A new feature of angiotensin-converting enzyme in the brain: hydrolysis of substance P

Highly purified rat brain angiotensin-converting enzyme hydrolyzes substance P which contains a C-terminal amino acid with an amidated carboxyl group. The hydrolysis of substance P verified by amino-group fluorometry and by high-performance liquid chromatography is inhibited by captopril, but not by phosphoramidon. The presence of sodium chloride is essential for the hydrolysis. The analyses of cleavage products indicate that the enzyme hydrolyzes substance P between Phe7-Phe8 and Phe8-Gly9 by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action.     

Enkephalin-degrading dipeptidyl carboxypeptidase in guinea pig serum: its properties and action on bioactive peptides

A dipeptidyl carboxypeptidase, which cleaved the Gly3-Phe4 bond of enkephalins, was purified from guinea pig serum 420-fold. The optimum pH of the enzyme was in the neutral range (pH 7.25), and the molecular weight was estimated to be approx. 280,000. The enzyme hydrolyzed Met- and Leu-enkephalin with Km values of 0.30 and 0.50 mM, respectively. The enzyme was inhibited by metal chelators and p-chloro-mercuribenzoate. Captopril showed high inhibitory potency, while phosphoramidon and Phe-Ala showed no effect on the enzyme activity. Therefore, the obtained enzyme can be classified as an angiotensin-converting enzyme (EC 3.4.15.1). Among the bioactive peptides examined, bradykinin and angiotensin I were hydrolyzed by the enzyme. Angiotensin III showed a stronger inhibitory effect than that of angiotensin II. Substance P, gastrin I, and secretin were also inhibitory toward the enzyme activity. On high-performance liquid chromatography analysis, Met-enkephalin-Arg6-Phe7 and Leu-enkephalin-Arg6 were cleaved sequentially at the second peptide bond of the C terminus. Thus, the dipeptidyl carboxypeptidase in guinea pig serum may play a role not only in the angiotensin-bradykinin system but also in the metabolism of circulating enkephalins and other bioactive peptides.     

Seasonal changes in photosynthetic capacity of leaves of kiwifruit (Actinidia deliciosa) vines

A simplified procedure for the assay and purification of an enzyme which activates a galactosyltransferase (EC 2.4.1.96) involved in volume regulation of the unicellular alga Poterioochromonas malhamensis (Peterfi) is described. The enzyme was extracted with water from membranes, followed by chromatography on DEAE-Sephacel, phenyl-Sepharose and fetuin-agarose. Its proteinase activity was demonstrated by cleavage of oxidized insulin A- and B-Chains. The predominant cleavage site of the oxidized A-chain is the peptide bond between ¹³Leu and ¹⁴Tyr whereas ¹⁶Leu-¹⁷Glu is also hydrolyzed with minor activity. Besides this chymotrypsin-like endopeptidase activity some carboxypeptidase activity was also observed.     

Rat insulin-degrading enzyme: cleavage pattern of the natriuretic peptide hormones ANP, BNP, and CNP revealed by HPLC and mass spectrometry.

The degradation of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) by insulin-degrading enzyme (IDE) has been investigated. As revealed by high-performance liquid chromatography, all three peptides are sequentially cleaved at a limited number of sites, the latter of which were identified by mass spectrometric analyses. The studies revealed that ANP is preferred as substrate over BNP and CNP. ANP degradation is rapidly initiated by hydrolysis at the Ser25-Phe26 bond. Three additional cleavage sites were identified in ANP after prolonged incubation with IDE; in contrast, three and two bonds were hydrolyzed in BNP and CNP, respectively. Analysis of the nine cleavage sites shows a preference for basic or hydrophobic amino acid residues on the carboxyl side of a cleaved peptide bond. In contrast to most of the peptide fragments generated by IDE activity, the initial ANP cleavage product, F-R-Y, is rapidly degraded further by cleavage of the R-Y bond. Cross-linking studies with 125I-ANP in the presence of sulfhydryl-modifying agent indicate that IDE activity is inhibited at the level of initial substrate binding whereas metal-ion chelating agents only prevent hydrolysis. On the basis of its structural and enzymatic properties, IDE exhibits striking similarity to a number of recently-described endopeptidases.     

