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Piero Andreuccetti - One of the best experts on this subject based on the ideXlab platform.

  • pituitary adenylate cyclase activating peptide pacap and pac1 receptor in the testis of cartilaginous fish Torpedo Marmorata a molecular and phylogenetic study
    Comparative Biochemistry and Physiology B, 2016
    Co-Authors: Marisa Agnese, Salvatore Valiante, Luigi Rosati, Piero Andreuccetti, Marina Prisco
    Abstract:

    The role of PACAP in spermatogenesis and steroidogenesis has been largely investigated in last years in mammals; conversely, a few studies have been performed in non mammalian vertebrates. In this paper we investigated the sequence, expression and localization of PACAP and its PAC1 receptor in the testis of the benthic elasmobranch Torpedo Marmorata, the marbled electric ray. Cloning a partial PACAP cDNA, we demonstrated for the first time in elasmobranches that PACAP shows a highly conserved sequence, compared with the PACAP of other chordates (tunicates and vertebrates). Moreover, the phylogenetic analysis revealed that PACAP has been well preserved during evolution and that a negative selection acts on PACAP sequence, leading to the conservation of the coding sites. The phylogenetic consensus tree showed also that Torpedo PACAP is more related with the amphibian PACAP than with the teleost one. Finally, we demonstrated that in T. Marmorata PACAP and its PAC1 receptor are synthesized directly in the testis, where they show a wider localization than mammals, suggesting that this neuropeptide is involved in the control of Torpedo spermatogenesis.

  • expression of prothymosin alpha during the spermatogenesis of the spotted ray Torpedo Marmorata
    General and Comparative Endocrinology, 2009
    Co-Authors: Marina Prisco, Marisa Agnese, Aldo Donizetti, Francesco Aniello, Annamaria Locascio, Giuseppina Del Giudice, Francesco Angelini, Piero Andreuccetti
    Abstract:

    In this study, we show that Prothymosin alpha (Ptma), a small, unfolded, negatively charged protein, is present in the cartilaginous fish Torpedo Marmorata. The ptma gene is functional and peculiarly controlled during the male spermatogenesis of T. Marmorata, as revealed by in situ hybridization and by immunocytochemistry studies. The data show that the ptma transcript is present in stage-specific germ cells, i.e. spermatocytes II and round spermatids. The Ptma protein is detectable in spermatocytes II, in round and elongated spermatids as well as in spermatozoa before their release from cysts, while it is not evident in spermatozoa located in male genital tracts. The ptma transcript and protein are also evident in some Leydig cells, located among maturing cysts containing meiotic and differentiating male cells. No expression for ptma is observed within Sertoli cells. Furthermore, immunolocalization procedures demonstrate that the protein is preferentially localized in the cytoplasm, whereas a nuclear localization is observed in round and elongated spermatids. The possibility that Ptma is involved in testis activity is discussed.

  • Oogenesis in the spotted ray Torpedo Marmorata
    Reviews in Fish Biology and Fisheries, 2007
    Co-Authors: Marina Prisco, Annamaria Liguoro, Loredana Ricchiari, Giuseppina Del Giudice, Piero Andreuccetti
    Abstract:

    Investigation of embryos, neonates, sub-adult and adult female Torpedo Marmorata showed that oogenesis is immediate. It begins very early in development and continues, in its proliferative phase after birth. In newborns, 3–4 month old, all germ cells present in the ovary have achieved diplotene stage and are surrounded by a single layer of small follicle cells (primordial follicles). Later with continued oocyte growth, the follicular epithelium (granulosa) progressively changes its organization and becomes multilayered and polymorphic with the presence of three types of cells: small, intermediate and pyriform cells. This organization lasts until the end of oocyte growth, but its activity changes significantly. Indeed, in the previtellogenic phase intermediate and pyriform-cells transfer to the oocyte mitochondria, ribosomes, vesicles, via intercellular bridges; during vitellogenesis, intermediate and pyriform cells, as well as small follicle cells, synthesize and transfer vitellogenin within the oocyte. During Torpedo oogenesis, in concomitance with the differentiation of the granulosa, the nucleolar apparatus is modified significantly: transcriptionally active in primary follicles, it becomes inactive during the subsequent stages of oocyte growth.

