On the subunit structure of particulate aminopeptidase from pig kidney.

Solubilization of particulate aminopeptidase (EC 3.4.11.2) from pig kidney with Triton X-100 yields an aggregate (mol. wt. approx. 10(6)) that decomposes into "free" aminopeptidase (mol. wt. 280 000) either upon autolysis at pH 5 or after exposure to trypsin. Both procedures yield free enzymes that are identical with respect to electrophoretic mobility, enzymatic activity and zinc content. After dissociation, the enzyme resulting from autolysis yields a single subunit of 140 000 molecular weight while the trypsin-treated enzyme produces three fragments (140 000, 95 000 and 48 000 mol. wt.). As the aggregate is formed by subunits 10 000 daltons heavier than those of the free enzyme, the existence of a hydrophobic portion anchoring the enzyme to the membrane might be postulated. Reactivation experiments carried out on the three purified fragments of urea-denatured aminopeptidase show that the 140 000 molecular weight subunit is the only one able to yield an active enzyme (after spontaneous dimerization). It can be concluded that the smaller fragments are artefacts resulting from trypsin degradation during purification.     

Purification and properties of a new aminopeptidase from Escherichia COLI K12.

An aminopeptidase (EC 3.4.11.-) capmable of hydrolyzing L-alanyl-beta-naphthyl-amide and certain other aminoacyl beta-naphthylamides was purified to homogeneity from extracts of Exherichia coli K-12. The enzyme, designated aminopeptidase II, is a monomeric protein of mol. wt. 100 000. It exhibits a broad pH optimum in the range pH 7.0--9.0. Although Zn2+, Fe3+ and Cr3+ are strong inhibitors of enzyme activity, a metal requirement for catalysis could not be firmly established. Neither sulfhydryl reagents nor serine protease inhibitors affected enzyme activity.     

Leucine aminopeptidase and exsheathing activity in preparations from Haemonchus contortus

Rogers W.P. and Brooks F. 1978. Leucine aminopeptidase and exsheathing activity in preparations from Haemonchus contortus. International Journal for Parasitology 8: 449–452. Exsheathing activity relative to leucine aminopeptidase activity (LAP) was greater in exsheathing fluid of infective juveniles of Haemonchus contortus than extracts of homogenates of the same organism. In both preparations the biological and enzyme activities were precipitated with acetone 20 v/v and ammonium sulphate, 40% saturation. Broad peaks of exsheathing and LAP activities obtained by sucrose density-gradient centrifugation and on Sephadex G150 overlapped but the peak of biological activity was always found on the low mol. wt. side of the LAP peak. LAP in exsheathing fluid was separated into two sharp peaks in polyacrylamide gradient-pore electrophoresis. In four experiments the major peak gave a mol. wt. within the limits 345,000–354,500. A minor peak was obtained at 1,800,000. Exsheathing activity remained broadly distributed but fell mostly on the low mol. wt. side of the major LAP peak.It is concluded that LAP cannot be the sole agent involved in exsheathment a lipase may be necessary to expose the substrate attacked by LAP.     

Purification and characterization of thiol aminopeptidase from the cytosolic fraction of human placenta

A thiol-dependent aminopeptidase was purified from the cytosolic fraction of human placenta. The purified enzyme consisted of a single polypeptide chain with a mol wt of 95,000. The enzyme was most active in the neutral region with Ala-pNA as substrate, and the activity was increased about 20-fold in the presence of some -SH compounds. The results of substrate specificity studies indicated that the enzyme hydrolyzes bonds involving the amino groups of neutral and basic amino acid residues. However, higher thiol-dependent activity was only detected with neutral ones. The enzyme was strongly inhibited by microbial aminopeptidase inhibitors, puromycin, o-phenanthroline, and sulfhydryl reactive-reagents. As to several naturally occurring peptides tested, the enzyme showed N-terminal Tyr-releasing activity toward enkephalins and kinin-converting activity.     

xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase.