Membrane bound pituitary metalloendopeptidase: Apparent identity to enkephalinase

A membrane bound zinc-metalloendopeptidase from bovine pituitaries with a specificity toward bonds on the amino side of hydrophobic amino acids, cleaves Met- and Leu-enkephalin at the Gly-Phe bond, releasing Phe-Met and Phe-Leu respectively. The enzyme also hydrolyzes bonds on the amino side of hydrophobic amino acids in oxytocin, bradykinin, neurotensin and several synthetic substrates. A free carboxyl group on a dipeptide C-terminal to the hydrolyzed bond is not a requirement for activity. The enzyme is also present in brain membrane fractions. The regional distribution of this enzyme in brain, its specificity toward natural and synthetic substrates, and its sensitivity to inhibitors, suggest that the enzyme is identical to an activity referred to as “enkephalinase”, which has been described as dipeptidyl carboxypeptidase. The data show that the enzyme is an endopeptidase with a specificity similar to that of a group of microbial proteases, one of which is thermolysin.     

N-terminal tetrapeptide of dermorphin and D-Arg-substituted tetrapeptides: inactivation process of the antinociceptive activity by peptidase

Degradation products of the N-terminal tetrapeptide of dermorphin, H-Tyr-D-Ala-Phe-Gly-OH (ALPG) and D-Arg2-substituted tetrapeptide analogs of dermorphin, H-Tyr-D-Arg-Phe-Gly-OH (ARPG), H-Tyr-D-Arg-Phe-Gly-NH2 (TDAPG-NH2) and H-Tyr-D-Arg-Phe-beta-Ala-OH (TDAPA) by enkephalin degrading enzymes were studied by using reversed-phase high-performance liquid chromatography. After 5 and 25 hr incubations of the peptides with solubilized enzymes of mouse brain or spinal cord, liberation of the appreciable Tyr1 residue was observed in ALPG but not in ARPG, TDAPG-NH2 and TDAPA. When ARPG and TDAPG-NH2 were incubated with enzymes for 25 hr, a main degradation product was the N-terminal tripeptide produced from the hydrolysis of Phe3-Gly4 bond. Conversely, TDAPA did not produce the N-terminal tripeptide after 25 hr incubation with enzymes. In the enzyme assay, Tyr1-D-Arg2 bond of ARPG, TDAPG-NH2 and TDAPA was more stable than that of ALPG to the cleavage by aminopeptidase M (AP-M). Phe3-Gly4 bond of ALPG, ARPG and TDAPG-NH2 were easily hydrolyzed by carboxypeptidase Y (CP-Y) within 3 hr incubation, whereas the hydrolysis of Phe3-beta-Ala4 bond of TDAPA by CP-Y was not observed after 3 hr incubation. The present results and previous behavioural data suggest that a potent and prolonged antinociceptive activity of the D-Arg-substituted tetrapeptides is mainly attributed to the stability of Tyr1-D-Arg2 bond against aminopeptidase of peptidases.     

Degradation of bradykinin and its metabolites by rat brain synaptic membranes

Bradykinin (BK) (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) was degraded by rat brain synaptic membranes at a rate comparable to that found for Met-enkephalin, but approximately 40 times the rate for vasopressin and oxytocin. The catabolic pathway for BK and its metabolites was elucidated through the use of high performance liquid chromatography for metabolite identification and peptidase inhibitors for blocking specific cleavage sites. BK was hydrolyzed at three sites: at the -Phe5-Ser6- bond by metalloendopeptidase 24.15, at the -Pro7-Phe8- bond by an apparently novel peptidyl dipeptidase, and at the -Phe8-Arg9 bond by a carboxypeptidase B-like enzyme. Each enzyme contributed about equally to BK degradation under the assay conditions used. Some of the resulting metabolites were further hydrolyzed: BK(1-8) to BK(1-7) + Phe by a DFP inhibitable prolyl carboxypeptidase-like enzyme, BK(1-8) to BK(1-5) + BK(6-8) by metalloendopeptidase 24.15, BK(1-7) slowly to BK(1-5) by a second peptidyl dipeptidase which was captopril inhibited, and Phe-Arg to Phe + Arg by a bestatin-inhibited dipeptidase. A number of properties of the individual enzymes were determined including sensitivity to a variety of peptidase inhibitors. These results provide a starting point for investigating the potential physiological role of each enzyme in BK function in the brain.     

