Membrane Aminopeptidase Research Articles

Release of GPI-anchored membrane aminopeptidase P by enzymes and detergents has some peculiarities

The enzyme aminopeptidase P (APP) of rat small intestine is an integral membrane protein. It is not released from the membrane by proteinases and is also resistant to bacterial inositol-specific phospholipases C (PI-PLC's). We show that this resistance is due to a hindered accessibility of the bond split by PI-PLC and not to a modified glycosylphosphatidyl inositol (GPI)-anchor.

Effects of Small Peptides as Intraluminal Substrates on Transport Carriers for Amino Acids and Peptides.

The effects of small peptides on brush-border membrane enzyme activities and on transport carriers for amino acids and dipeptides were investigated by measurement of these enzyme activities and the transmural potential difference in the small intestines of guinea pigs fed elemental diets containing either small peptides (SP) or amino acids (AA) as the nitrogen source. Brush-border membrane aminopeptidase activities were significantly higher in the group fed the diet containing SP. In addition, the transmural potential differences induced by L-leucine and glycyl-L-leucine (Gly-Leu) were significantly higher in the SP group. The maximum potential differences for L-leucine and Gly-Leu were significantly higher in the SP group, whereas the half saturation values did not differ between the two groups. The same effect of a SP diet was seen in rats, when the uptake of L-[U-14C]leucylglycine (L-[14C]Leu-Gly) into intestinal brush-border membrane vesicles was measured. Some 70% of L-[14C]Leu-Gly was not hydrolyzed by aminopeptidase, but was found as a dipeptide in the brush-border membrane vesicles. These results indicate that small peptides as intraluminal substrates increase brush-border membrane aminopeptidase activities and the activity of carriers for amino acids and dipeptides.

Divergent regulation of cell surface protease expression in HL-60 cells differentiated into macrophages with granulocyte macrophage colony stimulating factor or neutrophils with retinoic acid.

Taking advantage of the recently demonstrated presence of N-aminopeptidases and the serine protease dipeptidyl aminopeptidase IV (DPP IV) at the surface of human myeloblastic HL-60 cells, the regulation of these protease activities in HL-60 cell differentiation has been assessed using combined spectrophotometric and flow cytometric assays. Addition of human recombinant granulocyte macrophage colony stimulating factor (rHu-GM-CSF) to HL-60 cells to induce differentiation into macrophages led to a time- and dose-dependent increase in both cell surface N-aminopeptidase and DPP IV activities. Protease up-regulation was due to an enhancement in cell surface protease number, associated with a slight rise in apparent affinities of the enzymes for their substrates. In contrast, in HL-60 cells induced to differentiate into neutrophils in the presence of retinoic acid, expression of cell surface N-aminopeptidases was almost completely abolished in a time- and dose-dependent fashion, and this down-regulation was accompanied by a weak but significant decrease in affinity. However, no noticeable difference was seen in serine DPP IV expression between retinoic acid-treated and untreated HL-60 cells. Retinoic acid treatment also reduced soluble protease activity in vitro indicating that down-regulation of membrane aminopeptidases was not due to their proteolytic clip. No modulation in the activity of any of the enzymes tested was seen with human recombinant tumor necrosis factor-alpha or retinol which do not induce HL-60 cell differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of aminopeptidases from Aplysia californica.

