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.     

Monkey brain arylamidase. II. Further characterization and studies on mode of hydrolysis of physiologically active peptides.

A large-scale purification of monkey brain arylamidase was carried out. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cystine. The amino terminal residue was determined to be alanine by dansylation. The enzyme was activated by sulfhydryl compounds. Dithiothreitol was more effective than beta-mercaptoethanol. Bestatin competitively inhibited the enzyme activity and the Ki value was calculated to be 2.5 x 10(-7) M, which was of the same order as that of puromycin. The inhibitions by puromycin and bestatin were reversible. The enzyme hydrolyzed di-, tri-, and oligopeptides including physiologically active peptides. Of physiologically active peptides, enkephalins and Met-Lys-bradykinin, which possess a neutral amino acid at the N-terminal position, were more rapidly hydrolyzed by the enzyme. Peptides such as LH-RH and TRH, which possess a pyrrolidonecarboxylyl group at the N-terminal position, and substance P and bradykinin, which possess a proline residue adjacent to the N-terminal residue, were not hydrolyzed by the enzyme. The Km values for various peptides indicate that the enzyme has higher affinity for oligopeptides than di- and tripeptides. The aminopeptidase activity of the enzyme was also competitively inhibited by puromycin and bestatin. Analyses of the hydrolysis products of various peptides by the dansylation method indicate that the enzyme has both kinin-converting activity and angiotensinase activity.     

Purification and properties of an aminopeptidase from rat-liver cytosol.

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.     

Proteases of swine small intestine enterocytes. Kinetics of substrate hydrolysis and inhibition of aminopeptidase N from brush border vesicles

The kinetics of hydrolysis of L-leucine p-nitroanilide and some p-nitrophenylalanine dipeptides by vesicular aminopeptidase N from the porcine small intestine brush border membrane was studied. It was shown that the catalytic properties of the vesicular enzyme are very similar to those known for its solubilized counterpart. Both enzymes are inhibited by o-phenanthroline, ZnCl2 and puromycin with Ki = 10(-5)-10(-6) M. The data obtained offer new possibilities for investigating the role of aminopeptidase N in the amino acid and peptide transport across the enterocyte membrane.     

SYNTHESIS OF PARAMECIUM SURFACE PROTEINS : I. Puromycin Insensitive Amino Acid Incorporation

The synthesis of a surface protein has been studied in Paramecium through double-labeling experiments using [¹⁴C]- and [³H]leucine-labeled bacteria as the source of radioactive amino acid. Over a 4–5 h incubation period, the turnover rate was found to be higher than that of overall cell protein. In addition, the initial label is apparently utilized during the chase period, being incorporated into protein via a puromycin insensitive pathway.     

Aminopeptidase P from rat brain. Purification and action on bioactive peptides.

Aminopeptidase P (EC 3.4.11.9) was purified from rat brain cytosol. A subunit Mr of 71,000 was determined for the reduced, denaturated protein whereas an Mr of 143,000 was determined for the native enzyme. The purified aminopeptidase P selectively liberated all unblocked, preferentially basic or hydrophobic ultimate amino acids from di-, tri- and oligopeptides with N-terminal Xaa-Pro- sequences. Corresponding peptides with penultimate Ala instead of Pro were cleaved with much lower rates; oligopeptides with residues other than Pro or Ala in the penultimate position appeared not to be substrates for the enzyme. Several bioactive peptides with Xaa-Pro sequences, especially bradykinin, substance P, corticortropin-like intermediate lobe peptide, casomorphin and [Tyr]melanostatin were shortened by the N-terminal amino acid by aminopeptidase P action. Rat brain aminopeptidase P was optimally active at pH 7.6-8.0 in the presence of Mn2+. Chelating agents and SH-reacting reagents inhibited the enzyme, but common inhibitors of aminopeptidases, like amastatin or bestatin, of prolidase or of dipeptidyl peptidases II and IV, like N-benzoyloxycarbonyl-proline or epsilon-benzyl-oxycarbonyl-lysyl-proline, as well as antibiotics like beta-lactam ones, bacitracin or puromycin, had little or no effect.     