Enkephalin degrading enzymes in cerebrospinal fluid

Enkephalins were rapidly degraded by specific enzyme systems in vivo. In cerebrospinal fluid (CSF), however, it has been undefined whether these enzyme systems existed. Our experiments showed enkephalins were hydrolyzed by the enzymatic activity in both CSF of human and monkey. The results by the thin layer chromatography and the high performance liquid chromatography revealed the reaction products of CSF and enkephalin were tyrosine, tyrosyl-glycine and tyrosyl-glycyl-glycine. Therefore, the enzymes in CSF were considered to be an aminopeptidase, a dipeptidyl aminopeptidase and a dipeptidyl carboxypeptidase. Our results suggest that in the assay of enkephalin in CSF, the effects of these enzymes should be considered.     

Purification and characterization of cathepsin B from monkey skeletal muscle

Cathepsin B was purified about 11,000-fold from monkey skeletal muscle by ammonium sulfate fractionation and sequential column chromatographies monitored by assaying of Z-Phe-Arg-MCA hydrolase activity. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 24,000 by gel filtration. It had a pH optimum of 6.5, required a thiol reducing agent for activation, and was inhibited by various thiol protease inhibitors. These properties were similar to those reported for cathepsins B from other sources. Although the enzyme scarcely hydrolyzed ordinary proteins, such as casein, hemoglobin, and bovine serum albumin, it degraded myosin and actin among various myofibrillar proteins. These results strongly suggested that skeletal muscle cathepsin B may participate in the degradation of muscle proteins in vivo. In addition, cathepsin B was shown to hydrolyze various neuropeptides such as Leu-enkephalin, beta-neoendorphin, alpha-neoendorphin, dynorphin(1-13), and substance P. It appeared to act on these peptides mainly as a dipeptidyl carboxypeptidase, although not so rigorously, presumably due to its endopeptidase activity.     

A fluorescent oligopeptide energy transfer assay with broad applications for neutral proteases

A fluorescent peptide substrate to explore the protease specificity for the amino acid regions C- and N-terminal to the cleavage site has been designed. Intramolecular quenching of indole fluorescence by an N-terminal dansyl group separated by six amino acid residues forms the basis of this assay. For a particular enzyme, specificity can be designed into the peptide sequence by means of the number of residues that separate the two chromophores. In the present instance, the heptapeptide Dns-Gly-Lys-Tyr-Ala-Pro-Trp-Val is used to assay angiotensin converting enzyme (ACE), Astacus protease, carboxypeptidase A, alpha-chymotrypsin, and trypsin, all of which cleave the peptide in accord with their known specificity: Trypsin and Astacus protease hydrolyze only the Lys-Tyr and Tyr-Ala bonds, respectively. alpha-Chymotrypsin primarily cleaves the Tyr-Ala bond while ACE makes three successive dipeptidyl cleavages from the C-terminus. Carboxypeptidase rapidly hydrolyzes first the Trp-Val and then the Pro-Trp bond. For all of the enzymes, catalytic activity (kcat/Km) is in the range from 10(5) to 10(6) M-1 s-1. Hydrolysis causes a fluorescence increase in the 310 to 410 nm region of 8.6- to 13.6-fold depending on the enzyme that is assayed. Assays can be designed based on the increase in tryptophan fluorescence or by individual product analyses using thin-layer or high-performance liquid chromatography. The specificity and sensitivity of such internally quenched fluorescent oligopeptides would seem to be ideal for the assay of specific endoproteases.     

Oligo(methionyl) proteins. Enzymatic hydrolysis of the model isopeptides N epsilon-oligo(L-methionyl)-L-lysine

A number of model isopeptides containing oligo(methionine) chains varying in length (2-5 residues) covalently linked to the epsilon-amino group of lysine were synthesized by solid-phase procedures. Hydrolysis of these peptides by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high-performance liquid chromatography to identify and quantify the hydrolysis products. Methionine oligomers grafted onto lysine were cleaved to tripeptides by pepsin. Chymotrypsin preferentially hydrolyzed the methionyl-methionine bond preceding the isopeptide bond. Cathepsin C released dimethionyl units from the covalently attached polymers. Intestinal aminopeptidase caused efficient hydrolysis of both peptides and isopeptide bonds although free methionine decreased the cleavage of the latter bond. Hydrophobic characteristics of oligo(methionine) chains promoted enzyme-catalyzed transpeptidations resulting probably from acyl-transfer-type reactions. Complementary hydrolysis of the isopeptides by these digestive enzymes suggests that covalent attachment of oligo(amino acid)s to food proteins may improve their nutritional value.     

