Detergent solubilization of the endocytic Ca(2+)-independent hyaluronan receptor from rat liver endothelial cells and separation from a Ca(2+)-dependent hyaluronan-binding activity.
Biochemistry
; 31(2): 576-84, 1992 Jan 21.
Article
en En
| MEDLINE
| ID: mdl-1370630
Rat liver sinusoidal endothelial cells (LECs) mediate the removal of hyaluronan (HA) from the circulation via a specific Ca(2+)-independent endocytic receptor. To characterize the receptor biochemically, detergent-soluble extracts were prepared from crude LEC membranes. Using a dot blot assay to quantitate 125I-HA binding activity in CHAPS-solubilized membranes, we detected not only specific Ca(2+)-independent but also specific Ca(2+)-dependent HA-binding activity. Both HA-binding activities behave as integral membrane-associated proteins; they are not released from LEC membranes by treatment at pH 11, and they require detergent for extraction. The Ca(2+)-independent HA receptor was inactivated by treatment at 56 degrees C for 30 min or with 200 mM DTT at 4 degrees C for 30 min, whereas the Ca(2+)-dependent activity actually increased by 75% after treatment at 56 degrees C and only 20% of the Ca(2+)-dependent activity was lost after DTT treatment. A two-cycle membrane extraction protocol using CHAPS partially separated the two HA-binding activities. Eight millimolar KCl and 0.5% CHAPS extracted approximately 50% of the Ca(2+)-independent HA receptor, but only 4-11% of the Ca(2+)-dependent activity. When the KCl and CHAPS concentrations were increased to 2.0 M and 1.5%, respectively, the remaining HA receptor, as well as 89-96% of the Ca(2+)-dependent activity, was then extracted. The Ca(2+)-independent and Ca(2+)-dependent activities could also be further separated using Sephacryl S-400 gel filtration chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Portadoras
/
Calcio
/
Ácidos Cólicos
/
Receptores de Superficie Celular
/
Endocitosis
/
Endotelio
/
Ácido Hialurónico
/
Hígado
Tipo de estudio:
Guideline
Límite:
Animals
/
Humans
/
Male
Idioma:
En
Revista:
Biochemistry
Año:
1992
Tipo del documento:
Article
Pais de publicación:
Estados Unidos