Conclusions about aminopeptidase in tissue sections from studies of amino acid naphthylamide hydrolysis.

Catalytic properties (KM, Vmax) of aminopeptidase in pig kidney sections, in isolated membranes and in a solubilized purified form were investigated using amino acid 2-naphthylamides and 4-methoxy-2-naphthylamides. In the first case these properties were estimated on the basis of the stain intensity resulting from the coupling of product with Fast Blue B, in the latter two cases they were measured fluorometrically. The following observations were made: (1) In all three cases the substrate turnover was shown to be a direct function of time and enzyme concentration. (2) The values obtained for the solubilized and the membrane bound form were practically identical but differed from those found in tissue sections. (3) Each amino acid derivative had defined constants, but these were difficult to obtain in sections, especially if it was necessary, on account of poor solubilities, to use low substrate concentrations. (4) Hydrophilic amino acid derivatives were adsorbed to tissue membranes much less than hydrophobic ones. (5) Fast Blue B caused a non-competitive inhibition of enzymic activity. (6) Binding of antibody against pure aminopeptidase caused inhibition of the enzymic hydrolysis of all the naphthylamides. Thus, histochemical stain intensities per time and area derived from one substrate at a defined concentration are suitable for the determination of enzyme concentrations. However, no conclusions regarding the homogeneity of the enzyme in sections can be drawn by comparing the stain intensities obtained with different substrates in contrast to data from the inhibition of substrate hydrolysis by antibody.     

Bacillus subtilis aminopeptidase: Specificity toward amino acid amides,dipeptides, and oligopeptides

Bacillus subtilis aminopeptidase hydrolyzed amino acid amides with a specificity similar to that determined using amino acyl-β-naphthylamides, but at much greater catalytic rates. Neutral and basic amino acid amides were the best substrates. A series of Leu and Lys NH₂-terminal dipeptides hydrolyzed by Co²⁺-activated aminopeptidase showed that the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ ratios for the Lys substrates were fourfold greater than the corresponding Leu substrates and that catalytic differences reflected the identity of COOH terminal residues. Greatest catalytic rates were obtained when aromatic residues were in the COOH terminal position of the substrate (Trp, Tyr, Phe); but, significant hydrolysis was achieved when aliphatic residues were COOH-terminal in the dipeptide. The Co²⁺-activated enzyme would not hydrolyze peptide bonds composed of the imide nitrogen of Pro, thus, bradykinin was not a substrate. However, the Co²⁺-activated enzyme removed sequentially the first four residues from eledoisin-related peptide and the A chain of bovine insulin.     

Purification and Partial Characterization of an Aminopeptidase from Lactobacillus lactis

A surface-bound aminopeptidase of Lactobacillus lactis cells was solubilized with lysozyme, and the extract was subjected to streptomycin sulfate precipitation, ammonium sulfate fractionation, chromatography on Sephadex G-100 and diethylaminoethyl-Sephadex A-50, and preparative polyacrylamide gel electrophoresis. The purified enzyme was homogeneous in disc electrophoretic analysis and consisted of a single polypeptide chain with a molecular weight of 78,000 to 81,000. The optimal pH and optimal temperature for enzyme activity were 6.2 to 7.2 and 47.5 degrees C, respectively, for l-lysine-4-nitroanilide as the substrate. The enzyme was activated by Co and Zn ions and inhibited by Cu, Hg, and Fe ions and by the metal-complexing reagents ethylenediaminetetraacetic acid, 1,10-phenanthroline, and alpha,alpha'-dipyridyl. Higher concentrations of substrate and hydrolysis products also inhibited the activity of the enzyme. The aminopeptidase had broad substrate specificity and hydrolyzed many amino acid arylamides and many peptides with unsubstituted NH(2)-terminal amino acids.     

Mammalian lens dipeptidyl aminopeptidase III

An intracellular exopeptidase identified as dipeptidyl aminopeptidase III (DAP III) was found to be abundant in the bovine lens. The enzyme contained in aqueous extracts exhibited a marked preference, compared to other dipeptidyl-β-naphthylamides, for the release of Arg-Arg from Arg-Arg-2-NNap at the optimum pH 9.0 and 37°. The K_m for this substrate was estimated to be 2.83 × 10^?5M. Lens DAP III was inhibited by EDTA, p-chloromercuriphenyl sulfonate, and puromycin. Lens aminopeptidase activities measured at pH 7.5 on the β-naphthylamides of leucine, alanine, and arginine, included for comparison, suggested that not only is leucine aminopeptidase abundant, but also other aminopeptidases that appear to include alanine aminopeptidase and aminopeptidase B.     