Proctolin: a potent inhibitor of aminoenkephalinase

Proctolin is a potent selective inhibitor of aminoenkephalinase. The specificity of its inhibition of various aminopeptidases is similar to that of puromycin; it inhibits aminoenkephalinase, but not leucine aminopeptidase or aminopeptidase M. Enkephalin breakdown by synaptic plasma membrane, but not by brain slices, is sensitive to proctolin. The inhibition by proctolin is partially caused by its resistance to enzymatic breakdown. The inhibition is of mixed type and is concentration dependent, and the two amino acids at the N-terminal are important for its action. The minimal structure for inhibition is a dipeptide with a basic amino acid at the N-terminal and a basic or an aromatic amino acid at the C-terminal.     

Porcine liver aminopeptidase B. Substrate specificity and inhibition by amino acids

Porcine liver aminopeptidase B[EC 3.4.11.6] is highly specific for hydrolysis of beta-naphthylamides of basic L-amino acids; the Km values for L-arginine beta-naphthylamide and L-lysine beta-naphthylamide were 0.035 and 0.12 mM, respectively. The enzyme was inhibited by various alpha-amino acids. Among basic amino acids, L-homoarginine and L-arginine were the most potent inhibitors, L-lysine and L-norarginine (alpha-amino-gamma-guanidinobutyric acid) being less inhibitory. Hydrophobic amino acids also inhibited the enzyme competitively. This suggests that there is a hydrophobic region that binds the side chain of the substrates or inhibitors in the specificity site of the enzyme. Studies on the inhibitions by L-arginine derivatives showed that blocking of the alpha-carboxyl or the alpha-amino group reduced the inhibitory effect of L-arginine. Porcine liver aminopeptidase B was not inhibited by puromycin, whereas bestatin inhibited the enzyme competitively with a Ki value of 1.4 X 10(-8) M. This enzyme had no kinin-converting activity.     

The pur7 Gene from the Puromycin Biosynthetic pur Cluster of Streptomyces alboniger Encodes a Nudix Hydrolase

Pur7 is the product of a gene from the puromycin biosynthetic pur cluster of Streptomyces alboniger. It was expressed in Escherichia coli as a recombinant protein fused to a His tag and then was highly purified through a Ni(2+) column. It showed a 3'-amino-3'-dATP pyrophosphohydrolase (nudix) activity which produced 3'-amino-3'-dAMP and pyrophosphate. This is consistent with the presence of a nudix box in its amino acid sequence. As observed with other nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement for Mg(2+). Among a large variety of other nucleotides tested, only 3'-amino-3'-dTTP was a Pur7 substrate, although at lower reaction rates than 3'-amino-3'-dATP. These findings suggest that Pur7 has a high specificity for the 3' amino group at the ribofuranoside moiety of these two substrates. The K(m) and V(max) values for these dATP and dTTP derivatives were 120 microM and 17 microM/min and 3.45 mM and 12.5 microM/min, respectively. Since it is well known that 3'-amino-3'-dATP is a strong inhibitor of DNA-dependent RNA polymerase, whereas 3'-amino-3'-dAMP is not, Pur7 appears to be similar to other nudix enzymes in terms of being a housecleaning agent that permits puromycin biosynthesis to proceed through nontoxic intermediates. Finally, the identification of this activity has allowed a revision of the previously proposed puromycin biosynthetic pathway.     

Effect of Puromycin on Guinea Pig Lymphotoxin Release and Lectin-Induced Cellular Cytotoxicity

In order to confirm the role of guinea pig lymphotoxin (GLT) in lectin-induced cellular cytotoxicity (LICC), the effect of puromycin, a potent enhancer of GLT activity, on the LICC to target L · P3 cells induced by phytohemagglutinin (PHA) was investigated under serum-free conditions. LICC was completely inhibited by puromycin, when it was added at the initiation of LICC culture, because of the inhibition of the release of GLT from the effector lymph node cells. However, LICC was markedly enhanced when puromycin was added several hours after the initiation of LICC culture. The interpretation of these facts is that GLT release can be inhibited by puromycin, but that the GLT already released exerts an enhanced cytotoxic effect on the target cells in the presence of puromycin. Enhancement of the cytotoxicity by the addition of puromycin several hours after the initiation of LICC culture was observed even after the removal of the GLT present in the supernatant, suggesting that the morphologically intact target cells were already affected by GLT in the early stages of LICC culture.     