  • distribution of pacap in the brain of the cartilaginous fish Torpedo Marmorata
    Annals of the New York Academy of Sciences, 2006
    Co-Authors: Salvatore Valiante, Marina Prisco, Loredana Ricchiari, Vincenza Laforgia, Lorenzo Varano, Piero Andreuccetti
    Abstract:

    In this article, we investigated the distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) and its mRNA by immunohistochemistry, in situ hybridization, and RT-PCR techniques, in the central nervous system of the elasmobranch Torpedo Marmorata. RT-PCR analysis showed that the CNS of T. Marmorata expresses a messenger encoding PACAP. The immunohistochemistry and in situ hybridization patterns were partly overlapping, with a major expression in the hypothalamo-pituitary region and, surprisingly, in the saccus vasculosus. Our results show that, in T. Marmorata, PACAP is synthesized and widely distributed in the CNS, suggesting an as yet unidentified role for this peptide in elasmobranch brain physiology.

Prisco M. - One of the best experts on this subject based on the ideXlab platform.

  • Expression of VIP and its Receptors in the Testis of the Spotted Ray Torpedo Marmorata (Risso 1880).
    'Springer Science and Business Media LLC', 2012
    Co-Authors: Agnese M., Valiante S., Andreuccetti P., Rosati L., Muriano F., Prisco M.
    Abstract:

    The aim of this work was to study, by immunoprecipitation, in situ hybridization and immunohistochemistry, and the expression of the vasoactive intestinal peptide (VIP) and of its receptors (VPAC(1)R and VPAC(2)R) in the testis of a nonmammalian vertebrate, the cartilaginous fish Torpedo Marmorata. We demonstrated that, differently from mammals, VIP and VPAC(2)R were widely distributed in the testicular cells while the VPAC(1)R had a limited distribution. In details, we showed that VIP and VPAC(2)R were present in mitotic and differentiating germ cells as well as in the cells involved in the steroidogenesis, i.e., Leydig, Sertoli cells, and prespermatogonia and spermatogonia. The possibility that VIP is involved in the spermatogenesis and particularly in the steroidogenesis of T. Marmorata is discussed

  • Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo Marmorata Risso 1880.
    'Wiley', 2012
    Co-Authors: Del Giudice G., Prisco M., Agnese M., Verderame M., Limatola E., Rosati L., Andreuccetti P.
    Abstract:

    The aim of this investigation was to assess the effects of nonylphenol (NP), an oestrogen-like environmental pollutant, on the vitellogenin (VTG) synthesis in adult males of the aplacental viviparous cartilaginous fish Torpedo Marmorata. The VTG recovery in males is considered a biomarker of xeno-oestrogenic pollution as this lipophosphoglycoprotein is physiologically induced by oestrogens only in females of oviparous and ovoviparous vertebrates. Using in situ hybridization and immunohistochemistry, T. Marmorata males injected with nonylphenol showed the presence of VTG in the liver and the kidney. In particular, vtg messenger (m)RNA and VTG protein were expressed in the liver, whereas in the kidney cells only the presence of VTG was recorded. By contrast, no expression for VTG was detected in the testis. These results demonstrate that in T. Marmorata NP induces the expression of vtg only in the liver; the presence of VTG in the kidney and its absence in the testis are discussed

  • Expression of vitellogenin receptor in the ovarian follicles during the reproductive cycle of the spotted ray Torpedo Marmorata Risso 1880.
    'Wiley', 2011
    Co-Authors: Del Giudice G., Prisco M., Agnese M., Valiante S., Verderame M., Limatola E., Andreuccetti P.
    Abstract:

    The aim of this investigation was to identify the encoding sequence of vitellogenin receptor gene (vtgr), and its expression during the oogenesis in the spotted ray, Torpedo Marmorata, in different phases of reproductive cycle. From an ovarian cDNA of vitellogenic female, we obtained a fragment of 581 bp, which corresponds to a partial sequence encoding the vitellogenin receptor (VTGR) in Torpedo (accession number: gi/193244760). This sequence shows a high identity with the VTGR of other vertebrates, particularly Leucoraja erinacea (89% identity) and Squalus acanthias (84% identity). We also showed that vtgr mRNA expression in the ovary modifies during the oogenesis and throughout the reproductive cycle. Indeed, in immature females, whose ovary contains only previtellogenic follicles, vtgr mRNA occurred in the oocyte cortex as well as within intermediate and pyriform cells. In mature females, whose ovary contains pre- and vitellogenic follicles, vtgr mRNA was detectable not only in the oocyte cortex and in intermediate and pyriform cells but also in small follicle cells present in the follicular epithelium of vitellogenic follicles. In ovulating females, that, as pregnant ones, show pre-and vitellogenic follicles, vtgr mRNA was evident in the oocyte cortex only, whereas in pregnant females, no vtgr mRNA was evident. The role of VTGR in the control of Torpedo vitellogenesis is discussed

