases are present. A biologically active Ha-tagged Aut10p, chromosomally expressed from its endogenous promoter, localizes in indirect immunofluorescence microscopy in the cytosol and on granulated structures, which appear clustered around the vacuolar membrane. This localization differs from known autophagy proteins.

Lovastatin treatment caused down-regulation of the insulin-responsive glucose transporter 4 (Glut4) and up-regulation of Glut1 in 3T3-L1 adipocytes. These changes in protein expression were associated with a marked inhibition of insulin-stimulated glucose transport. Lovastatin had no effect on cell cholesterol levels, but its effects were reversed by mevalonate, demonstrating that inhibition of isoprenoid biosynthesis causes insulin resistance in 3T3-L1 adipocytes. These findings support the notion that whole body insulin resistance may arise as a result of perturbations in general biochemical pathways, rather than primary defects in insulin signalling.

Eukaryotic 70 kDa heat shock proteins (Hsp70s) are localized in various cellular compartments and exhibit functions such as protein translocation across membranes, protein folding and assembly. Here we demonstrate that the constitutively expressed members of the yeast cytoplasmic Ssa subfamily, Ssa1/2p, are involved in the transport of the vacuolar hydrolase aminopeptidase 1 from the cytoplasm into the vacuole. The Ssap family members displayed overlapping functions in the transport of aminopeptidase 1. In SSAI and SSAII deletion mutants the precursor of aminopeptidase 1 accumulated in a dodecameric complex that is packaged in prevacuolar transport vesicles. Ssa1/2p was prominently localized to the vacuolar membrane, consistent with the role we propose for Ssa proteins in the fusion of transport vesicles with the vacuolar membrane.

Tyrosine phosphorylation of Cbl and its association with signal-transducing molecules in response to macrophage colony-stimulating factor (M-CSF) were analyzed by using cell lines which express the wild-type and a mutant M-CSF receptor, Fms. We found that in a clone, F723 TF-1 cells expressing mutant Fms in which tyrosine 723 had been substituted with phenylalanine, the M-CSF stimulation-dependent association between Cbl and Fms was markedly impaired. However, phosphorylation of Cbl and its association with the p85 subunit of phosphatidylinositol 3-kinase were induced in these mutant cells as seen in the wild-type fms transfectant. These results suggest that phosphorylation of tyrosine 723 is particularly important for the recruitment of Cbl to the M-CSF receptor, but is not required for the phosphorylation and binding of Cbl to signal-transducing molecules such as p85.

Tripeptidyl peptidase I (TPP-I) is a lysosomal enzyme that cleaves tripeptides from the N-terminus of polypeptides. A comparison of TPP-I amino acid sequences with sequences derived from an EST database suggested that TPP-I is identical to a pepstatin-insensitive carboxyl proteinase of unknown specificity which is mutated in classical late infantile neuronal ceroid lipofuscinosis (LINCL), a lysosomal storage disease. Both TPP-I and the carboxyl proteinase have an M(r) of about 46 kDa and are, or are predicted to be, resistant to inhibitors of the four major classes of proteinases. Fibroblasts from LINCL patients have less than 5% of the normal TPP-I activity. The activities of other lysosomal enzymes, including proteinases, are in the normal range. LINCL fibroblasts are also defective at degrading short polypeptides and this defect can be induced in normal fibroblasts by treatment with a specific inhibitor or TPP-I. These results suggest that the cell damage, especially neuronal, observed in LINCL results from the defective degradation and consequent lysosomal storage of small peptides.

The regularities of their functioning of enzyme, water-soluble and membrane forms, in the systems of the reversed micelles of surfactants in organic solvents are compared. Using as examples γ-glutamyltransferase (in AOT reversed micelles in octane) and aminopeptidase (in Brij 96 reversed micelles in cyclohexane), the principal difference in the catalytic activity regulation of water-soluble and membrane forms is demonstrated. The catalytic activity of the membrane form depends considerably on the surfactant concentration at the constant degree of hydration, whereas the activity of the water-soluble form is constant under these conditions. The catalytic activity dependence on the surfactant concentration is regarded as a test for enzyme membrane activity.

Proline iminopeptidase from Xanthomonas campestris pv. citri , displaying no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapour diffusion method. Two different orthorhombic crystal forms (space group C222 and I222) were obtained from a solution containing NaCl or polyethylene glycol monomethyl ether (MW 5000) as precipitating agent for the native and lanthanum-derivatized protein, respectively. Complete diffraction data sets have been collected up to 2.6 A (native) and 3.0 A (lanthanum derivative) resolution. Cell dimensions are a =147.2 A, b =167.8 A, and c =85.6 A (C222) and a =146.7 A, b =167.7 A, and c =171.4 A (I222), respectively. Considerations of the possible values of V m and analysis of the self-rotation function of the native crystals account for the presence of one dimer per asymmetric unit, whereas a tetramer probably would occupy the smallest crystallographically independent crystal portion in the lanthanum-derivatized protein crystals.

