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Title: Photolabeling of the alpha-neuraminidase/beta-galactosidase complex from human placenta with a photoreactive neuraminidase inhibitor

Abstract

Photolabeling of the alpha-neuraminidase/beta-galactosidase complex in human placenta was carried out using the radioactive photoprobe, 9-S-(4-azido-3,5-3H-2-nitrophenyl)-5-acetamido-2,6 anhydro-2,3,5,9- tetradeoxy-9- thio-D-glycero-D-galacto-non-2-enonic acid. Two intensely labeled bands at 61 and 46 kD were detected with autoradiography. Labeling of the 46 kD protein was blocked with the inclusion of the surfactant Triton X-100 in the photolysis mixture, indicating a nonspecific, hydrophobic interaction. The 61 kD protein was protected from labeling only when the neuraminidase inhibitor 2,3 dehydro N-acetyl neuraminic acid (1 mM) was present during photolysis. These results suggest that the neuraminidase activity resides among the proteins in the 61 kD molecular weight range comigrating with the lysosomal beta-galactosidase, under denaturing conditions.

Authors:
; ;  [1]
  1. (Univ. of Tennessee, Memphis (USA))
Publication Date:
OSTI Identifier:
6072439
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; (USA); Journal Volume: 173:1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; GALACTOSIDASE; LABELLING; GLYCOSYL HYDROLASES; AFFINITY; AUTORADIOGRAPHY; COMPLEXES; ELECTROPHORESIS; ENZYME ACTIVITY; ENZYME INHIBITORS; MAN; MOLECULAR WEIGHT; PLACENTA; TRITIUM COMPOUNDS; ANIMALS; ENZYMES; FETAL MEMBRANES; HYDROGEN COMPOUNDS; HYDROLASES; MAMMALS; MEMBRANES; O-GLYCOSYL HYDROLASES; PRIMATES; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Warner, T.G., Louie, A., and Potier, M. Photolabeling of the alpha-neuraminidase/beta-galactosidase complex from human placenta with a photoreactive neuraminidase inhibitor. United States: N. p., 1990. Web. doi:10.1016/S0006-291X(05)81014-9.
Warner, T.G., Louie, A., & Potier, M. Photolabeling of the alpha-neuraminidase/beta-galactosidase complex from human placenta with a photoreactive neuraminidase inhibitor. United States. doi:10.1016/S0006-291X(05)81014-9.
Warner, T.G., Louie, A., and Potier, M. 1990. "Photolabeling of the alpha-neuraminidase/beta-galactosidase complex from human placenta with a photoreactive neuraminidase inhibitor". United States. doi:10.1016/S0006-291X(05)81014-9.
@article{osti_6072439,
title = {Photolabeling of the alpha-neuraminidase/beta-galactosidase complex from human placenta with a photoreactive neuraminidase inhibitor},
author = {Warner, T.G. and Louie, A. and Potier, M.},
abstractNote = {Photolabeling of the alpha-neuraminidase/beta-galactosidase complex in human placenta was carried out using the radioactive photoprobe, 9-S-(4-azido-3,5-3H-2-nitrophenyl)-5-acetamido-2,6 anhydro-2,3,5,9- tetradeoxy-9- thio-D-glycero-D-galacto-non-2-enonic acid. Two intensely labeled bands at 61 and 46 kD were detected with autoradiography. Labeling of the 46 kD protein was blocked with the inclusion of the surfactant Triton X-100 in the photolysis mixture, indicating a nonspecific, hydrophobic interaction. The 61 kD protein was protected from labeling only when the neuraminidase inhibitor 2,3 dehydro N-acetyl neuraminic acid (1 mM) was present during photolysis. These results suggest that the neuraminidase activity resides among the proteins in the 61 kD molecular weight range comigrating with the lysosomal beta-galactosidase, under denaturing conditions.},
doi = {10.1016/S0006-291X(05)81014-9},
journal = {Biochemical and Biophysical Research Communications; (USA)},
number = ,
volume = 173:1,
place = {United States},
year = 1990,
month =
}
  • Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta..more » heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.« less
  • The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na{sup +}/H{sup +} antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na{sup +}/H{sup +} exchange in a competitive manner with a K{sub i} of 2.5 and 5.9 micromolar for {Delta}pH-dependent {sup 22}Na{sup +} influx in tonoplast vesicles and Na{sup +}-dependent H{sup +} efflux in intact vacuoles, respectively. Scatchard analysis of the binding of ({sup 3}H)MIA to tonoplast membranes revealed a high affinity binding component with a K{sub d} of 1.3 micromolar. The close relationship between themore » dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na{sup +}/H{sup +} antiport. Photolabeling of the tonoplast with ({sup 3}H)MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.« less
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