  • Expression of vitellogenin in the testis and kidney of the spotted ray Torpedo Marmorata exposed to 17-βestradiol
    'Elsevier BV', 2011
    Co-Authors: Del Giudice G., Prisco M., Agnese M., Verderame M., Limatola E., Andreuccetti P.
    Abstract:

    In vertebrates, the liver was long thought to be the only site of vitellogenin (Vtg) production, but recent studies demonstrated that Vtg is also expressed in extrahepatic districts. The aim of this paper is to assess, by in situ hybridization and immunohistochemistry, the expression of Vtg in the testis and kidney of Torpedo Marmorata exposed to 17β-estradiol (E(2)). In treated samples vtg mRNA and Vtg were detected contemporaneously only in the testis; differently the kidney cells were positive to Vtg antibody, but negative to vtg mRNA. This is the first study to assess that male germ cells, after an exposure to E(2), synthesize Vtg in a stage-dependent manner. The presence of Vtg and the modifications observed in the kidney after E(2) treatment are discussed

  • Immunolocalization of 3beta-HSD and 17beta-HSD in the testis of the spotted ray Torpedo Marmorata
    'Elsevier BV', 2008
    Co-Authors: Prisco M., Del Giudice G., Ricchiari L, Liguoro A, Angelini F, Andreuccetti P.
    Abstract:

    Using polyclonal antibodies, we examined the localization of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) as markers of the site of steroidogenetic activity during the spermatogenesis of Torpedo Marmorata. These enzymes play a central role in the biosynthesis of steroid hormones, including androgen and oestrogen production. We demonstrated that in the spotted ray testis, Sertoli and Leydig cells, as well as spermatogonia, show a positive reaction to anti 3beta-HSD and 17beta-HSD antibodies. In particular, we demonstrated that Sertoli cells show a positive reaction to anti 3beta-HSD and 17beta-HSD antibodies in cysts containing spermatogonia and spermatozoa, while Leydig cells present a positive reaction only when they are located between cysts containing meiotic cells. This study strongly suggests that, as hypothesised in our previous study [Prisco, M., Liguoro, A., D'Onghia, B., Ricchiari, L., Andreuccetti, P., Angelini, F., 2002. Fine structure of Leydig and Sertoli cells in the testis of immature and mature spotted ray Torpedo Marmorata. Mol. Reprod. Dev. 63, 192-201.], Sertoli and Leydig cells are differently involved in the hormonal control of spermatogenesis: Sertoli cells before the beginning of meiosis and after spermiation, Leydig cells only during meiosis phase. Moreover, the present paper deals with the possibility that also spermatogonia are engaged in the production of androgen hormones, as they are characterized by the presence of 3beta-HSD and 17beta-HSD enzymes, and show the ultrastructural features of steroid hormone-producing cells

Andreuccetti P. - One of the best experts on this subject based on the ideXlab platform.

  • Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo Marmorata Risso 1880.
    'Wiley', 2012
    Co-Authors: Del Giudice G., Prisco M., Agnese M., Verderame M., Limatola E., Rosati L., Andreuccetti P.
    Abstract:

    The aim of this investigation was to assess the effects of nonylphenol (NP), an oestrogen-like environmental pollutant, on the vitellogenin (VTG) synthesis in adult males of the aplacental viviparous cartilaginous fish Torpedo Marmorata. The VTG recovery in males is considered a biomarker of xeno-oestrogenic pollution as this lipophosphoglycoprotein is physiologically induced by oestrogens only in females of oviparous and ovoviparous vertebrates. Using in situ hybridization and immunohistochemistry, T. Marmorata males injected with nonylphenol showed the presence of VTG in the liver and the kidney. In particular, vtg messenger (m)RNA and VTG protein were expressed in the liver, whereas in the kidney cells only the presence of VTG was recorded. By contrast, no expression for VTG was detected in the testis. These results demonstrate that in T. Marmorata NP induces the expression of vtg only in the liver; the presence of VTG in the kidney and its absence in the testis are discussed