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.     

Molecular cloning and expression of aminopeptidase A isoforms from rat hippocampus

The full-length cDNA encoding aminopeptidase A (APAL) was cloned from a rat hippocampus cDNA library. A short variant aminopeptidase A (APAS), produced by deletion, was also cloned. In the case of APAL, the longest open reading frame encodes 945 amino acid residues with a calculated molecular mass of 108 kDa, and the deduced amino acid sequence shows 76, 86 and 78% identity with its human, murine and porcine counterparts, respectively. Rat aminopeptidase A mRNAs were detected in the kidney, liver, heart and brain by Northern blot analysis. When overexpressed in COS-1 cells, APAL shows apparent aminopeptidase A activity, whereas APAS does not.     

Self-Association of Puromycin as studied by Proton Magnetic Resonance Spectroscopy

Abstract

The self-association of puromycin has been studied using proton magnetic resonance spectroscopy. The concentration, temperature and pH dependence studies of the proton chemical shifts of the adenine protons indicate that puromycin in aqueous solution at pD 7.4 self associates predominantly through adenine-adenine interaction. At this pD, the amino group of the aminoacyl segment of puromycin has been demonstrated to exist in a equilibrium blend of protonated and non-protonated forms. At pD 2.6, PM is found to exist predominantly in the monomeric from in which the methyl groups of the 6N-dimethyladenine are found to be non-equivalent due to hindered rotation about the C6-N6 bond.     

Amino acid uptake in the developing chick embryo heart. The effect of insulin on glycine and leucine accumulation

1. The accumulation of [1-(14)C]glycine and the uptake, accumulation, incorporation (into protein, lipid, glycogen) and oxidation of l-[1-(14)C]leucine in 5-day-old chick embryo hearts were investigated in vitro, and the effects of insulin, puromycin and 4-methyl-2-oxopentanoic acid on these processes were studied. 2. With glycine, the ratio of concentration of the labelled amino acid in the cell water to that in medium markedly exceeded unity. Insulin significantly increased this ratio. Puromycin did not prevent the insulin effect. 3. With leucine, the concentration ratio of the labelled amino acid between intracellular and extracellular water approached unity in the absence of puromycin and was doubled by its presence. In neither case did insulin substantially alter this ratio. The addition of 4-methyl-2-oxopentanoic acid had no effect in the absence of insulin, but produced a significant increase of the concentration ratio in the presence of the hormone. 4. Leucine uptake was increased slightly by insulin in all experimental conditions except in the presence of puromycin, where a more pronounced stimulation was observed. The hormone had no effect on the incorporation of the labelled amino acid into protein, but accelerated its oxidation to carbon dioxide; the latter effect was particularly evident in the presence of puromycin and disappeared after the addition of 4-methyl-2-oxopentanoic acid.     

Puromycin inhibition of eucaryotic ribosomes. Differences in sensitivity between polypeptide synthesis directed by endogenous mRNA and synthetic templates including poly(U).

In a protein synthesis sytem derived from porcine uteri we have made the following observations: 1. Synthesis directed by the endogenous mRNA of the system is more sensitive to inhibition by puromycin than poly(U) directed synthesis. 2. Synthesis directed by the synthetic templates poly(G,U) and poly(C,U) is more sensitive to inhibition by puromycin than poly(U) directed synthesis and frequently shows a sensitivity to puromycin similar to that directed by endogenous mRNA. 3. Protein synthesis was inhibited by three aminoacyloligonucleotides (C-A-Phe, C-A-Asp, and C-A-Pro) which are analogs of the 3' terminus of aminoacyl tRNAs. Of the three, C-A-Phe was the most active and C-A-Asp the least active but, unlike puromycin, inhibition by these compounds was the same for endogenous and poly(U) directed synthesis. These results are interpreted as supporting the proposal that the acceptor site of ribosomes contains an hydrophobic binding region which interacts with the side chain of aliphatic amino acids, and particularly with the aromatic side chain of phenylalanine.     

