Our group previously described a new type of G protein, the 78-kDa XL alphas (extra large as) (Kehlenbach, R. H., Matthey, J., and Huttner, W. B. (1994) Nature 372, 804-809 and (1995) Nature 375, 253). Upon subcellula...
详细信息
Our group previously described a new type of G protein, the 78-kDa XL alphas (extra large as) (Kehlenbach, R. H., Matthey, J., and Huttner, W. B. (1994) Nature 372, 804-809 and (1995) Nature 375, 253). Upon subcellular fractionation, XLas labeled by ADP-ribosylation with cholera toxin was previously mainly detected in the bottom fractions of a velocity sucrose gradient that contained trans-Golgi network and was differentially distributed to G alphas, which also peaked in the top fractions containing plasma membrane. Here, we investigate, using a new antibody specific for the XL domain, the tissue distribution and subcellular localization of XL alphas and novel splice variants referred to as XLN1. Upon immunoblotting and immunofluorescence analysis of various adult rat tissues, XLas and XLN1 were found to be enriched in neuroendocrine tissues, with a particularly high level of expression in the pituitary, By both immunofluorescence and immunogold electron microscopy, endogenous as well as transfected XL alphas and XLN1 were found to be predominantly associated with the plasma membrane, with only little immunoreactivity on internal, perinuclear membranes. Upon subcellular fractionation, immunoreactive XL alphas behaved similarly to G alphas but was differentially distributed to ADP-ribosylated XL alphas. Moreover, the bottom fractions of the velocity sucrose gradient were found to contain not only trans-Golgi network membranes but also certain subdomains of the plasma membrane, which reconciles the present with the previous observations. To further investigate the molecular basis of the association of XL alphas with the plasma membrane, chimeric proteins consisting of the YL domain or portions thereof fused to green fluorescent protein were analyzed by fluorescence and subcellular fractionation, In both neuroendocrine and non-neuroendocrine cells, a fusion protein containing the entire XL domain, in contrast to one containing only the proline-rich and cysteine-rich re
Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the productio...
详细信息
Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as "pro-hormone thiol protease," demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue, suggesting inhibition of basic residue-cleaving proteases;indeed, trypsin was potently inhibited (K7(i(app)) of 5 nM), and plasmin was moderately inhibited. Although endopin 1 possesses homology with alpha(1)-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing), These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.
In order to ascertain that alpha-subunit of guanine nucleotide-binding protein Go (Go alpha)-positive cells in the lung epithelia are pulmonary neuroendocrine cells (PNECs), we carried out an immunohistochemical study...
详细信息
In order to ascertain that alpha-subunit of guanine nucleotide-binding protein Go (Go alpha)-positive cells in the lung epithelia are pulmonary neuroendocrine cells (PNECs), we carried out an immunohistochemical study in young adult and fetal lungs of rodents and in cultured fetal lung explants. Serial sections showed that Goa-positive cells were immunostained for calcitonin gene-related peptide and serotonin in young adult mouse, rat, and hamster lungs and that these cells are, therefore, PNECs. In the fetal lungs of hamster and mouse, Go alpha-positive PNECs appeared in the epithelium of the lobar bronchus by gestational day 13 in hamster and by day 15.5 in mouse, and they increased with a proximal-to-distal wave during the late fetal period. Explants of immature lung from the fetal hamster on gestational day 11 were cultured. After 2 days of culture, Go alpha-positive PNEC clusters appeared in the main and lobar bronchi and many PNEC clusters were seen after 4 days of culture. To determine the functional significance of Go in the development of the fetal lung, pertussis toxin, a Go inhibitor, was added to the medium, and changes in branching morphogenesis and PNEC development were studied. Although branching morphogenesis was not disturbed by pertussis toxin, the toxin treatment induced large PNEC clusters in the cultured lung explant. In summary, we showed that Goa is a neuroendocrine marker for PNECs and that Goa-positive cells appear along with development of PNECs in fetal hamster lung in vivo and in vitro. The functional significance of Go in the development of fetal lung is obscure, but signals mediated through this GTP-binding protein could be related to some functions of PNECs.
We successfully isolated and identified the abundant neuropeptides of the abdominal perisympathetic organs of the American cockroach, including all myoactive compounds. Peptide sequence analysis and mass spectrometry ...
详细信息
We successfully isolated and identified the abundant neuropeptides of the abdominal perisympathetic organs of the American cockroach, including all myoactive compounds. Peptide sequence analysis and mass spectrometry of abundant substances that were not bioactive in different muscle assays yielded the following sequences: TDPLWQLPGAHLEQYLS-NH2 (Pea-YLS-amide), AFLTLTPGSHVDSYVEA-OH (Pea-VEAacid), and SDLTWTYQSPGDPTNSKN-OH (Pea-SKNacid). The given structures led to the conclusion of an unique neuropeptide pattern in abdominal perisympathetic organs. We confirmed this assumption with immunocytochemical studies, using antisera raised against different myotropic neuropeptides of the abdominal perisympathetic organs. Moreover, mass spectrometric methods, developed for the investigation of single neurohemal organs, confirmed the neuropeptide pattern in these organs.
暂无评论