  • Expression of VIP and its Receptors in the Testis of the Spotted Ray Torpedo Marmorata (Risso 1880).
    'Springer Science and Business Media LLC', 2012
    Co-Authors: Agnese M., Valiante S., Andreuccetti P., Rosati L., Muriano F., Prisco M.
    Abstract:

    The aim of this work was to study, by immunoprecipitation, in situ hybridization and immunohistochemistry, and the expression of the vasoactive intestinal peptide (VIP) and of its receptors (VPAC(1)R and VPAC(2)R) in the testis of a nonmammalian vertebrate, the cartilaginous fish Torpedo Marmorata. We demonstrated that, differently from mammals, VIP and VPAC(2)R were widely distributed in the testicular cells while the VPAC(1)R had a limited distribution. In details, we showed that VIP and VPAC(2)R were present in mitotic and differentiating germ cells as well as in the cells involved in the steroidogenesis, i.e., Leydig, Sertoli cells, and prespermatogonia and spermatogonia. The possibility that VIP is involved in the spermatogenesis and particularly in the steroidogenesis of T. Marmorata is discussed

  • Expression of vitellogenin receptor in the ovarian follicles during the reproductive cycle of the spotted ray Torpedo Marmorata Risso 1880.
    'Wiley', 2011
    Co-Authors: Del Giudice G., Prisco M., Agnese M., Valiante S., Verderame M., Limatola E., Andreuccetti P.
    Abstract:

    The aim of this investigation was to identify the encoding sequence of vitellogenin receptor gene (vtgr), and its expression during the oogenesis in the spotted ray, Torpedo Marmorata, in different phases of reproductive cycle. From an ovarian cDNA of vitellogenic female, we obtained a fragment of 581 bp, which corresponds to a partial sequence encoding the vitellogenin receptor (VTGR) in Torpedo (accession number: gi/193244760). This sequence shows a high identity with the VTGR of other vertebrates, particularly Leucoraja erinacea (89% identity) and Squalus acanthias (84% identity). We also showed that vtgr mRNA expression in the ovary modifies during the oogenesis and throughout the reproductive cycle. Indeed, in immature females, whose ovary contains only previtellogenic follicles, vtgr mRNA occurred in the oocyte cortex as well as within intermediate and pyriform cells. In mature females, whose ovary contains pre- and vitellogenic follicles, vtgr mRNA was detectable not only in the oocyte cortex and in intermediate and pyriform cells but also in small follicle cells present in the follicular epithelium of vitellogenic follicles. In ovulating females, that, as pregnant ones, show pre-and vitellogenic follicles, vtgr mRNA was evident in the oocyte cortex only, whereas in pregnant females, no vtgr mRNA was evident. The role of VTGR in the control of Torpedo vitellogenesis is discussed

  • Expression of vitellogenin in the testis and kidney of the spotted ray Torpedo Marmorata exposed to 17-βestradiol
    'Elsevier BV', 2011
    Co-Authors: Del Giudice G., Prisco M., Agnese M., Verderame M., Limatola E., Andreuccetti P.
    Abstract:

    In vertebrates, the liver was long thought to be the only site of vitellogenin (Vtg) production, but recent studies demonstrated that Vtg is also expressed in extrahepatic districts. The aim of this paper is to assess, by in situ hybridization and immunohistochemistry, the expression of Vtg in the testis and kidney of Torpedo Marmorata exposed to 17β-estradiol (E(2)). In treated samples vtg mRNA and Vtg were detected contemporaneously only in the testis; differently the kidney cells were positive to Vtg antibody, but negative to vtg mRNA. This is the first study to assess that male germ cells, after an exposure to E(2), synthesize Vtg in a stage-dependent manner. The presence of Vtg and the modifications observed in the kidney after E(2) treatment are discussed

  • Immunolocalization of 3beta-HSD and 17beta-HSD in the testis of the spotted ray Torpedo Marmorata
    'Elsevier BV', 2008
    Co-Authors: Prisco M., Del Giudice G., Ricchiari L, Liguoro A, Angelini F, Andreuccetti P.
    Abstract:

