Dipeptidyl aminopeptidase activities of guinea-pig brain

Smyth, Maria; O'cuinn, Gerard

doi:10.1016/0020-711x(94)90085-x

Cited by 16 publications

(8 citation statements)

References 25 publications

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“…These results are in line with the detection of native DPP III after Western blot analysis of soluble and membrane samples prepared from D. melanogaster [12] and with the purification of a presumed DPP III from gut membranes in cockroach [12,14]. Our results also agree with the dual localization of DPP III previously reported in rat [6] and guinea-pig [5] brain cytosol and membranes. The partial purification of D. melanogaster DPP III expressed in transfected S 2 cells was achieved from both cytosol and solubilized membranes after thorough washing, and the characterization of both D. melanogaster DPP III partially purified from cytosol and membrane of transfected cells showed similar K m values to met-enkephalin and proctolin.…”

Section: Discussionsupporting

confidence: 92%

“…At the end of incubation, the supernatant (incubation medium) was separated from the cells by centrifugation (1000 g for 8 min at room temperature) 4 and added with 5 lL HCl (2 M). Then, the cell pellet was rinsed twice before cell disruption and centrifugation (13 000 g for 15 min) to isolate the cytosolic sample also added with 5 lL HCl (2 M) 5 . Proctolin degradation was analyzed by reversed-phase separation of the incubation medium in order to estimate the metabolism of proctolin at the cell surface.…”

Section: Immunocytochemistry Of Stable Transfected S 2 Cellsmentioning

confidence: 99%

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Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster

Mazzocco¹,

Fukasawa²,

Auguste³

et al. 2003

European Journal of Biochemistry

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A Drosophila melanogaster cDNA clone (GH01916) encoding a putative 723-residue long (82 kDa) protein (CG 7415) and displaying 50% identity with mammalian cytosolic dipeptidyl aminopeptidase (DPP) III was functionally expressed in Schneider S 2 cells. Immunocytochemical studies using anti-(rat liver DPP III) Ig indicated the expression of this putative DPP III at the outer cell membrane and into the cytosol of transfected cells. Two protein bands (82 and 86 kDa) were immunologically detected after PAGE and Western blot of cytosol or membrane prepared from transfected cells. Western blot analysis of partially purified D. melanogaster DPP III confirmed the overexpression of these two protein bands into the cytosol and on the membranes of transfected cells. Despite the identification of six potential glycosylation sites, PAGE showed that these protein bands were not shifted after deglycosylation experiments. The partially purified enzyme hydrolysed the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) at the Tyr-Leu bond (K m 4 lM). In addition, low concentration of the specific DPP III inhibitor tynorphin prevented proctolin degradation (IC 50 ¼ 0.62 ± 0.15 lM). These results constitute the first characterization of an evolutionarily conserved insect DPP III that is expressed as a cytosolic and a membrane peptidase involved in proctolin degradation.Keywords: enkephalinase; genome sequencing; insects; neuropeptides; proctolin.Mammalian DPP III was first discovered in the bovine anterior pituitary gland [1] and it has been recently cloned from rat liver as a 738-residue (82 kDa) cytosolic protein [2,3]. This enzyme (EC 3.4.14.4) is a zinc metallopeptidase containing a specific domain HELLGH-18X-E where a zinc molecule is bound to both histidines [4]. DPP III is mainly identified as a cytosolic peptidase, but DPP III was also detected on membranes prepared from the brain of guineapig [5] and rat [6]. Angiotensins and enkephalins constitute the preferred substrates of the rat brain cytosolic DPP III [7]. The routes of degradation of the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) have been compared to those of enkephalins. Indeed, a dipeptidyl aminopeptidase activity appears as one major proctolindegrading peptidase, liberating the N-terminal Arg-Tyr dipeptide [8][9][10][11]. This dipeptidyl aminopeptidase activity was compared to the vertebrate DPP III [11] and is mainly recovered as a cytosolic enzyme [8]. Interestingly, a proctolin-degrading DPP activity is also measured on membranes [8,9] especially those obtained from insect proctolin-rich tissues such as hindgut [10]. None of the presumed proctolinases has been fully characterized yet.We recently purified [12] from hindgut membranes of the cockroach, Blaberus craniifer, a proctolin-degrading protein (76 and 80 kDa) that removes the N-terminal dipeptide from proctolin (K m ¼ 3.8 ± 1.1 lM) and enkephalins (K m ¼ 4.2 ± 0.8 lM). The partial sequencing of this purified protein revealed a significant homology with the rat liver cytosolic ...

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Section: Discussionsupporting

confidence: 92%

Section: Immunocytochemistry Of Stable Transfected S 2 Cellsmentioning

confidence: 99%

Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster

Mazzocco¹,

Fukasawa²,

Auguste³

et al. 2003

European Journal of Biochemistry

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“…Prolidase is also capable of hydrolysing Pro-Pro. The other imide-bondhydrolysing peptidase in mammalian cells is DAP I1 (EC 3.4.14.2), which has not been identified in guinea-pig brain [126]. DAP IV, which removes aminoacyl proline moieties from oligopeptides, generates potential substrates for prolidase.…”

Section: Ilipeptidr Rrtrtabolisrrtmentioning

confidence: 99%

“…DAP 111 (EC 3.4.14.4) cleaves dipeptidyl moieties from peptides provided that the peptide contains between four and eight or nine amino acids and provided also that proline does not occur in the first, second or third position from the N-terminus [70,71]. The activity has a pH optimum of 8.5 and can be assayed using Arg-Arg-NH-Mec, substrates for DAP I and DAP I1…”

Section: Dipeptidyl Aminopeptidases (Daps)mentioning

confidence: 99%

Peptide Metabolism in Cytoplasm of Brain Cells

O'cuinn

1998

Biochemical Society Transactions

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“…In addition there was no hydrolysis of peptides containing proline residues at both positions two and three from the N-terminus. There was no evidence of a dipeptidyl aminopeptidase I1 type activity [2]. It was thus possible to conclude that the Gly-Pro-NHMec hydrolase isolated from the soluble fraction of guinea-pig brain was a dipeptidyl aminopeptidase IV type activity.…”

mentioning

confidence: 97%

The Complementary Actions Of Proline Specific Enzymes of Guinea-Pig Brain

Gilmartin

1995

Biochemical Society Transactions

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Dipeptidyl aminopeptidase activities of guinea-pig brain

Cited by 16 publications

References 25 publications

Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster

Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster

Peptide Metabolism in Cytoplasm of Brain Cells

The Complementary Actions Of Proline Specific Enzymes of Guinea-Pig Brain

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