Aminopeptidase activity of elastotic fibres--a histochemical artifact?

Synopsis Elastotic fibres of human skin and of elastofibroma were found to be stained when sections of these tissues had been incubated for aminopeptidase using leucine naphthylamide as substrate and Fast Blue B as the coupling agent. Sections that had been inactivated (with formalin or mercuric chloride) and partially covered with an intact kidney section were stained identically. A dialysing membrane placed between the inactivated skin section and the intact kidney section did not prevent staining of the elastotic fibres. The fibres were also stained when sections were incubated in an aqueous mixture of naphthylamine and an excess of Fast Blue B. It is concluded that the staining of elastotic fibres in histochemical amino-peptidase reactions may be due to adsorption of coloured azo derivatives and may not indicate enzyme activity in the fibres.     

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.     

A membrane-bound aminopeptidase isolated from monkey brain and its action on enkephalin

A membrane-bound aminopeptidase which cleaves the tyrosin-glycine bond of enkephalin was purified about 1600-fold from monkey brain. This aminopeptidase hydrolyzed Leu-enkephalin with a K_m value of 35 μM and also hydrolyzed basic, neutral and aromatic amino acid β-naphthylamides. An apparently homogeneous enzyme consisted of a single polypeptide chain with a molecular weight of approx. 100 000. The optimum pH was in the neutral region. From the analysis of the reaction products, only aminopeptidase activity was detected. The enzyme was inactivated by metal chelators, but the activity could be restored by the addition of divalent cations, such as Co²⁺, Mg²⁺ and Zn²⁺. Puromycin, bestatin and amastatin, which are aminopeptidase inhibitors derived from microorganism, showed strong competitive inhibition of the enzyme, the most potent being amastatin, with a K_i value of 0.02 μM.     

Purification and characterization of human placental aminopeptidase A

Human placental aminopeptidase A (AAP) was purified 3,900-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100, then subjected to trypsin digestion, zinc sulfate fractionation, chromatographies with DE-52, Sephacryl S-300, and hydroxylapatite, affinity chromatography with Bestatin-Sepharose 4B, and finally immunoaffinity chromatography with the antibody against microsomal leucine aminopeptidase (LAP). Aminopeptidase A was completely separated from leucine aminopeptidase by the immunoaffinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 280,000 by gel filtration. The purified enzyme was most active at pH 7.1 with L-aspartyl-beta-naphthylamide (L-Asp-NA) as substrate; the Km value for this substrate was 4.0 mmol/l in the presence of Ca2+. Human placental aminopeptidase A was markedly activated by alkaline earth metals (Ca2+, Sr2+, Ba2+), but strongly inhibited by metal chelating agents such as EDTA and o-phenanthroline. The highest activity was observed with L-glutamyl-beta-naphthylamide, while only minimal hydrolysis was found with some neutral and basic amino acid beta-naphthylamides.     

An Aminopeptidase from Bovine Brain Which Catalyzes the Hydrolysis of Enkephalin

An aminopeptidase from bovine brain which catalyzes the hydrolysis of the tyrosyl¹-glycine² bond of methionine⁵-enkephalin has been purified to electrophoretic homogeneity. The enzyme also catalyzes the hydrolysis of di-peptides, tripeptides, and amino acid β-naphthylamides. The enzyme can be inactivated by dialysis against EDTA, and reconstituted with divalent metal ions. Inhibition of the enzyme is observed in the presence of p-chloromercuribenzoate and puromycin, the latter compound not being hydro-lyzed by the enzyme. The enzyme is composed of a single polypeptide chain of molecular weight approx. 100,000. The properties of this enzyme are similar to those reported for other brain aminopeptidases active on enkephalin, although distinct differences are observed.     