    Using polyclonal antibodies, we examined the localization of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) as markers of the site of steroidogenetic activity during the spermatogenesis of Torpedo Marmorata. These enzymes play a central role in the biosynthesis of steroid hormones, including androgen and oestrogen production. We demonstrated that in the spotted ray testis, Sertoli and Leydig cells, as well as spermatogonia, show a positive reaction to anti 3beta-HSD and 17beta-HSD antibodies. In particular, we demonstrated that Sertoli cells show a positive reaction to anti 3beta-HSD and 17beta-HSD antibodies in cysts containing spermatogonia and spermatozoa, while Leydig cells present a positive reaction only when they are located between cysts containing meiotic cells. This study strongly suggests that, as hypothesised in our previous study [Prisco, M., Liguoro, A., D'Onghia, B., Ricchiari, L., Andreuccetti, P., Angelini, F., 2002. Fine structure of Leydig and Sertoli cells in the testis of immature and mature spotted ray Torpedo Marmorata. Mol. Reprod. Dev. 63, 192-201.], Sertoli and Leydig cells are differently involved in the hormonal control of spermatogenesis: Sertoli cells before the beginning of meiosis and after spermiation, Leydig cells only during meiosis phase. Moreover, the present paper deals with the possibility that also spermatogonia are engaged in the production of androgen hormones, as they are characterized by the presence of 3beta-HSD and 17beta-HSD enzymes, and show the ultrastructural features of steroid hormone-producing cells

Marina Prisco - One of the best experts on this subject based on the ideXlab platform.

  • pituitary adenylate cyclase activating peptide pacap and pac1 receptor in the testis of cartilaginous fish Torpedo Marmorata a molecular and phylogenetic study
    Comparative Biochemistry and Physiology B, 2016
    Co-Authors: Marisa Agnese, Salvatore Valiante, Luigi Rosati, Piero Andreuccetti, Marina Prisco
    Abstract:

    The role of PACAP in spermatogenesis and steroidogenesis has been largely investigated in last years in mammals; conversely, a few studies have been performed in non mammalian vertebrates. In this paper we investigated the sequence, expression and localization of PACAP and its PAC1 receptor in the testis of the benthic elasmobranch Torpedo Marmorata, the marbled electric ray. Cloning a partial PACAP cDNA, we demonstrated for the first time in elasmobranches that PACAP shows a highly conserved sequence, compared with the PACAP of other chordates (tunicates and vertebrates). Moreover, the phylogenetic analysis revealed that PACAP has been well preserved during evolution and that a negative selection acts on PACAP sequence, leading to the conservation of the coding sites. The phylogenetic consensus tree showed also that Torpedo PACAP is more related with the amphibian PACAP than with the teleost one. Finally, we demonstrated that in T. Marmorata PACAP and its PAC1 receptor are synthesized directly in the testis, where they show a wider localization than mammals, suggesting that this neuropeptide is involved in the control of Torpedo spermatogenesis.

  • expression of prothymosin alpha during the spermatogenesis of the spotted ray Torpedo Marmorata
    General and Comparative Endocrinology, 2009
    Co-Authors: Marina Prisco, Marisa Agnese, Aldo Donizetti, Francesco Aniello, Annamaria Locascio, Giuseppina Del Giudice, Francesco Angelini, Piero Andreuccetti
    Abstract:

    In this study, we show that Prothymosin alpha (Ptma), a small, unfolded, negatively charged protein, is present in the cartilaginous fish Torpedo Marmorata. The ptma gene is functional and peculiarly controlled during the male spermatogenesis of T. Marmorata, as revealed by in situ hybridization and by immunocytochemistry studies. The data show that the ptma transcript is present in stage-specific germ cells, i.e. spermatocytes II and round spermatids. The Ptma protein is detectable in spermatocytes II, in round and elongated spermatids as well as in spermatozoa before their release from cysts, while it is not evident in spermatozoa located in male genital tracts. The ptma transcript and protein are also evident in some Leydig cells, located among maturing cysts containing meiotic and differentiating male cells. No expression for ptma is observed within Sertoli cells. Furthermore, immunolocalization procedures demonstrate that the protein is preferentially localized in the cytoplasm, whereas a nuclear localization is observed in round and elongated spermatids. The possibility that Ptma is involved in testis activity is discussed.