Aminopeptidase activities were identified in extracts of kidney, ovotestis, head ganglia, heart and haemolymph of Aplysia californica. These enzyme preparations hydrolysed [3H][Leu]enkephalin at the Try-1-Gly-2 bond as determined by h.p.l.c. analysis of cleavage products. In all these tissues, enkephalin-degrading aminopeptidase activities were present both in membrane-bound and cytosolic fractions. The bivalent-cation-chelating agent, 1,10-phenanthroline, inhibited kidney membrane aminopeptidase activity with an IC50 of 30 microM, suggesting that this enzyme is a metalloproteinase. The aminopeptidase inhibitor amastatin was the most potent inhibitor of [Leu]enkephalin degradation (IC50 25 nM) by membrane-bound aminopeptidase, and bacitracin, bestatin and puromycin were about 100-1000 times less potent. In contrast with membrane-bound aminopeptidase, the cytosolic form is sensitive to puromycin. Angiotensin-converting enzyme inhibitor had no effect on [Leu]enkephalin degradation by kidney membranes, while the neutral endopeptidase inhibitors were poor inhibitors of the enzymes in this preparation. The Km values of the aminopeptidase in the kidney membranes and cytosolic fractions for the [Leu]enkephalin substrate were 2.4 and 7.4 microM respectively. The aminopeptidase present in the kidney membranes also hydrolysed endogenous Phe-Met-Arg-Phe-amide peptide at the Phe-1-Met-2 bond as well as synthetic alanine p-nitroanilide and leucine p-nitroanilide. When used in a competition assay, these substrates inhibited hydrolysis of [3H][Leu]enkephalin, suggesting that the same enzyme degraded all these substrates. Taken together, these results suggest that Aplysia tissues contain both a membrane-bound aminopeptidase related to the mammalian aminopeptidase N and a cytosolic puromycin-sensitive aminopeptidase.

Transport of N-α(or N-ϵ)- L-methionyl- L-lysine and acetylated derivatives across the rabbit intestinal epithelium

Intestinal absorption constitutes an important step in the biological utilization of L-Methionine and N-Acetyl- L-methionine covalently bound to ϵ-NH 2 groups of food protein lysyl residues. The transport of synthesized and purified N-α-(or N-ϵ-) L-Methionyl- L-lysine and N-α-(or N-ϵ-)Acetyl- L-methionyl- L-lysine was studied in vitro in rabbit ileum mounted in the Ussing chamber and compared to that of both free L-Methionine and L-Lysine or both free N-Acetyl- L-methionine and L-Lysine. Addition of all solutes to the mucosal reservoir, except N-ϵ-Acetyl- L-methionyl- L-lysine, led to an increase in the short-circuit current, the lowest response obtained by N-α-Acetyl- L-methionyl- L-lysine. In all cases, only the constitutive amino acids were recovered in the serosal chamber. When free L-Methionine and L-Lysine were added together to the mucosal reservoir, comparable fluxes were obtained: when any of the dipeptides were added, transport of lysine was the highest. Although the mucosal reservoir disappearance of N-α- L-Methionyl- L-lysine was faster than that of N-ϵ- L-Methionyl- L-lysine, the mucosal to serosal fluxes of their constitutive amino acids were not significantly different. However, the transepithelial flux of L-Methionine originating from the dipeptides was 40–50% less than that of the free amino acid, whereas L-Lysine flux was unchanged. Substantial tissue oxidation of L-Methionine unlike L-Lysine, which are slowly released from peptide hydrolysis by the brush border membrane aminopeptidases, likely altered the amino acid transport. Moreover, the observed higher transepithelial flux for L-Lysine relative to L-Methionine when both were derived from the dipeptides may have resulted from intracellular L-Methionine stimulation of L-Lysine absorption. As compared to the normal di- and isodipeptides, lower net fluxes of L-Methionine, and to a lesser extent of L-Lysine, were observed from the acetylated peptides, the former being zero even in the case of N-ϵ-Acetyl- L-methionyl- L-lysine. The results demonstrate the significant contribution of brush border aminopeptidases to the intestinal absorption of N-ϵ- L-Methionyl- L-lysine in the rabbit. Our findings also suggest that the availability of amino acids from acetylated peptides is limited by their transport rates and/ or their hydrolysis from cytosolic N-acylpeptide hydrolase and N-acylase.