An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro

The degradation of thyroliberin (less than Glu-His-Pro-NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. 1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed. 2. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyse the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. 3. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalysed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin but did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. 4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 microM), the post-proline cleaving enzyme by bacitracin (IC50 = 42 microM) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 microM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.     

Purification of three aminopeptidases from human maternal serum

Three aminopeptidases (I--III) were purified from maternal serum using sequential chromatographic fractionations. Aminopeptidase I was specific for N-terminal alpha-L-dicarboxylic acid residues and activated by alkaline earth metals (Ba2+, Ca2+, Sr2+). It is concluded that aminopeptidase I is aminopeptidase A (L-alpha-aspartyl-(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7). Aminopeptidase II hydrolysed all tested substrates including L-cystine and Bz-L-cysteine derivatives but preferred L-leucine derivatives. The properties of aminopeptidase II are equal to those described for the cystine aminopeptidase (oxytocinase) (EC 3.4.11.3.). Aminopeptidase III preferred L-alanine derivatives as substrates. It was activated by Co2+, but strongly inhibited by amastatin, puromycin and L-methionine. The characteristics are reminiscent of those of alanine aminopeptidase (EC 3.4.11.-).     

Puromycin dependent formation of initial peptides as a method to measure the initiation

Amino acid incorporating mitochondria, supplied with formyl-group donor, show a puromycin dependent formation of initial peptides. When the puromycin concentration is increased from 1 to 10 mM the length of the puromycin peptides formed is reduced up to the predominant formation of formyl-methionyl-puromycin. A method for measuring the total production of these peptides has been developed, allowing an assay of the initiation reaction. The number of initiation acts is higher than that of mitochondrial ribosomes present. Under suitable conditions the rate of formation of puromycin peptides is constant and largely accounts for the rate of synthesis of the polypeptide chains.     

Purification and characterization of an enkephalin-degrading aminopeptidase from guinea pig serum

An enkaphalin-degrading aminopeptidase using Leu-enkephalin as a substrate was purified about 4100-fold from guinea pig serum. The purified preparation was apparently homogenous, showing on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was approx. 92 000. The amino-peptidase had a pH optimum of 7.0 with K_m values of 0.12 mM and 0.18 mM for Leu- and Met-enkephalin, respectively. The enzyme hydrolyzed neutral, basic and aromatic amino acid β-naphthylamides, but did not the acidic one. The enzyme was inhibited strongly by metal-chelating agents, bestatin and amastatin and weakly by puromycin. Among several biologically active peptides, angiotensin III and substance P strongly inhibited the enzyme.     

Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition

Peptidyl transferase inhibitors have generally been studied using simple systems and remain largely unexamined In in vitro translation extracts. Here, we investigate the potency, product distribution, and mechanism of various puromycin-oligonucleotide conjugates (1 to 44 nt with 3'-puromycin) In a reticulocyte lysate cell-free translation system. Surprisingly, the potency decreases as the chain length of the oligonucleotide is increased in this series, and only very short puromycin conjugates function efficiently (IC50 < 50 microM). This observation stands in contrast with work on isolated large ribosomal subunits, which Indicates that many of the puromycin-oligonucleotide conjugates we studied should have higher affinity for the peptidyl transferase center than puromycin itself. Two tRNA(Al)-derived minihelices containing puromycin provide an exception to the size trend, and are the only constructs longer than 4 nt with any appreciable potency (IC50 = 40-56 microM). However, the puromycin minihelices inhibit translation by sequestering one or more soluble translation factors, and do not appear to participate in detectable peptide bond formation with the nascent chain. In contrast, puromycin and other short derivatives act in a factor-independent fashion at the peptidyl transferase center and readily become conjugated to the nascent protein chain. However, even for the short derivatives, much of the translation inhibition occurs without peptide bond formation between puromycin and the nascent chain, a revision of the classical model for puromycin function. This peptide bond-independent mode is likely a combination of multiple effects including inhibition of initiation and failure to properly recycle translation complexes that have reacted with puromycin.