Purification and properties of a proteolytic enzyme from French beans.

1. A proteolytic enzyme with some features of a carboxypeptidase has been purified some 1180-fold from the sap of French beans (Phaseolus vulgaris var. Prince). A bright blue protein, plastocyanin, was separated from the enzyme by DEAE-cellulose chromatography. 2. Unlike carboxypeptidase A or B of animal origin, there is no evidence that the enzyme is a metalloprotein. There was no stimulation of activity by a number of metal ions, reducing agents or 2-mercapto-ethanol. Neither EDTA nor 1,10-o-phenanthroline inhibited the enzyme. 3. The proteolytic enzyme from beans, readily soluble at neutral or slightly acidic pH values, has a pH optimum of pH5.6 for the hydrolysis of leucine from benzyloxy-carbonylglycyl-l-leucine. Solutions of the enzyme in 0.1m-sodium acetate, pH5.5, lose about 2% of their activity/week at 4 degrees . Virtually no loss of activity results after prolonged storage at -15 degrees . 4. Incubation of the bean enzyme with peptides indicates that the enzyme will release acidic, neutral and basic amino acid residues as well as proline, although adjacent acidic residues in a peptide appear to inhibit the enzyme. The possibility of endopeptidase activity in the purified preparation requires further examination.     

Interdependency of sequence and positional specificities for cysteine proteases of the papain family

The specificity of cysteine proteases is characterized by the nature of the amino acid sequence recognized by the enzymes (sequence specificity) as well as by the position of the scissile peptide bond (positional specificity, i.e., endopeptidase, aminopeptidase, or carboxypeptidase). In this paper, the interdependency of sequence and positional specificities for selected members of this class of enzymes has been investigated using fluorogenic substrates where both the position of the cleavable peptide bond and the nature of the sequence of residues in P2-P1 are varied. The results show that cathepsins K and L and papain, typically considered to act strictly as endopeptidases, can also display dipeptidyl carboxypeptidase activity against the substrate Abz-FRF(4NO2)A and dipeptidyl aminopeptidase activity against FR-MCA. In some cases the activity is even equal to or greater than that observed with cathepsin B and DPP-I (dipeptidyl peptidase I), which have been characterized previously as exopeptidases. In contrast, the exopeptidase activities of cathepsins K and L and papain are extremely low when the P2-P1 residues are A-A, indicating that, as observed for the normal endopeptidase activity, the exopeptidase activities rely heavily on interactions in subsite S2 (and possibly S1). However, cathepsin B and DPP-I are able to hydrolyze substrates through the exopeptidase route even in absence of preferred interactions in subsites S2 and S1. This is attributed to the presence in cathepsin B and DPP-I of specific structural elements which serve as an anchor for the C- or N-terminus of a substrate, thereby allowing favorable enzyme-substrate interaction independently of the P2-P1 sequence. As a consequence, the nature of the residue at position P2 of a substrate, which is usually the main factor determining the specificity for cysteine proteases of the papain family, does not have the same contribution for the exopeptidase activities of cathepsin B and DPP-I.     

Differentiation of endopeptidases and aminopeptidases by high-performance liquid chromatography of reaction products from chromogenic peptide p-nitroanilines as substrates

A method for differentiating endopeptidases and aminopeptidases on the basis of substrate specificity is presented. Various synthetic chromogenic substrates, succinyl-(Ala)3-p-nitroaniline, succinyl-(Ala)2-p-nitroaniline, (Ala)3-p-nitroaniline, and (Ala)2-p-nitroaniline, were incubated with various peptidases and the incubation mixtures were directly analyzed by high-performance liquid chromatography to determine the splitting patterns of these substrates by the enzymes. The substrates and hydrolyzed products containing the chromophore were separated on a reverse-phase column under isocratic conditions, and the chromophore was specifically detected in the effluent fractions by absorbance measurement at 314 nm. Endopeptidases, leucine aminopeptidase, and dipeptidyl aminopeptidase showed different patterns of cleavage of the substrates. This simple and rapid high-performance liquid chromatographic procedure is suitable for identifying the above activities in different fractions obtained during separation and purification studies. The same approach was applied to the simultaneous determination of three types of endopeptidase activities in rat tissues based on the ability of the enzymes to hydrolyze different sites in succinyl-(Ala)3-p-nitroaniline.     