  • Oogenesis in the spotted ray Torpedo Marmorata
    Reviews in Fish Biology and Fisheries, 2007
    Co-Authors: Marina Prisco, Annamaria Liguoro, Loredana Ricchiari, Giuseppina Del Giudice, Piero Andreuccetti
    Abstract:

    Investigation of embryos, neonates, sub-adult and adult female Torpedo Marmorata showed that oogenesis is immediate. It begins very early in development and continues, in its proliferative phase after birth. In newborns, 3–4 month old, all germ cells present in the ovary have achieved diplotene stage and are surrounded by a single layer of small follicle cells (primordial follicles). Later with continued oocyte growth, the follicular epithelium (granulosa) progressively changes its organization and becomes multilayered and polymorphic with the presence of three types of cells: small, intermediate and pyriform cells. This organization lasts until the end of oocyte growth, but its activity changes significantly. Indeed, in the previtellogenic phase intermediate and pyriform-cells transfer to the oocyte mitochondria, ribosomes, vesicles, via intercellular bridges; during vitellogenesis, intermediate and pyriform cells, as well as small follicle cells, synthesize and transfer vitellogenin within the oocyte. During Torpedo oogenesis, in concomitance with the differentiation of the granulosa, the nucleolar apparatus is modified significantly: transcriptionally active in primary follicles, it becomes inactive during the subsequent stages of oocyte growth.

  • distribution of pacap in the brain of the cartilaginous fish Torpedo Marmorata
    Annals of the New York Academy of Sciences, 2006
    Co-Authors: Salvatore Valiante, Marina Prisco, Loredana Ricchiari, Vincenza Laforgia, Lorenzo Varano, Piero Andreuccetti
    Abstract:

    In this article, we investigated the distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) and its mRNA by immunohistochemistry, in situ hybridization, and RT-PCR techniques, in the central nervous system of the elasmobranch Torpedo Marmorata. RT-PCR analysis showed that the CNS of T. Marmorata expresses a messenger encoding PACAP. The immunohistochemistry and in situ hybridization patterns were partly overlapping, with a major expression in the hypothalamo-pituitary region and, surprisingly, in the saccus vasculosus. Our results show that, in T. Marmorata, PACAP is synthesized and widely distributed in the CNS, suggesting an as yet unidentified role for this peptide in elasmobranch brain physiology.

C. Hirth - One of the best experts on this subject based on the ideXlab platform.

  • synthesis of an acetylcholine receptor specific toxin derivative regioselectively labeled with an undecagold cluster
    Bioconjugate Chemistry, 1994
    Co-Authors: Pascal Kessler, Maurice Goeldner, F. Bouet, Florence Kotzybahibert, M Leonetti, Philippe Ringler, Alain Brisson, Andre Menez, C. Hirth
    Abstract:

    The regioselective modification of a snake curaremimetic toxin is described. Toxin-alpha from Naja nigricollis was derivatized with an electron-dense maleimido undecagold cluster 5. The cluster-bound obtained toxin 8 has a very high affinity for the cholinergic binding site (Ki = 70 pM) of Torpedo Marmorata nicotinic receptor. It is harboring a compact heavy atom core of about 8 A which makes it very useful for electron microscopy experiments.

  • Trp279 is involved in the binding of quaternary ammonium at the peripheral site of Torpedo Marmorata acetylcholinesterase.
    European Journal of Biochemistry, 1994
    Co-Authors: I. Schalk, L. Ehret-sabatier, F. Bouet, M. Goeldner, C. Hirth
    Abstract:

    Specific photoaffinity labelling of purified acetylcholinesterase from Torpedo Marmorata by p-N,N-[3H]dimethylamino benzenediazonium and p-N,N-[3H]dibutylamino benzenediazonium derivatives was demonstrated. This occurred at the active site of the enzyme for lower concentrations of the probes and at the peripheral ammonium binding site for higher concentrations. The affinities and the rate constants of alkylation for each probe on both sites have been established. Specific labelling at the peripheral site of the enzyme with both probes allowed the identification of radio-labelled peptides having the common sequence K270PQELIDVEW. The radioactivity was always associated with the residue Trp279 indicating the preferential ammonium complexation with this aromatic residue.