scholarly journals Neuropeptide-degrading endopeptidase activity of locust (Schistocerca gregaria) synaptic membranes

1988 ◽  
Vol 255 (3) ◽  
pp. 843-847 ◽  
Author(s):  
R E Isaac
Keyword(s):  
Schistocerca Gregaria ◽  
Physiological Role ◽  
Aminopeptidase Activity ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Adipokinetic Hormone ◽  
Peptide Bonds ◽  
Endopeptidase 24.11 ◽  
Endopeptidase Activity ◽  
Neural Membranes

Locust adipokinetic hormone (AKH, pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) was used as the substrate to measure neuropeptide-degrading endopeptidase activity in neutral membranes from ganglia of the locust Schistocerca gregaria. Initial hydrolysis of AKH at neural pH by peptidases of washed neural membranes generated pGlu-Leu-Asn and Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 as primary metabolites, demonstrating that degradation was initiated by cleavage of the Asn-Phe bond. Amastatin protected the C-terminal fragment from further metabolism by aminopeptidase activity without inhibiting AKH degradation. The same fragments were generated on incubation of AKH with purified pig kidney endopeptidase 24.11, and enzyme known to cleave peptide bonds that involve the amino group of hydrophobic amino acids. Phosphoramidon (10 microM), a selective inhibitor of mammalian endopeptidase 24.11, partially inhibited the endopeptidase activity of locust neural membranes. This phosphoramidon-sensitive activity was shown to enriched in a synaptic membrane preparation with around 80% of the activity being inhibited by 10 microM-phosphoramidon (IC50 = 0.2 microM). The synaptic endopeptidase was also inhibited by 1 mM-EDTA, 1 mM-1,10-phenanthroline and 1 microM-thiorphan, and the activity was maximal between pH 7.3 and 8.0. Localization of the phosphoramidon-sensitive enzyme in synaptic membranes is consistent with a physiological role for this endopeptidase in the metabolism of insect peptides at the synapse.

scholarly journals Proctolin degradation by membrane peptidases from nervous tissues of the desert locust (Schistocerca gregaria)

1987 ◽  
Vol 245 (2) ◽  
pp. 365-370 ◽  
Author(s):  
R E Isaac
Keyword(s):  
Schistocerca Gregaria ◽  
Physiological Role ◽  
Hydrolytic Activity ◽  
Mitochondrial Fraction ◽  
Membrane Preparation ◽  
Aminopeptidase Activity ◽  
Synaptic Membrane ◽  
Desert Locust ◽  
Ph Optimum ◽  
Enzyme Preparations

The hydrolysis of the insect neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) by enzyme preparations from the nervous tissue of the desert locust (Schistocerca gregaria) was investigated. Neural homogenate degraded proctolin (100 microM) at neutral pH by cleavage of the Arg-Tyr and Tyr-Leu bonds to yield Tyr-Leu-Pro-Thr, Arg-Tyr and free tyrosine. Arg-Tyr was detected as a major metabolite when the aminopeptidase inhibitors amastatin and bestatin were present to prevent Arg-Tyr breakdown. Around 50% of the proctolin-degrading activity was isolated in a 30,000 g membrane fraction and was shown to be almost entirely due to aminopeptidase activity. The aminopeptidase had an apparent Km of 23 microM, a pH optimum of 7.0 and was inhibited by 1 mM-EDTA and amastatin [IC50 = 0.3 microM], but was relatively insensitive to bestatin, actinonin and puromycin. Phenylmethanesulphonyl fluoride (1 mM) and p-chloromercuriphenylsulphonic acid (1 mM) had no effect on this enzyme activity. Although the bulk of the Tyr-Leu hydrolytic activity was located in the 30,000 g supernatant, some weak activity was detected in a washed membrane preparation. This peptidase displayed a high affinity for proctolin (Km = 0.35 microM) and optimal activity at around pH 7.0. Synaptosome- and mitochondria-rich fractions were prepared from crude neural membranes. The aminopeptidase activity was concentrated in the synaptic-membrane preparation, whereas activity giving rise to Arg-Tyr was predominantly localized in the mitochondrial fraction. The subcellular localization of the membrane aminopeptidase is consistent with a possible physiological role for this enzyme in the inactivation of synaptically released proctolin.