The heritable stability of ColE1 is dependent on a site-specific recombination system which acts to resolve plasmid multimers into monomers. This plasmid stabilizing recombination system requires the presence in cis of the ColE1 cer region, plus at least two trans-acting factors encoded by the xerA and xerB genes of Escherichia coli. The xerB gene has been cloned and sequenced and found to encode a polypeptide with a calculated mol. wt of 55.3 kd. The predicted amino acid sequence of this protein exhibits striking similarity to that of bovine lens leucine aminopeptidase (53 kd). The biological significance of this similarity is corroborated by genetic and biochemical evidence which suggests that xerB is identical to the E.coli and S.typhimurium pepA genes that encode aminopeptidase A.     

The Caenorhabditis elegans orthologue of mammalian puromycin-sensitive aminopeptidase has roles in embryogenesis and reproduction

Mammals possess membrane-associated and cytosolic forms of the puromycin-sensitive aminopeptidase (PSA; EC 3.4.11.14). Increasing evidence suggests the membrane PSA is involved in neuromodulation within the central nervous system and in reproductive biology. The functional roles of the cytosolic PSA are less clear. The genome of the nematode Caenorhabditis elegans encodes an aminopeptidase, F49E8.3 (PAM-1), that is orthologous to PSA, and sequence analysis predicts it to be cytosolic. We have determined the spatio/temporal gene expression pattern of pam-1 by using the promoter region of F49E8.3 to control expression in the nematode of a second exon translational fusion of the aminopeptidase to green fluorescent protein. Cytosolic fluorescence was observed throughout development in the intestine and nerve cells of the head. Neuronal expression was also observed in the tail of adult males. Recombinant PAM-1, expressed and purified from Escherichia coli, hydrolyzed the N-terminal amino acid from peptide substrates. Favored substrates had positively charged or small neutral amino acids in the N-terminal position. Peptide hydrolysis was inhibited by the metal-chelating agent 1,10-phenanthroline and by the aminopeptidase inhibitors actinonin, amastatin, and leuhistin. However, the enzyme was approximately 100-fold less sensitive toward puromycin (IC50, 135 mum) than other PSA homologues. Following inactivation of the enzyme, aminopeptidase activity was recovered with Zn2+, Co2+, and Ni2+. Silencing expression of pam-1 by RNA interference resulted in 30% embryonic lethality. Surviving adult hermaphrodites deposited large numbers of oocytes throughout the self-fertile period. The overall brood size was, however, unaffected. We conclude that pam-1 encodes an aminopeptidase that clusters phylogenetically with the PSAs, despite attenuated sensitivity toward puromycin, and that it functions in embryo development and reproduction of the nematode.     

Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulin-regulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.     

The influenza virus M2 protein cytoplasmic tail interacts with the M1 protein and influences virus assembly at the site of virus budding

The cytoplasmic tail of the influenza A virus M2 proton-selective ion channel has been shown to be important for virus replication. Previous analysis of M2 cytoplasmic tail truncation mutants demonstrated a defect in incorporation of viral RNA (vRNA) into virions, suggesting a role for M2 in the recruitment of M1-vRNA complexes. To further characterize the effect of the M2 cytoplasmic tail mutations on virus assembly and budding, we constructed a series of alanine substitution mutants of M2 with mutations in the cytoplasmic tail, from residues 71 to 97. Mutant proteins M2-Mut1 and M2-Mut2, with mutations of residues 71 to 73 and 74 to 76, respectively, appeared to have the greatest effect on virus-like particle and virus budding, showing a defect in M1 incorporation. Mutant viruses containing M2-Mut1 and M2-Mut2 failed to replicate in multistep growth analyses on wild-type (wt) MDCK cells and were able to form plaques only on MDCK cells stably expressing wt M2 protein. Compared to wt M2 protein, M2-Mut1 and M2-Mut2 were unable to efficiently coimmunoprecipitate with M1. Furthermore, statistical analysis of planar sheets of membrane from cells infected by virus containing M2-Mut1 revealed a reduction in M1-hemagglutinin (HA) and M2-HA clustering as well as a severe loss of clustering between M1 and M2. These results suggest an essential, direct interaction between the cytoplasmic tail of M2 and M1 that promotes the recruitment of the internal viral proteins and vRNA to the plasma membrane for efficient virus assembly to occur.     

Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes

Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.     