ラット小腸サイトゾルアミノペプチダーゼの精製とその性質

The isolation of cytosol aminopeptidase (CAP) from intestinal mucosa has involved difficult procedures because of low yield, instability of the enzyme and many other factors related to purification. The authors resolved these difficulties in the purification of the enzyme from intestinal mucosa of rats using hydrophobic chromatography on phenyl-TOYOPEARL 650S and ion exchange HPLC on Mono Q 5/5. In this procedure, a 60-fold purification was achieved and the purified enzyme revealed a single band with 56kDa molecular weight by SDS polyacrylamide gel electrophoresis. The purified enzyme preferentially hydrolyzed the Leucyl-containing peptides of Leu-Gly and Leu-Gly-Gly. However, it showed no activity on synthetic substrates of Leu-beta-naphthylamide, Gly-p-nitroanilide, Phe-p-nitroanilide or Met-beta-naphthylamide. The Km and Vmax for Leu-Gly were 0.91 mmol/l and 16.4 mol/min/mg protein, while for Leu-Gly-Gly they were 0.77 mmol/l and 20.6 mol/min/mg protein. The purified enzyme was heat-labile and quickly became less active in highly concentrated ammonium sulfate. The optimum pH was 6.5-10. The enzyme activity was stimulated by Mn2+ and Mg2+, while it was inhibited by EDTA, 1,10-phenanthroline, 2-mercaptoethanol and p-hydroxymercuribenzoate. The results suggested the participation of metal ions and the SH group in the enzyme activity. Furthermore the cytosol aminopeptidase was distinct from the brush-border membrane aminopeptidase in molecular weight, immunoreaction to cytosol aminopeptidase antiserum and the specificities to the substrates.

Localization of aminopeptidases inStreptococcus sanguis strain 903

The presence of aminopeptidases in the cytoplasm, in the cell wall, and in the cytoplasmic membrane fractions ofStreptococcus sanguis 903 was demonstrated by isoelectric focusing in combination with enzyme-staining procedures. The cytoplasm and the cell wall both had two aminopeptidases (pI 4.25 and 4.3) with broad substrate specificities and one enzyme (pI 4.2) specific for arginine substrates. The former enzymes were both stimulated by Co2+ ions; the latter enzyme had no metal cofactor. The cytoplasmic membrane aminopeptidase (pI 4.65) was arginine specific and was not stimulated by metal ions.

An aminoboronic acid derivative inhibits thymopentin metabolism by mucosal membrane aminopeptidases

Thymopentin is a pentapeptide with immunomodulatory activity. Transmucosal delivery may offer advantages over other routes, but published data have shown relatively poor efficacy when dosed nasally. Metabolism of thymopentin by the rat nasal mucosa and the effects of an aminoboronic acid aminopeptidase inhibitor, boroleucine, were evaluated. Thymopentin concentrations in a solution perfused through the isolated nasal cavity decayed with a first-order half-life of 12 minutes. Thymopentin was metabolized primarily by aminopeptidases, based on the amount of tetrapeptide metabolite formed. In the presence of boroleucine, at an inhibitor/substrate molar concentration ratio of 0.015 1 , the degradation half-life was prolonged to 37 minutes. Appearance of the tetrapeptide metabolite of aminopeptidases was delayed. Boroleucine and other aminoboronic acid peptidase inhibitors may be useful for improving thymopentin delivery.

Metabolism of vasoactive peptides by vascular endothelium and smooth muscle aminopeptidase M