Tricorn protease (TRI), a high molecular mass complex from the archaeon T. acidophilum, forms the core of a modular proteolytic system; upon interacting with low molecular mass factors intrinsic activities are enhanced and novel activities are generated. Here we characterize the first factor, F1, which turns out to be homologous with several bacterial proline iminopeptidases (PIPs). Surprisingly, it cleaves not only typical PIP substrates such as H-Pro-AMC, but a wide spectrum of amino acid substrates and several peptide substrates without a proline at the N-terminus. The pip gene encodes a 293 amino acid residue protein with a molecular mass of 33487 Da. By means of site-directed mutagenesis we identified Ser105 and His271 as the active site nucleophile and proton donor, respectively. Experiments with inactive mutant PIPs indicate that the activities elicited by interacting with TRI are contributed by PIP.

Upon trypsinolysis, the 18 C-terminal residues of Escherichia coli peptide deformylase were removed but the resulting form exhibited full activity. Moreover, a mutant fms gene encoding the first 145 out of the 168 residues of the enzyme was able to complement a fms(Ts) strain and exhibited full activity. Upon progressive truncation up to residue 139, both activity and stability decreased up to complete inactivation. Mutagenesis of residues of the 138–145 region highlights the importance of Leu-141 and Phe-142. N-Terminal deletions were also carried out. Beyond two residues off, the enzyme showed a dramatic instability. Finally, NMR and thermostability studies of the full-length enzyme and comparison to the 1–147 form strongly suggest that the dispensable residues are disordered in solution.

A scheme based on the zinc binding site [1992, FEBS Lett. 312, 110–114] has been extended to classify zinc metalloproteases into distinct families. The gluzincins, defined by the HEXXH motif and a glutamic acid as the third zinc ligand, include the thermolysin, endopeptidase-24.11, aminopeptidase, angiotensin converting enzyme, endopeptidase-24.15, and tetanus and botulinum neurotoxin families. The metzincins, defined by the HEXXH motif, a histidine as the third zinc ligand and a Met-turn, include the astacin, serralysin, reprolysin and matrixin families. The inverted zincin motif, HXXEH, defines the inverzincin family of insulin-degrading enzymes, the HXXE motif defines the carboxypeptidase family, and the HXH Motif dd-carboxypeptidase.

The active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis (PepX) was identified. The enzyme was labeled by [3H]DFP, treated by CNBr and the resulting peptides were separated by reverse-phase-HPLC. The main radiolabeled peptide was sequenced. Ser-348, in the following sequence, Gly-Lys-Ser-Tyr-Leu-Gly, was identified as the active site serine. A sequence comparison between the active site of PepX and other serine proteases was made, showing only limited sequence homologies in this area. The consensus sequence surrounding the active site serine in the three known X-prolyl dipeptidyl aminopeptidases (mammalian DPPIV, yeast DPAB and PepX) is G-X-S-Y-X-G, where X is a non-conserved amino acid.

Membrane-bound peptidases are critical regulators of peptide hormones. We therefore characterized aminopeptidase A (APA) activity in human glomerular epithelial cells (HGEC) and studied the control of its expression. APA, which splits off the N-terminal Asp from angiotensin II (AII), was present at the surface of HGECs (55% of the total enzyme). APA activity was calcium-dependent and was inhibited by amastatin. Treatment of HGECs by dexamethasone (DEX) increased ecto-APA activity in a dose- and time-dependent manner. Maximal increase of APA activity (× 2) occurred after treatment with 0.5 μM DEX for 5 days. Higher concentrations (1·10 μM) of aldosterone (ALD) stimulated APA activity to a lesser extent (× 1.25). Actinomycin D and cycloheximide prevented and RU 38486, a glucocorticoid receptor antagonist, suppressed the DEX-induced increase in APA activity. These results indicate that AII availability at glomerular receptor sites may be reduced by DEX and suggest a role for glucocorticoids in AII-dependent changes of glomerular filtration rate.

Using the active-site-directed irreversible inhibitor bromohydroxy acetone phosphate (BHAP) Coulson et al. [l] isolated a hexapeptide from the active centre of chicken triose phosphate isomerase to which the inhibitor was bound covalently. Waley et al. [2] isolated a peptide of homologous sequence from the rabbit enzyme. Later de la Mare et al. [3] elucidated the residue to which the inhibitor moiety became attached and that to which it subsequently migrated. We have isolated from the chicken enzyme the tryptic peptide of fifteen residues that include the former hexapeptide sequence and have established its sequence by a combination of conventional and massspectrometric techniques. The sequence is identical to that derived for rabbit enzyme by Corran and Waley [4], but differs from those put forward, also for the rabbit enzyme, by Hartman [S, 61. Preliminary results bbtained by conventional techniques were reported by Offord et al. [7].