Evidence for conversion of N-Tyr-MIF-1 into MIF-1 by a specific brain aminopeptidase

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly·NH₂), an immunoreactive neuropeptide exhibiting saturable high affinity binding in rat brain was found to be converted into MIF-1 (Pro-Leu-Gly·NH₂) by a specific brain aminopeptidase present in rat brain homogenates or cytosol, but with low activity associated with synaptosomal plasma membranes and microsomes. Conversion occurred at a rate of 16 μmol per g w/wt per h and was unaffected by puromycin but inhibited by bestatin (I₅₀, 5 × 10^?5 M). Aminopeptidases purified from cytosolic fractions of rat brain (arylamidase), mouse brain (Mn²⁺-activated aminopeptidase) or porcine kidney (leucine aminopeptidase) were inactive towards N-Tyr-MIF-1 but degraded MIF-1 with release of Leu-Gly·NH₂ as detected by RP-HPLC procedures. Morphiceptin (Tyr-Pro-Phe-Pro·NH₂), a μ opioid agonist, also acted as a substrate for the N-Tyr-MIF-1 converting enzyme with cleavage of the Tyr-Pro bond. These tetrapeptides, but not MIF-1 or its N-blocked analogs, were degraded in vitro by a metalloendopeptidase purified from kidney membranes. Since dipeptide products were not detected for crude extracts, a significant role for brain metalloendopeptidase on turnover can be excluded. Thus the results point to the presence of a specific (X-Pro-degrading) aminopeptidase in brain cytosol as an enzyme responsible for converting N-Tyr-MIF-1 and inactivating morphiceptin.     

Bacteriophage T4 tail assembly: proteins of the sheath, core and baseplate

Structural intermediates in phage tail formation have been isolated by sucrose gradient centrifugation from cells infected with mutants blocked at various stages in tail assembly. The polypeptide chains of these structures containing ¹⁴C-labeled amino acids have been analyzed by sodium dodecyl sulfate—acrylamide gel electrophoresis, enabling us to identify the proteins forming the various morphological components of the tail. Comparison of sheathed tails with corebaseplates shows that the contractile sheath is composed of a single species of subunit, the product of gene 18 (mol.wt 80,000). The site for head attachment terminating the tail is composed of the product of gene 15 (mol.wt 35,000). Comparison of core-baseplates with free baseplates shows that the tail core is composed of a single species of subunit, the product of gene 19 (mol.wt 21,000).Free baseplates are composed of at least twelve species of proteins: the products of genes 6, 7, 8, 9, 10, 11, 12 and 29, and four genetically unidentified species.The incomplete tails which accumulate in cells infected with mutants defective in genes 9, 11 and 12, which specify proteins on the outside of the baseplate, have also been characterized. Tails from 9^? lysates lack only P9. Tails from 11^? lysates lack both Pll and P12. Tails from 12^? infection lack only P12. Incorporation of P12 into the baseplate requires the function of gene 57, which is also required for tail fiber assembly. P57 thus appears to take part in the maturation of three different phage structural proteins.The sequential nature of the protein interactions in tail formation is discussed in terms of the regulation of morphogenesis at the level of assembly.     

Dopamine D5 receptor agonist high affinity and constitutive activity profile conferred by carboxyl-terminal tail sequence

The mammalian dopamine D1-like receptor gene family is comprised of two members, termed D1/D1A and D5/D1B. In an attempt to define the role of the carboxyl terminal (CT) tail in the expression of D5 subtype-specific pharmacological and constitutive activity profiles, we examined a series of D5 receptor chimeras in which only the CT tail was swapped with corresponding sequences encoding human/vertebrate D1-like receptors. D5/D1(CT) or D5/D1D(CT) tail substitution mutants displayed a rank order of potency and agonist affinities virtually mimicking wild-type (wt) D1 receptors, as indexed by both ligand binding and dopamine-stimulated cAMP accumulation assays, and, similar to wt D1 receptors, did not exhibit receptor constitutive activity or responsiveness to inverse agonists. D1/D5(CT) or D1/D1D(CT) tail receptor mutants displayed agonist pharmacological and functional characteristics not significantly different from parental D1 or mutant D5/D1(CT) and D5/D1D(CT) receptors. The affinities for numerous antagonists remained essentially unchanged for all receptor chimeras relative to parental wt receptors. A series of stepwise D5-CT-tail truncation/deletion mutants identified the region encoded by amino acids 438-448 and particularly Gln(439), as necessary and sufficient for the full expression of high affinity agonist and functional D5 receptor characteristics. Site-directed mutagenesis of the highly conserved D5/D1B receptor residue Gln(439)-(Ala/Ile), converts the full-length D5 receptor to one displaying "super" D5 characteristics with expressed affinities for discriminating agonists approximately 4- to 5-fold higher than wt D5 but without any concomitant increases of agonist-independent basal cAMP accumulation or intrinsic activity. Taken together, these data suggest that, in addition to other well characterized receptor domains, the agonist pharmacological and functional signature of the D5/D1B receptor is modulated by sequence-specific motifs within the CT tail and that one conserved amino acid in this region can further regulate D5 agonist high affinity binding interactions independent of receptor constitutive activity.     

