Isolation of surface binding protein specific for advanced glycosylation end products from mouse macrophage-derived cell line RAW 264.7
Isolation of surface binding protein specific for advanced glycosylation end products from mouse macrophage-derived cell line RAW 264.7. S Radoff , A Cerami and H Vlassara Laboratory of Medical Biochemistry, Rockefeller University, New York, New York 10021. Abstract Macrophages internalize and degra...
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Published in | Diabetes (New York, N.Y.) Vol. 39; no. 12; pp. 1510 - 1518 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
American Diabetes Association
01.12.1990
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Online Access | Get full text |
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Summary: | Isolation of surface binding protein specific for advanced glycosylation end products from mouse macrophage-derived cell line
RAW 264.7.
S Radoff ,
A Cerami and
H Vlassara
Laboratory of Medical Biochemistry, Rockefeller University, New York, New York 10021.
Abstract
Macrophages internalize and degrade proteins modified by advanced glycosylation end products (AGEs) via a specific receptor
(AGE-R). Chemical cross-linking studies with AGE-bovine serum albumin have demonstrated that the molecular weight of this
receptor is approximately 90,000. We previously established that the binding constant (Ka) of this receptor site for the chemically
synthesized model AGE, 2-(2-furoyl)-4(5)-(2-furanyl)-1H- imidazole-butyric acid (FFI-BA), on cells of the mouse macrophagelike
cell line RAW 264.7 is identical to that for AGE proteins. Therefore, FFI was used as an affinity matrix in the first purification
step of the AGE-R. The membranes of RAW 264.7 cells were solubilized in octyl-beta-glucoside and subjected to affinity chromatography
on FFI-sepharose and gel permeation on Superose 6 fast protein liquid chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis analysis of this material revealed a high enrichment of a 90,000-Mr protein that had AGE binding activity.
Approximately 25% of the protein at this step was the 90,000-Mr protein. The 90,000-Mr membrane protein was purified to homogeneity
by rechromatographing the material on Superose 12 in the presence of SDS before and after reduction with 2-mercaptoethanol.
After these harsh conditions, the 90,000-Mr protein lost AGE binding activity. Additional cross-linking studies on human peripheral
monocytes revealed an AGE-R protein of identical size to that on RAW 264.7 cells, suggesting the relatively highly conserved
nature of this molecule. |
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ISSN: | 0012-1797 1939-327X 0012-1797 |
DOI: | 10.2337/diabetes.39.12.1510 |