Degradation of tritiated Leu-enkephalin was studied in cultures of primary astrocytes from rat brain. The incubation experiments with a cell suspension revealed Tyr as the main tritiated metabolite; however, Tyr—Gly—Gly and Tyr—Gly were detectable as well. Using a crude membrane prepartion of the astrocytes, we found about equal amount of Tyr and Tyr—Gly—Gly but only trace quantities of Tyr—Gly. The production of Tyr was completely inhibited by bestatin, an inhibitor of aminopeptidases, that of Tyr—Gly—Gly by thiorphan, a specific inhibitor of enkephalinase A. The results prove the ability of glial cells to degrade enkephalin by aminopeptidase and a membrane-bound enkephalinase A.

Endopeptidase-24.11 (EC 3.4.24.11) from pig kidney hydrolysed CCK-8 (sulphated) at two distinct sites: Asp-Tyr(SO3H)-Met-Gly↓Trp-Met-Asp↓PheNH2. Under initial conditions, the splitting of the Asp7-Phe8NH2 bond proceeded 4-times more rapidly than the Gly4-Trp5 bond. Pig brain striatal synaptic membranes attacked this substrate at the same sites and this activity was inhibited by phosphoramidon. However, other products were detected even in the presence of phosphoramidon. One of these products was identified as free tryptophan. Since their formation was inhibited by bestatin, one or more membrane aminopeptidases is also implicated in the degradation of CCK-8.

The effect of tunicamycin on synthesis and intracellular transport of pig small intestinal aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48–10) and maltase-glucoamylase (EC 3.2.1.20) was studied by labelling of mucosal explants with [35S]methionine. The expression of the microvillar enzymes was greatly reduced by tunicamycin but could be partially restored by leupeptin, suggesting the existence of a mechanism whereby newly synthesized, malprocessed enzymes are recognized and degraded. In the presence of tunicamycin, polypeptides likely to represent non-glycosylated forms of the enzymes persisted in the Mg2+-precipitated membrane fraction, indicating that high mannose glycosylation is essential for transport to the microvillar membrane. Treatment of aminopeptidase N and sucrase-isomaltase with endo F reduced the size of the high mannose forms approximately to those seen in the presence of tunicamycin. The complex forms were also sensitive to endo F but did not coincide with the high mannose forms after treatment, indicating that the size difference cannot alone be ascribed to processing of N-linked carbohydrate.

[Cyt6]AVP-(3–9), an intermediate in the metabolism of AVP-(1–9) in vitro, was used to investigate the mechanism of formation of centrally active AVP metabolites. Exposure of [Cyt6]AVP-(3–9) to rat brain membranes resulted in formation of [Cyt6]AVP-(4–9),-(5–9), [pGlu6,Cyt6]AVP-(4–9) and AVP-(3–5), which were isolated and chemically identified. Products derived from cleavage of the C-terminus of the substrate were absent. Time-course experiments further indicated that the conversion process is predominantly mediby an aminopeptidase-like mechanism. The conversion of [Cyt6]AVP-(3–9) by membranes from hippocampus, amygdala and septum was quantitatively and qualitatively similar. The results point to a major role of aminopeptidase activity in the metabolic conversion of AVP and the formation of centrally active AVP metabolites.

The mode of membrane anchorage of three kidney microvillar membrane ectoenzymes has been examined. The release of aminopeptidase P (EC 3.4.11.9) from kidney membranes by bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and the pattern of detergent solubilization of this ectoenzyme implies that it is anchored to the membrane via a covalently attached glycosyl-phosphatidylinositol moiety. As deduced by phase separation in Triton X-114, octyl-glucoside solubilized the amphipathic form of aminopeptidase P, whereas the PI-PLC-released form displayed hydrophilic properties. In contrast, the pattern of detergent solubilization of two microvillar carboxypeptidases and their resistance to release from the membrane by bacterial PI-PLC suggest that these two ectoenzymes are not anchored via phosphatidylinositol.

The structural gene, APE1, (LAP4), for the vacuolar aminopeptidase I of Saccharomyces cerevisiae was cloned with the aid of a staining technique which permitted monitoring of aminopeptidase activity in yeast colonies. Genetic linkage data demonstrate that integrated copies of the cloned gene map to the APEI locus. The nucleotide sequence of the cloned gene was determined. The open reading frame of APEI consists of 514 codons and, therefore, encodes a larger protein (MW 57 003) than the reported mature aminopeptidase yscI (MW 44 800), suggesting that proteolytic processing must occur. A 1.75-kb mRNA, which is made in substantial amounts only when yeast cells have exhausted the glucose supply, was identified.