Degradation of Neurotensin by Rat Brain Synaptic Membranes: Involvement of a Thermolysin-Like Metalloendopeptidase (Enkephalinase), Angiotensin-Converting Enzyme, and Other Unidentified Peptidases

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.     

Kinetic-controlled hydrolysis of Leu-Val-Val-hemorphin-7 catalyzed by angiotensin-converting enzyme from rat brain

Leu-Val-Val-hemorphin-7 (LVV-H7, LVVYPWTQRY), an opioid peptide, was found to be hydrolyzed sequentially by rat brain angiotensin-converting enzyme (ACE) in three steps through dipeptidyl carboxypeptidase activity. The kinetic constants evaluated were in order of: k(1) (0.19 min(-1))>k(2) (0.0008 min(-1)) approximately k(3) (0.0006 min(-1)) in 10 mM NaCl at pH 7.5 giving rise to LVV-H5 almost quantitatively. The decapeptide was noted to be hydrolyzed 164- and 346-fold more efficiently than angiotensin I (Ang I) in k(cat) and kcat/Km values, respectively, at their optimal conditions. The kinetic-controlled preferential action of the brain enzyme on LVV-H7 is suggestive of its multiple roles in vivo.     

Specificity of action on neuropeptides of an endopeptidase from the synaptosomal membranes of guinea pig brain

An endopeptidase was solubilized and highly purified from the synaptosomal membrane fraction of guinea pig brain, and its specificity of action on various neuropeptides was investigated. It hydrolyzed specifically the Pro10-Tyr11 bond of neurotensin and showed a marked specificity toward Pro-X bonds present in the interior parts of various neuropeptides and related peptides. No cleavage, however, was observed at the first and second peptide bonds from the NH2-termini or from the COOH-termini of the peptides examined, suggesting that the enzyme requires both NH2- and COOH-terminal extentions of at least 3 residues from the scissile bond for its action. In addition, a limited number of other peptide bonds were cleaved, indicating that the enzyme is not strictly specific to Pro-X bonds. These results suggest the possible implication of this enzyme in the specific degradation of neurotensin and other peptide neurotransmitters in the synaptic cleft.     

Identification of enzymatically produced peptide fragments by liquid chromatography and mass spectrometry

The qualitative and quantitative determination of peptide fragments of angiotensin I generated by rat lung dipeptidyl carboxypeptidase (angiotensin converting enzyme, EC 3.4.15.1) is described. Enzymatically formed peptide fragments, after derivatization with fluorescamine, were separated and isolated by reverse-phase high-performance liquid chromatography. The recovered fluorescamine derivative of histidyl-leucine was then further identified by mass spectrometry. It is anticipated that this approach would be widely applicable to other enzyme systems.     

Porcine spleen cathepsin B is an exopeptidase

The major cathepsin B isozyme CB-I purified from porcine spleens was studied for its specificity against various peptide and denatured protein substrates. The enzyme degraded all the peptide substrates by an exopeptidase activity. The substrates were degraded mainly by a dipeptidyl carboxypeptidase activity of the enzyme except for angiotensin I, from which a COOH-terminal leucine residue was released. The enzyme failed to hydrolyze peptides having a proline or cysteic acid in the COOH-terminal, penultimate, and antepenultimate positions. Reduced and carboxymethylated soybean trypsin inhibitor was degraded by the same dipeptidyl carboxypeptidase action of cathepsin B. No significant endopeptidase activity was observed. These results do not support the general assumption that cathepsin B has both endo- and exopeptidase activities, neither do these observations support the postulation that cathepsin B might be involved in the in vivo proteolytic processing of protein precursors. We propose that the biological role of this enzyme is mainly the degradation of tissue proteins in lysosomes.