Comparison of aminopeptidase inhibition by amino acids in human and porcine skeletal muscle tissues in vitro

We compared the inhibitory action of individual amino acids in vitro on the activities of alanyl-, arginyl-, leucyl- and pyroglutamyl aminopeptidases purified from human and porcine skeletal muscle tissues. The range of susceptibility to inhibition by individual amino acids (<25 mM) for different aminopeptidase types broadly paralleled that for the respective substrate specificities (in terms of relative rates of hydrolysis of amino acyl-AMC derivatives) for these enzymes. Thus, alanyl aminopeptidase (which hydrolyses a broad range of aminoacyl-AMC substrates) was inhibited by a correspondingly broad range of amino acids (although the respective ranking order of amino acids was not identical in each case), whereas pyroglutamyl aminopeptidase (which hydrolyses only pyroglutamyl AMC as substrate) was inhibited by pyroglutamic acid only. The mode of inhibition (competitive/non-competitive) varied for different enzyme types, both within and between each species. For enzymes purified from human muscle, alanyl, arginyl and leucyl aminopeptidases were inhibited by amino acids via the non-competitive mode (pyroglutamyl aminopeptidase via the competitive mode), whereas corresponding enzymes purified from porcine muscle were inhibited via the competitive mode. The data obtained indicate that the same aminopeptidase types are present in human and porcine skeletal muscle tissues, with corresponding enzymes having broadly similar assay characteristics and susceptibilities to inhibition by amino acids (although the mode of inhibition for corresponding enzymes may differ in each species). Such data obtained in vitro may prove of value in devising experimental strategies to manipulate protein turnover/muscle deposition in vivo, via inhibition of aminopeptidase action after administration of an appropriate admixture of amino acids.     

An investigation of arterial insufficiency in rat hindlimb. An enzymic, mitochondrial and histological study.

A membrane-bound aminopeptidase able to remove methionine from haemoglobin nascent peptides is described. The enzyme also hydrolyses methionine from methionyl-lysyl-bradykinin but not lysine from lysyl-bradykinin. The tripeptide Met-Ala-Ser is poorly hydrolysed. This aminopeptidase also splits amino acid 2-naphthylamides, being, however, less specific with respect to these synthetic substrates.     

Intramolecularly-quenched fluorescent peptides as fluorogenic substrates ofleucine aminopeptidase and inhibitors of clostridial aminopeptidase.

Fluorogenic oligopeptide derivatives of the type Lys(ABz)-ONBzl, where ABz iso-aminobenzoyl (anthraniloyl), X stands for Ala Phe, or Ala-Ala, and ONBzlis p-nitrobenzyloxy, were synthesized and shown to be hydrolyzed by leucine aminopeptidase. The hydrolysis is accompanied by an increase in fluorescence due to disruptionof the intramolecular quenching of the fluorescent anthraniloyl moiety by the nitrobenzyester group. The spectral characteristics of the compounds are not consistent withan energy transfer mechanism according to F?rster, therefore the quenching isassumed to be caused by a direct encouter between the quenching and the fluorecentgroups. The change in fluorescence that accompanies the enzymic hydrolysis ofthe first peptide bound was used for quantitative measurement of the activity ofthe activity of leucine aminopeptidase and for the determination of some of itskinetic parameters. A bacterial aminopeptidase from Clostrdium histolyticumthat is very similar to leucine aminopeptidase in its substrate specificity inits substrate specificity did not hydrolyze the above peptidederivatives. Thehydrolysis of leucine p-nitroanilide by this enzyme was found to be inhibitedby the three peptides and the corresponding inhibition constants were determined.     

Human liver alanine aminopeptidase. Inhibition by amino acids.

Human liver alanine aminopeptidase is inhibited by L-amino acids having hydrophobic side chains such as Phe, Tyr, Trp, Met, and Leu. Blocking of the amino group or the carboxyl group greatly reduces the inhibitory capacity of the amino acid. Kinetic studies demonstrate that inhibition of hydrolysis of the substrate L-Ala-beta-naphthylamide is of the noncompetitive type. Inhibition of the substrate L-Leu-L-Leu is of the mixed type. Inhibition of the substrate L-Ala-L-Ala-L-Ala is of the competitive type. These changes in the mechanism of inhibition are thought to be the result of the binding of the amino acid to the third residue binding site on the enzyme. This is the part of the active center to which the third residue from the amino end of a peptide substrate is normally bound. The inhibitor constants of several alanine oligopeptides are shown to decrease with increasing length through L-Ala-L-Ala-L-Ala-L-Ala, demonstrating that alanine aminopeptidase is a multisited enzyme with three and possibly four residue sites per active center. The inhibitor constant for Gly-Gly--Phe suggesting that indeed the third residue site preferentially binds large hydrophobic residues.     