scholarly journals The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N

1985 ◽  
Vol 231 (2) ◽  
pp. 445-449 ◽  
Author(s):  
R Matsas ◽  
S L Stephenson ◽  
J Hryszko ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Phase Separation ◽  
Membrane Protein ◽  
Total Activity ◽  
Aminopeptidase N ◽  
The Other ◽  
Aminopeptidase Activity ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Rich Phase ◽  
Triton X

The property of solutions of Triton X-114 to separate into detergent-rich and detergent-poor phases at 30 degrees C has been exploited to investigate the identities of the aminopeptidases in synaptic membrane preparations from pig striatum. When titrated with an antiserum to aminopeptidase N (EC 3.4.11.2), synaptic membranes solubilized with Triton X-100 revealed that this enzyme apparently comprises no more than 5% of the activity releasing tyrosine from [Leu]enkephalin. When assayed in the presence of puromycin, this proportion increased to 20%. Three integral membrane proteins were fractionated by phase separation in Triton X-114. Aminopeptidase activity, endopeptidase-24.11 and peptidyl dipeptidase A partitioned predominantly into the detergent-rich phase when kidney microvillar membranes were so treated. However, only 5.5% of synaptic membrane aminopeptidase activity partitioned into this phase, although the other peptidases behaved predictably. About half of the aminopeptidase activity in the detergent-rich phase could now be titrated with the antiserum, showing that aminopeptidase N is an integral membrane protein of this preparation. Three aminopeptidase inhibitors were investigated for their ability to discriminate between the different activities revealed by these experiments. Although amastatin was the most potent (IC50 = 5 × 10(−7) M) it failed to discriminate between pure kidney aminopeptidase N, the total activity of solubilized synaptic membranes and that in the Triton X-114-rich phase. Bestatin was slightly more potent for total activity (IC50 = 6.3 × 10(−6) M) than for the other two forms (IC50 = 1.6 × 10(−5) M). Puromycin was a weak inhibitor, but was more selective. The activity of solubilized membranes was more sensitive (IC50 = 1.6 × 10(−5) M) than that of the pure enzyme or the Triton X-114-rich phase (IC50 = 4 × 10(−4) M). We suggest that the puromycin-sensitive aminopeptidase activity that predominates in crude synaptic membrane preparations may be a cytosolic contaminant or peripheral membrane protein rather than an integral membrane component. Aminopeptidase N may contribute to the extracellular metabolism of enkephalin and other susceptible neuropeptides in the brain.

scholarly journals The metabolism of neuropeptides. Endopeptidase-24.11 in human synaptic membrane preparations hydrolyses substance P

1985 ◽  
Vol 228 (2) ◽  
pp. 487-492 ◽  
Author(s):  
R Matsas ◽  
M Rattray ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Substance P ◽  
Human Brain ◽  
Choroid Plexus ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Immunoradiometric Assay ◽  
Endopeptidase 24.11 ◽  
Pig Brain ◽  
Enzymic Assay

Synaptic membrane preparations from human striatum and human diencephalon were shown to contain a phosphoramidon-sensitive metalloendopeptidase that appeared identical with endopeptidase-24.11. The activity of endopeptidase-24.11 was determined with an enzymic assay employing [D-Ala2,Leu5]enkephalin as substrate, and its distribution in human brain was similar to that in pig brain, with the striatum containing the highest levels. The choroid plexus and pons also contained substantial activity. A good correlation (r = 0.97) was obtained for the distribution of the endopeptidase in pig brain and pituitary by the enzymic assay and by an immunoradiometric assay specific for pig endopeptidase-24.11. Synaptic membrane preparations from human striatum and diencephalon hydrolysed substance P at the same sites as did preparations of pig striatal synaptic membranes, and hydrolysis was substantially abolished by phosphoramidon. These results suggest that endopeptidase-24.11 is the principal enzyme hydrolysing substance P in human synaptic membrane preparations.

