Among amino acids leucine is the most effective inhibitor of liver autophagy induced by nutrient deprivation. To elucidate the site of action of leucine we used a branched leucine-mimetic peptide named Leu8-MAP, it was synthesized by attaching 8 residues of leucine to the 4 alfa + 4 epsilon amino termini of the branched Lys core (termed MAP for Multiple Antigen Peptide), thus creating a compact molecule of about 1900 Da with leucine groups arranged peripherally. When compared on a molar basis Leu8-MAP was as effective in suppressing autophagy as leucine and had the same apparent Km (0.1 mM). Inhibition was specific for leucine since Ile8-MAP evoked no response. Because it is not transported into the cytosolic compartment, it very likely mediates its effect through a plasma membrane site. In an attempt to isolate the site of interaction we photoreacted intact hepatocytes with a biologically active, biotin conjugated, azide derivative of Leu8-MAP [Leu7-(ASA)MAP- Biotin]. An approximately 103 kD protein whose labeling was protected 90% with 5 mM Leucine was found in plasma membrane enriched fractions when electrophoresed in 4-8% gradient gels, blotted on PVDF membrane and detected by chemiluminescence after labeling with Avidin-HRP. Valine and isoleucine did not compete, thus the photobinding was leucine specific. We exploited the biotin moiety to set up a protocol for isolation/purification of the photolabeled protein(s). The photolabeled plasma membrane enriched fractions were solubilized with zwitterionic detergents and incubated with a support immobilized monomeric avidin, then the biotinilated proteins were recovered under mild conditions with a yield of about 50% and with a high degree of purification. Furthermore the recovered proteins were compatible with bidimensional electrophoresis protocols. These findings show that Leu7-(ASA)MAP-Biotin is a valuable tool for isolation of the putative plasmalemma leucine receptor.

Photoreactive- Leu7-MAP-biotin: a strategic tool for the isolation of leucine receptor involved in regulation of autophagy in isolated hepatocytes

MIOTTO, GIOVANNI;CARAMPIN, PAOLO;VENERANDO, RINA;MARIN, ORIANO
2002

Abstract

Among amino acids leucine is the most effective inhibitor of liver autophagy induced by nutrient deprivation. To elucidate the site of action of leucine we used a branched leucine-mimetic peptide named Leu8-MAP, it was synthesized by attaching 8 residues of leucine to the 4 alfa + 4 epsilon amino termini of the branched Lys core (termed MAP for Multiple Antigen Peptide), thus creating a compact molecule of about 1900 Da with leucine groups arranged peripherally. When compared on a molar basis Leu8-MAP was as effective in suppressing autophagy as leucine and had the same apparent Km (0.1 mM). Inhibition was specific for leucine since Ile8-MAP evoked no response. Because it is not transported into the cytosolic compartment, it very likely mediates its effect through a plasma membrane site. In an attempt to isolate the site of interaction we photoreacted intact hepatocytes with a biologically active, biotin conjugated, azide derivative of Leu8-MAP [Leu7-(ASA)MAP- Biotin]. An approximately 103 kD protein whose labeling was protected 90% with 5 mM Leucine was found in plasma membrane enriched fractions when electrophoresed in 4-8% gradient gels, blotted on PVDF membrane and detected by chemiluminescence after labeling with Avidin-HRP. Valine and isoleucine did not compete, thus the photobinding was leucine specific. We exploited the biotin moiety to set up a protocol for isolation/purification of the photolabeled protein(s). The photolabeled plasma membrane enriched fractions were solubilized with zwitterionic detergents and incubated with a support immobilized monomeric avidin, then the biotinilated proteins were recovered under mild conditions with a yield of about 50% and with a high degree of purification. Furthermore the recovered proteins were compatible with bidimensional electrophoresis protocols. These findings show that Leu7-(ASA)MAP-Biotin is a valuable tool for isolation of the putative plasmalemma leucine receptor.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/1357256
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