An intracellular aminopeptidase from Streptomyces rimosus that prefers basic amino acids

An aminopeptidase from the mycelia of Streptomyces rimosus was isolated in an electrophoretically homogeneous form. It was shown to be a monomeric, acidic protein (pI = 4.4, mol. wt. approx. 83,000), with optimal activity at pH 7.1–7.8 and at 35–41° C. The enzyme was fully inhibited by 0.1 mM EDTA or 1 mM o-phenanthroline; the activity was restored upon addition of 0.05 mM Co²⁺, Zn²⁺, or Ni²⁺. Amastatin, bestatin, and puromycin also inhibited the enzyme. The aminopeptidase hydrolyzed amino-acid-2-naphthylamides and various di- to heptapeptides. The highest catalytic coefficients (23 and 19 μM^–1 s^–1) were obtained with Arg- and Lys-2-naphthylamide, followed by Leu-, Phe- and Met-derivatives with one order of magnitude lower catalytic coefficients. Basic or bulky hydrophobic amino acids at the P₁ and/or P₁′ position of peptide substrates were preferred. Acidic amino acids and proline were not accepted. The affinity of the enzyme increased with the length of peptide. According to these properties, S. rimosus intracellular aminopeptidase is distinct from the extracellular leucine aminopeptidase of the same organism and can be classified as an Arg(Lys)-preferring metalloaminopeptidase. Received: 18 January 1996 / Accepted: 19 March 1996     

Is there correlation between analgesic potency and biodegradation of enkephalin analogs?

Met-enkephalin and its Pro⁵ analogs containing Gly or D amino acids at position 2 were subjected to digestion with aminopeptidase M, rat brain extracts and human sera. The enzyme resistance of these peptides was compared with their analgesic activity determined in tail flick test after central and intravenous administrations. Our data did not reveal an unambigous correlation between the analgesic potency and metabolic stability of the analogs. This suggest that analgesic activity of synthetic peptides should be due to factors other than enzyme resistance /e.g. receptor binding and transport properties/.     

Triiodothyronine induces an increase in cyclic GMP in bullfrog tadpole tissues

Cyclic GMP (cGMP) and cyclic AMP (cAMP) were determined in bullfrog tadpole liver and tail fin using 125I-RIA. cGMP increased approx. 100% 1-6 h after the injection of T3 (3 X 10(-10) mol/g body wt.). Reducing the dose of T3 to 1, 3, and 10 X 10(-11) mol/g body wt. provided increases in cGMP of 50-100% above the control value after 2 h. In contrast, only small increases (less than 20%) in cAMP were observed 2-24 h after T3 injection. We conclude that T3 produces a rapid and significant increase in cGMP in the liver and tail fins of premetamorphic tadpoles. These results suggest that thyroid hormones in amphibia may not be an exclusively nucleus-mediated hormone.     

Characterization of proteolytic activities in embryos of Xenopus laevis

1. Proteolytic activities in early embryos of Xenopus laevis exhibited maximum levels at pH 3.2, 5.6 and 7.2 when 3H-BSA was used as substrate, and the maximum proteolytic activity at pH 3.2 was several thousand-fold higher during the tail bud stage than in the unfertilized egg. 2. The proteolytic activity at pH 3.2 was separated into two fractions by gel chromatography. One fraction corresponded to a mol. wt of about 40,000 and its activity was inhibited by thiol protease inhibitors. The other appeared to be a protease of much higher mol. wt. 3. The maximum activities at pH 5.6 and 7.2 appear to correspond to proteins of mol. wt greater than 1,000,000.     