The cellular localization of vascular plasma membrane aminopeptidase M (AmM;EC 3.4.11.2) was examined in cultured porcine aorta endothelium and smooth muscle cells. AmM was 14-fold higher on smooth muscle (117 ± 16 units/mg) than on endothelium (8.4 ± 0.2). Proportional to its cellular distribution, AmM hydrolyzed the N-terminus of kallidin to produce bradykinin, and degraded des(Asp 1)angiotensin I, angiotensin III, hepta(5-ll)substance P and Met 5-enkephalin. In contrast, bradykinin, angiotensin II and substance P were resistant to AmM-mediated hydrolysis. Peptide metabolism was optimal at pH7.0 and was inhibited by o-phenanthroline, bestatin ( K i = 2.2 ± 0.1 μM) and amastatin ( K i, = 25 ±5 nM). Des(Asp 1)angiotensin I and angiotensin III had the highest affinity (lowest K m) for AmM ( K m = 2.2 ± 0.5 and 2.0 ±0.4 μM respectively), followed by hepta(5–11)substance P (53.9 ± 1.7 μM) and Met 5-enkephalin (75.7 ± 3.5 μM). In contrast, maximal velocities of hydrolysis were higher for Met 5-enkephalin (313 ± 2 nmol/min/mg) than for hepta(5–11)substance P (109 ± 18 nmol/min/mg) or angiotensin III (26.5 ±1.0 nmol/min/mg). As expected for hydrolysis by a common enzyme, AmM-mediated enkephalin degradation was inhibited competitively by angiotensin III ( K i = 0.34 ± 0.04 μM), hepta(5-ll)substance P (43.7 ± 6.3 μM) and kallidin (62 μM). These data suggest that vascular AmM may modulate vasoactive peptide levels in vivo, particularly within the microenvironment of endothelial and smooth muscle cell surface receptors.

In vivo effect of tunicamycin on the expression of rat small intestinal brush border membrane glycoproteins and glycoenzymes

Tunicamycin, a known inhibitor of the lipid-dependent glycosylation of proteins, was used in vivo to study the biosynthesis of rat intestinal brush border membrane aminopeptidase N and dipeptidyl aminopeptidase IV. The incorporation of [ 3H]glucosamine into newly synthesized total protein of mucosal cell homogenates was inhibited by 60%, whereas incorporation of [ 3H]leucine was decreased only 21% by tunicamycin. This effect was much more pronounced in the brush border membrane fraction isolated from intestinal mucosal cells where incorporation of radiolabled leucine and glucosamine was reduced to 50 and 82% of control values respectively. An examination of the brush border membrane protein profile by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that there was a marked selective decrease in the amount of glycoproteins of molecular weights greater than 130 kD. In addition, there were decreased levels of assayable aminopeptidase N, dipeptidyl aminopeptidase IV and disaccharidase activity in intestinal mucosal cell homogenates and brush border membranes of tunicamycin-treated rats. Though tunicamycin decreased incorporation of newly synthesized aminopeptidase N and dipeptidyl aminopeptidase IV protein into brush border membranes by 70–75%, the newly synthesized enzyme that was incorporated was indistinguishable from that of controls. Further, non-glycoslyated forms of both enzymes were not detected in any other subcellular fractions. These results show that tunicamycin, an inhibitor of glycosylation, significantly affected the expression of brush border membrane glycoproteins, suggesting that both polypeptide synthesis and degradation of these proteins may be altered in the presence of this drug.

Ectoenzymes of the kidney microvillar membrane Aminopeptidase P is anchored by a glycosyl-phosphatidylinositol moiety

The mode of membrane anchorage of three kidney microvillar membrane ectoenzymes has been examined. The release of aminopeptidase P (EC 3.4.11.9) from kidney membranes by bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and the pattern of detergent solubilization of this ectoenzyme implies that it is anchored to the membrane via a covalently attached glycosyl-phosphatidylinositol moiety. As deduced by phase separation in Triton X-114, octyl-glucoside solubilized the amphipathic form of aminopeptidase P, whereas the PI-PLC-released form displayed hydrophilic properties. In contrast, the pattern of detergent solubilization of two microvillar carboxypeptidases and their resistance to release from the membrane by bacterial PI-PLC suggest that these two ectoenzymes are not anchored via phosphatidylinositol.

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

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.