scholarly journals The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme)

1985 ◽  
Vol 231 (2) ◽  
pp. 357-361 ◽  
Author(s):  
N M Hooper ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Substance P ◽  
Selective Inhibitor ◽  
Synaptic Membranes ◽  
Neurokinin A ◽  
Pig Kidney ◽  
Endopeptidase 24.11 ◽  
The Family ◽  
General Capacity ◽  
Substance K ◽  
Hydrolysis Of

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A.

In vitrometabolism of an insect neuropeptide by homogenates of the nematodeCaenorhabditis elegans

2003 ◽  
Vol 77 (1) ◽  
pp. 43-48 ◽  
Author(s):  
E.P. Masler
Keyword(s):  
General Pattern ◽  
Residue Analysis ◽  
Aminopeptidase Activity ◽  
Soil Nematode ◽  
Peptide Fragments ◽  
Adipokinetic Hormone ◽  
Initial Product ◽  
Insect Neuropeptide ◽  
Endoproteolytic Cleavage ◽  
N Terminus

AbstractThe cytosolic fraction of homogenates from the free-living soil nematodeCaenorhabditis elegansis capable of metabolizing the insect neuropeptide adipokinetic hormone, a decapeptide blocked at the N-terminus by a pGlu residue. Analysis of digests by RP-HPLC and LC-MS revealed that an initial endoproteolytic cleavage step produced a heptapeptide with an unblocked N-terminus that can serve as a substrate for aminopeptidases. The aminopeptidase activity is depressed in the presence of the inhibitor amastatin; the initial product of the endoproteolytic step accumulates during incubation, and expected aminopeptidase product peptides are reduced in amount, as assessed by chromatographic peak size. The absence of some expected peptide fragments in the reaction mixtures suggests that multiple proteases contribute to short peptide half-lives. Comparison of the adipokinetic hormone digestion inC. elegansto that reported previously for insects reveals the same general pattern of peptide fragment production.

scholarly journals A monoclonal antibody to kidney endopeptidase-24.11. Its application in immunoadsorbent purification of the enzyme and immunofluorescent microscopy of kidney and intestine

1983 ◽  
Vol 214 (2) ◽  
pp. 377-386 ◽  
Author(s):  
N S Gee ◽  
R Matsas ◽  
A J Kenny
Keyword(s):  
Monoclonal Antibody ◽  
Fluorescence Microscopy ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Specific Activity ◽  
Cell Types ◽  
Kidney Tubules ◽  
Pig Kidney ◽  
Endopeptidase 24.11 ◽  
Endopeptidase Activity

Hybridoma methodology has been used to produce a monoclonal antibody, GK 7C2, that binds specifically to microvillar endopeptidase-24.11 (EC 3.4.24.11). The antibody (an immunoglobulin G) was generated by fusion of mouse plasmacytoma cells with splenocytes from a Balb/c mouse immunized with pig kidney microvillar membranes. The identity of the antigen recognized by GK 7C2 was established by immuno-precipitation from detergent-solubilized pig kidney microvilli. The protein had an apparent Mr of 90 000 and contained endopeptidase activity sensitive to phosphoramidon. The identity was confirmed by immunoadsorbent purification of endopeptidase-24.11 by a column to which GK 7C2 had been attached. The endopeptidase, purified in a yield of 40%, was electrophoretically homogeneous and of specific activity comparable with that purified by other means. Fluorescence microscopy established that GK 7C2 bound specifically to the luminal membranes of kidney tubules and the intestinal mucosa. Thus endopeptidase-24.11 is located in the brush-border membranes of both cell types.

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