Purification and Characterisation of a Membrane‐Bound Substance‐P‐Degrading Enzyme from Human Brain

Abstract

A membrane-bound enzyme which degrades substance P (an undecapeptide) has been purified from human brain. The properties of this enzyme suggest that it may be involved in the physiological inactivation of the peptide by neural tissues. Enzyme activity was extracted from a membrane fraction of human diencephalon with a non-ionic detergent, Brij 35, and activity was monitored by measuring the disappearance of added substance P using radioimmunoassay, bioassay or radiochemical assay. The enzyme was purified about 1000-fold by chromatography on DEAE-cellulose, hydroxyapatite and Sephadex gel filtration columns. To identify the cleavage sites in substance P, the peptide was incubated with the purified enzyme and the breakdown products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis. The results suggested that the enzyme preparation was functionally homogeneous and it cleaved substance P between Gln6-Phe', Phe7-Phe' and Phe'-Gly', with no exopeptidase action. The enzyme had a pH optimum in the range 7-9 and was strongly inhibited by metal-chelating agents, but not affected by most other peptidase inhibitors; it can thus be classified as a neutral metallo-endopeptidase. The enzyme was thermolabile and had a molecular weight of 40000 -50000 as estimated by gel filtration, density-gradient ultracentrifugation and sodium dodecylsulphate gel electrophoresis. The highly purified substance-P-degrading enzyme could be distinguished from previously described peptidases for which substance P is a substrate. An important feature was that substance P was the preferred substrate among various other neuropeptides tested.Substance P, an undecapeptide (Fig.l) The mechanism involved in the inactivation of substance P after its synaptic release is unknown, although a number of enzymes capable of degrading this peptide have been reported to exist in brain [I0 -121. These include both cytosolic [12-151 and membrane-bound [13,14,16-181 substance-Pdegrading enzyme activities. A cytosolic enzyme has been partially purified from rat brain and shown to release phenylalanine and leucine preferentially from substance P, suggesting cleavage at two or more internal peptide bonds (Gln6-Phe7 or Phe7-Phe' and Glyg-Leu") [19]. Akopyan and coworkers [20] purified a neutral endopeptidase from bovine hypothalamus which degraded luteinizing-hormone-releasing hormone (luliberin) and somatostatin and also cleaved substance P, probably between Gln6-Phe' and Phe7-Phe'. In addition, a prolyl endopeptidase has been purified from rabbit brain and shown to cleave substance P between Abbreviations. Substance P, the undecapeptide Arg-Pro-Lys-ProGln-Gln-Phe-Phe-Gly-Leu-MetNHz; [3H]substance P, [PheK-3H]substance P ; Ala-Ala-Phe-Nan, alanyl-alanyl-phenylalanyl p-nitroanilide; Fao-Gly-LeuNHZ, 3-(2-furylacryloyl)-glycyl-~-leucine amide; Hepes, 4-(2-hydroxyethyl)-l -piperazineethanesulphonic acid.Enzymes. Substance-P-degrading enzyme (EC 3.4.24.-) ; angiotensinconverting enzyme or dipeptidyl carboxypeptidase...

Subcellular distribution of particle-bound neutral peptidases capable of hydrolyzing gonadoliberin, thyroliberin, enkephalin and substance P

“…When the incubation mixtures were resolved by thinlayer chromatography on silica gel plates, three radioactive fragments with R, values of O.25,0.83 and 0.97 were observed. These values are similar to those reported for the substance P fragments (1 -8)octapeptide, (7-1l)pentapeptide and (8 -Il)tetrapeptide, which are released from substance P by the brain enzyme [16]. In addition [3H]Phe and another radioactive fragment (presumably [3H]Phe-Phe) were also obtained, which most likely represent the degradation products of the primary cleavage products.…”

“…Recently an enzyme, which seems to be the major substance-P-degrading peptidase in brain, has been isolated from a crude membrane preparation [16]. Using the same conditions as described in that report, the hydrolysis of substance P was found to be associated exclusively with the mitochondria.…”

“…For determining the activity of the substance-P-degrading enzyme, aliquots of the fractions were incubated with [3H]substance P under the same conditions as described [16].…”

“…Substance P is known to be susceptible to cleavage by the dipeptidyl peptidase IV (post-proline dipeptidyl aminopeptidase), which releases Arg-Pro and subsequently Lys-Pro [13], the thermolysin-like metalloendopeptidase, which hydrolyzes peptide bonds at the amino side of hydrophobic amino acids [14, 151 and a substance-P-degrading enzyme, which has been isolated from brain and cleaves the peptide between Gln6-Phe7, Phe7-Phe' and Phe'-Gly' [16]. As shown above, the thermolysin-like enzyme is associated with the plasma membrane.…”

“…The identity of this activity with the enzyme from brain is further confirmed by the tentative identification of the same cleavage products. It is tempting to speculate that this substance-P-degrading enzyme may be identical to the mitochondria1 gonadoliberin-degrading enzyme, because both enzymes are known to hydrolyze peptide bonds at the amino and carboxy sides of hydrophobic amino acids and exhibit comparable chemical properties [16,321. Furthermore, the phosphoramidon-resistant release of AcTyr-Gly-Gly from N-acetyl-enkephalin and degradation of Suc-Ala-Ala-PheAmc by the mitochondrial fractions may be attributed to this enzyme, but proof for this hypothesis can only be obtained after extensive enzyme purification and characterization.…”