Exopeptidase activity in eggs of Trichostrongylus colubriformis (Nematoda)

Summary

A supernatant from eggs of the ruminant nematode Trichostrongylus colubriformis contained an enzyme that was similar to leucine aminopeptidase (LAP), based on hydrolysis of the substrate L-leucine β-naphthylamide to β-naphthylamine. A Michaelis-Menten constant (K _m) of 0.155 mM was obtained. Rate of hydrolysis of 16 substrates revealed that L-phenylalanine and L-tyrosine β-naphthylamides were hydrolyzed most readily while seven additional substrates were hydrolyzed at lesser rates. The optimum pH for enzymatic activity was 6.75–7.5. Enzymatic activity was lost by heating the egg supernatant to 60°C for 5 min or freezing at 0°C for 28 days. Addition of millimolar concentrations of the chlorides of zinc, manganese and magnesium to the egg supernatant had no stimulatory effect on enzyme activity while 10 and 100 mM concentrations significantly reduced activity. Ethylenediamine tetraacetic acid at 10^?4 M had no effect on enzymatic activity. Activity was inhibited by 10^?4 M 1,10-phenanthroline, but the inhibition was reversed by zinc chloride at 10^?3 M. Di-isopropylphosphofluoridate at 10^?3 M reduced enzymatic activity moderately. Enzyme activity in egg supernatant increased 2.2-fold from 21 days to 60–90 days of a primary infection in the host while a 3.3-fold increase was found in primary versus secondary infections.     

The hydrolysis of new lipid-like substrates and trilaurin in monolayers catalyzed with the lipase fromPseudomonas fluorescens

A simple method for determining the enzymic hydrolysis parameters of lipid-like substrates and trilaurin assembled in monolayers at the water-air interface was suggested. At a surface pressure of 10 mN/m, the initial rates of lipolysis were found to be proportional to the decrease in area of the substrate monolayer caused by the enzymic hydrolysis in a single-compartment Langmuir balance. The kinetic parameters for the hydrolysis of trilaurin and three 1,3-dilaurylpseudoglycerides acetylated in position 2 with an amino acid (phenylalanine, leucine, or valine) catalyzed with lipase fromPseudomonas fluorescens were determined. Unlike models of enzymic hydrolysis that neglect the thickness of the substrate monolayer, our method allows the determination of kinetic parameters in standard dimensions. The values ofk _cat for the synthetic pseudoglycerides were found to be significantly higher than that for trilaurin, while the values ofK _m(app) were close. This may be due to the presence of positively charged primary amino groups in the molecules of pseudoglycerides.     

Specificity and inhibition studies of human renal dipeptidase

Purified human renal dipeptidase was shown to exhibit no detectable activity against substrates that are characteristic for other known mammalian peptidases. The enzymic activities that were assayed were: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminopeptidase P, and tripeptidase. A quantitative assay for renal dipeptidase was developed which measures the rate of release of glycine from glycylpeptides by pre-column derivatization of the amino acid with phenylisothiocyanate followed by high-performance liquid chromatography. The ratio of Vmax/Km for a series of dipeptides was used as an index of the enzyme's preference for substrates. According to the data obtained, the enzyme prefers that a bulky, hydrophobic group of the dipeptide be located at the N-terminal position. This suggests that the substrate-binding site of the enzyme may provide a hydrophobic pocket to accommodate the hydrophobic moiety at the N-terminus of the dipeptide. The unsaturated dipeptide substrate, glycyldehydrophenylalanine, was employed in spectrophotometric assays to provide kinetic analyses of enzymic inhibition. The inhibitory effect of dithiothreitol was immediate, and the kinetic data indicated reversible, competitive inhibition. These results suggest that the inhibitor competes with substrate for a coordination site of zinc within the active site of the enzyme. The reaction of renal dipeptidase with the transition-state peptide analog, bestatin, was time dependent, and velocity measurements were made after the inhibitor had been incubated with the enzyme until constant rates were observed. These steady-state rate measurements, made following preincubation of enzyme with inhibitor, were employed to show that bestatin caused apparent non-competitive inhibition of the enzyme. The inhibitory effect of the beta-lactam inhibitor, cilastatin, upon the oligomeric dipeptidase was shown to be competitive. Graphical analysis of this inhibition indicated that the subunits of the enzyme react independently during enzymic catalysis and that the catalytic event is not influenced by cooperativity between sites on the subunits. The conversion of leukotriene D4 to leukotriene E4 in the presence of human renal dipeptidase was demonstrated by HPLC procedures. This bioconversion reaction was quantitated by derivatizing the glycine produced by cleavage of the cysteinylglycine bond and isolating this derivative as a function of time. The relationship between the purified enzyme concentration and enzyme activity against leukotriene D4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay.(ABSTRACT TRUNCATED AT 400 WORDS)     