Analgesic effect of actinonin, a new potent inhibitor of multiple enkephalin degrading enzymes

Actinonin, previously isolated as an antibiotic and shown to be an inhibitor of aminopeptidase M (EC 3.4.11.2), has now been shown to inhibit three enkephalin-degrading enzymes from guinea-pig striatum. The values of IC 50 were 0.39 μM for striatal membrane aminopeptidase (“enkephalin-aminopeptidase”) and 5.6 μM striatal membrane neutral endopeptidase (“enkephalinase A”). Furthermore, soluble dipeptidylaminopeptidase in a rat whole brain homogenate was also inhibited by actinonin with the IC 50 value of 1.1 μM. Actinonin administered intracisternally (i.cist., 50 ug) or intraperitoneally (i.p., 100 mg/kg), potentiated the analgesic action of met-enkephalin (50 μg i.cist.) analgesia by a tail-flick test. The potentiating activity of actinonin i.p. to met-enkephalin analgesia was almost the same potency as that of thiorphan, whereas the inhibitory activity of actinonin against enkephalinase A was 1/1000 that of thiorphan. Actinonin alone, administered either i.cist. or i.p., showed an analgesic action as estimated by the tail-flick test.

Enzymatic and morphological response of the thymus to drugs in normal and zinc-deficient pregnant rats and their fetuses.

In the thymus of normally fed pregnant rats the plasma membrane enzymes dipeptidyl peptidase IV (DPP IV) and alkaline phosphatase (alP) were found in cortical and medullary lymphocytes (thymocytes). Plasma membrane aminopeptidase A (APA) and adenosine monophosphate hydrolysing phosphatase (AMPP) were present in cortical reticular cells. In medullary reticular cells, aminopeptidase M (APM), gamma-glutamyl transferase (GGT), adenosine triphosphate (ATPP) and thiamine pyrophosphate (TPPP) cleaving phosphatases were detected. Medullary reticular cells did not contain APA. Lysosomal DPP I and II, acid phosphatase, acid beta-D-galactosidase, beta-D-N-acetyl-glucosaminidase, beta-D-glucuronidase and non-specific esterases occurred especially in macrophages at the corticomedullary junction. The 21-day-old fetal thymus showed a similar reaction pattern as the maternal organ except for APA which was absent before birth. After treatment of the pregnant rats with valproic acid (VPA), salicylic acid (SA), streptozotocin (ST) and retinoic acid (RA) APA showed an increase in activity in the thymic cortex. In addition, ST and RA induced AMPP, ATPP and TPPP activity in cortical reticular cells up to the same pattern as in medullary reticular cells. After ethanol (ET) administration severe damages occurred. The thymic cortex was free of DPP IV-positive lymphocytes; the medullary reticular cells showed reduced or no GGT and occasionally an increased APM activity. Dexamethasone (DEXA) given to normal or zinc-deficient rats produced the most severe lesions; thymocytes with DPP IV activity were completely absent in the cortex and medulla. In Zn-deficient pregnant rats similar alterations were observed as after ET.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and Characterization of Brush-Border Membrane-Bound Neutral Metalloendopeptidases From Rat Small Intestine

Neutral metalloendopeptidase enzymes were identified and partially characterized in the brush-border membranes of rat small intestinal mucosal cells using insulin B chain and glutaryl-trialanine-4-methoxy-beta-naphthylamide as substrates. Three different molecular species of endopeptidase were identified by disc gel electrophoresis. These enzymes were shown to be distinct from pancreatic endopeptidases on the basis of the following: enrichment in the brush-border membrane fraction, site of hydrolysis of peptide substrates, sensitivity to specific proteinase inhibitors, and the presence of brush-border membrane-associated endopeptidase activity in mucosal cells of Thirty-Vella loops. Hydrolysis of the substrates was shown to be a two-step process involving initial cleavage by endopeptidase with secondary hydrolysis of the peptide products by brush-border membrane aminopeptidase N. Hydrolysis of both substrates was maximum at a neutral pH and was strongly inhibited by metal chelating agents, phosphoramidone, and amastatin. Intestinal perfusion studies using glutaryl-trialanine-4-methoxy-beta-naphthylamide suggest that these enzymes play a physiologic role in protein digestion. It was concluded that neutral endopeptidases are integral components of the intestinal brush-border membrane and work in concert with aminopeptidase N to hydrolyze dietary protein. This process may be of nutritional importance in normal subjects and those with diminished exocrine pancreatic function.

Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M

Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [ D-Ala 2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1-Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin ( I 50 = 0.08 μM), bestatin (9.0 μM) and puromycin (80 μM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met 5-enkephalin, Leu 5-enkephalin and Leu 5-enkephalin-Arg 6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin(1–13)). No significant hydrolysis of β- and γ-endorphin was detected. K m values decreased significantly with increasing peptide length ( K m = 72.9 ± 2.7, 43.6 ± 4.7 and 21.4 ± 0.9 μM for Met 5-enkephalin, Leu 5-enkephalin-Arg 6 and Met 5-enkephalin-Arg 6-Phe 7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1–13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5–11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.

Histochemistry of some proteases in the normal rabbit, pig and ox corneas.

The distribution of activities of membrane aminopeptidases (aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), gamma-glutamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II)) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4 degrees C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4 degrees C) for the demonstration of lysosomal enzymes. In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand, APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity. Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.

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

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 X 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 X 10(-6) M) than for the other two forms (IC50 = 1.6 X 10(-5) M). Puromycin was a weak inhibitor, but was more selective. The activity of solubilized membranes was more sensitive (IC50 = 1.6 X 10(-5) M) than that of the pure enzyme or the Triton X-114-rich phase (IC50 = 4 X 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.

Possible involvement of aminopeptidase, an ecto-enzyme, in the inactivation of bradykinin by intact neutrophils

The subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig neutrophils and the following results were obtained. 1. The bradykinin-inactivating activities were found to be present in the cytosol and membrane fractions but not in the granular and nuclear fractions. 2. The bradykinin-inactivating activity of the cytosol fraction was inhibited by N-carbobenzoxy-Gly-Pro, an inhibitor of prolyl endopeptidase, whereas that of the membrane fraction was inhibited by bestatin, an inhibitor of aminopeptidase. 3. Prolyl endopeptidase and aminopeptidase activities were located predominantly in the cytosol and membrane fractions, respectively, and their activities were inhibited by their respective inhibitors. 4. Prolyl endopeptidase and aminopeptidase activities measured with synthetic substrates were competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for prolyl endopeptidase and aminopeptidase. 5. Intact neutrophils inactivated bradykinin rapidly. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates ecto-enzymes selectively, both the bradykinin-inactivating activity and aminopeptidase activity of neutrophils decreased significantly without any inhibition of cytosol prolyl endopeptidase. The possibility that aminopeptidase, an ecto-enzyme, would be responsible for the inactivation of bradykinin by intact neutrophils was deduced from the results above, although both cytosol prolyl endopeptidase and membrane aminopeptidase could inactivate bradykinin.

Endopeptidase-24.11 and aminopeptidase activity in brain synaptic membranes are jointly responsible for the hydrolysis of cholecystokinin octapeptide (CCK-8)

Endopeptidase-24.11 (EC 3.4.24.11) from pig kidney hydrolysed CCK-8 (sulphated) at two distinct sites: Asp-Tyr(SO 3H)-Met-Gly↓Trp-Met-Asp↓PheNH 2. Under initial conditions, the splitting of the Asp 7-Phe 8NH 2 bond proceeded 4-times more rapidly than the Gly 4-Trp 5 bond. Pig brain striatal synaptic membranes attacked this substrate at the same sites and this activity was inhibited by phosphoramidon. However, other products were detected even in the presence of phosphoramidon. One of these products was identified as free tryptophan. Since their formation was inhibited by bestatin, one or more membrane aminopeptidases is also implicated in the degradation of CCK-8.