The crystal structure of AF1521 a protein from Archaeoglobus fulgidus with homology to the non-histone domain of macroH2A

MacroH2A is an unusual histone H2A variant that has an extensive C-terminal tail that comprises approximately two thirds of the protein. The C-terminal non-histone domain of macroH2A is also found in a number of other proteins and has been termed the macro domain. Here we report the crystal structure to 1.7A of AF1521, a protein consisting of a stand-alone macro domain from Archaeoglobus fulgidus. The structure has a mixed alpha/beta fold that closely resembles the N-terminal DNA binding domain of the Escherichia coli leucine aminopeptidase PepA. The structure also shows some similarity to members of the P-loop family of nucleotide hydrolases.     

Novel inhibitors of enkephalin-degrading enzymes. III: 4-Carboxymethylamino-4-oxo-3 (phenylamino) butanoic acids as enkephalinase inhibitors.

4-Carboxymethylamino-4-oxo-3-(4'-aminophenylamino) butanoic acid (25), its ethyl ester (26) and the corresponding unsubstituted-aryl analogues (17) and (16) are fairly potent inhibitors of enkephalinase (neutral endopeptidase; EC 3.4.24.11), Ki = 0.14-0.39 microM, with weak inhibitory potency, Ki = 15-75 microM, towards aminopeptidase MII. In the mouse abdominal constriction test, the esters (26) and (16) showed systemic inhibitory (antinociceptive) activity with ED50 values 62 +/- 3.05 and 81 +/- 1.74 mg/kg respectively. In the mouse tail immersion test, both (26) and (16) exhibited antinociceptive activity when administered intracerebroventricularly and (26) also exhibited a systemic effect which was only partially reversed by naltrexone. The antinociceptive effect seen with (26) reflects its ranking in vitro as an inhibitor of enkephalinase (Ki = 0.14 microM) but it is possible that this effect is not totally opioid-mediated. Compounds (26) and (16) represent the first combined inhibitors of enkephalinase and aminopeptidase MII.     

Cloning of the pig aminopeptidase N gene. Identification of possible regulatory elements and the exon distribution in relation to the membrane-spanning region

We have isolated four lambda-phages covering the complete pig aminopeptidase N/CD13 gene. The sequence of 2.85 kbp encompasses 1.18 kbp of the 5' upstream region and 1.67 kbp of the structural gene. In the promoter region we find a TATA box and potential binding sites for CTF-1/NF-1 and AP-2. By sequence comparisons we have found three domains showing similarity to promoter regions of the genes encoding human alpha 1-antitrypsin and human intestinal alkaline phosphatase. The gene sequence includes the first three exons and two introns. It shows that a single exon encodes the cytoplasmic tail, the membrane anchor and the junctional peptide.     

Cardiovascular and sympathoadrenal responses to stress in swim-trained rats

Chronic exposure to swim stress (i.e., training) is associated with functional adaptations of the cardiovascular system. On the other hand, repeated exposure to tail shock, an emotional stress, often results in deleterious changes in resting blood pressure and myocardial pathology. We hypothesized that the pathological adaptation following chronic exposure to tail shock was associated with a larger acute physiological response compared with swim stress. Therefore, acute responses to swim and shock stress were compared. A second concern of this study examined the extent to which adaptation to swim training influences responses to predictable tail shock stress. The cardiovascular and sympathoadrenal responses to swim stress, using 1% body wt attached to the tail, were compared with predictable tail shock (0.2-0.4 mA intensity, 1-s duration, 1/min) in two groups of Long-Evans male rats. In the first, 11 rats were studied following 5-7 wk of swim training, consisting of daily 1-h sessions of swimming with 2% body wt attached to their tails. They were compared with an age-matched nontrained (NT) group (n = 8). During swimming, the trained animals showed significantly lower heart rate (387 +/- 10 vs. 449 +/- 18 beats/min) and significantly lower lactate (0.9 +/- 0.09 vs. 2.0 +/- 0.24 mmol/l), epinephrine (332 +/- 57 vs. 739 pg/ml), and corticosterone (32 +/- 10 vs. 62 +/- 9 micrograms/dl) responses. Systolic and diastolic blood pressures were elevated in swim stress by the same degree in trained (167/110 mmHg) and NT (177/116 mmHg) rats.(ABSTRACT TRUNCATED AT 250 WORDS)