Separation of rat muscle aminopeptidases.

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.     

BRAIN PEPTIDASES: CONVERSION AND INACTIVATION OF KININ HORMONES

Abstract— Two enzymes that selectively hydrolyse kinins at pH 7.5 were obtained in partially purified form from the supernatant fraction of homogenates of previously frozen rabbit brain by gel filtration on Sephadex G-100. The enzymes were detected and their activity estimated by bioassay with the isolated guinea pig ileum The products of the enzymic reactions were identified by high voltage electrophoresis at pH 3.5 and by the determination with the amino acid analyser of the amino acids released from the kinins.
One enzyme, kinin-converting enzyme, catalyses the hydrolysis of kinin-10 (Lysbradykinin) and kinin-11 (Met-Lys-bradykinin) into kinin-9 (bradykinin). It also hydrolyses the aminoacyl-8-naphthylamides of methionine, lysine, arginine and leucine. The conversion of kinin-10 to kinin-9 was inhibited by puromycin (K_i 3.5 × 10⁻⁵ M) These properties are similar to those of brain arylamidases described in the literature.
Kininase, the second enzyme, inactives kinins 9, 10 and 11 by peptide-bond hydrolysis. Similar rates of release of arginine and phenylalanine were observed for the three kinins, suggesting that kininase acts at the carboxy-terminus of these peptides.
Our results suggest that brain contains proteases which apparently selectively metabolize polypeptide hormones that exert definite pharmacological effects on the central and peripheral nervous systems.     

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.     

A novel aminopeptidase from Clostridium histolyticum

An aminopeptidase was found in the culture filtrate of Cl. histolyticum and purified to homogeneity (130 times) in a two-step procedure. All types of N-terminal amino acids, including proline and hydroxyproline are cleaved by the enzyme from small peptides and from polypeptides. A low rate of hydrolysis was observed for β-naphthylamides and for alanine amide; p-nitroanilides were not hydrolyzed. Kinetic parameters (K_m and V_max) for several tripeptides and the tetrapeptide Pro-Gly-Pro-Pro were determined. The enzyme has a pH optimum at 8.6. The presence of either Mn⁺⁺ or Co⁺⁺ is essential for its activity. Only slight activation was observed with Ni⁺⁺ and Cd⁺⁺, while Zn⁺⁺ and Cu⁺⁺ were inhibitory. The molecular weight of the native enzyme is about 340,000, and a molecular weight of about 60,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate (SDS).The culture filtrate of Cl. histolyticum has been shown to contain various proteolytic enzymes, in addition to collagenase^1–5. In a search for enzymes acting on proline-rich peptides, we tested the crude filtrate with (Pro-Gly-Pro)_n, (Pro-Gly-Pro)_n-OMe, α,DNP-(Pro-Gly-Pro)_n and poly-L-proline as substrates. Proline was formed only from (Pro-Gly-Pro)_n and its methyl ester. This showed the presence in Cl. histolyticum filtrate of an aminopeptidase which cleaves N-terminal proline from polypeptides but not from polyproline. The purification and some of the properties of this clostridial aminopeptidase (CAP) are described in this communication.