Purification and characterization of adipocyte heparan sulfate proteoglycans with affinity for lipoprotein lipase.
Lipoprotein lipase (LPL)-binding heparan sulfate proteoglycans (HSPGs) were isolated from cell extracts and conditioned media of cultured adipocytes treated with phosphatidylinositol-specific phospholipase C (PIPLC). The methodology employed included anion exchange chromatography, affinity chromatography on LPL Affi-Prep 10 and hydrophobic chromatography. HSPGs were resolved into two distinct fractions on the Octyl-Sepharose CL-4B matrix. Treatment of the eluted fractions with heparinase and heparitinase yielded core proteins of 48.4 and 39 kDa. The 39-kDa core protein is anchored to the cell surface by a glycosyl phosphatidylinositol anchor as evidenced by 1) release of the HSPG with the 39-kDa core protein into media by PIPLC treatment and 2) biosynthetic incorporation of [3H]ethanolamine and [32P]orthophosphate into the PIPLC-releasable 39-kDa core protein. PIPLC released 23% of the total heparin-releasable LPL. A similar percentage (24.5%) of the total heparan sulfate chains was released by PIPLC. Over 96% of the total adipocyte heparan sulfate chains bound to LPL Affi-Prep 10 column. The heterogeneity of core proteins of HSPGs with affinity for LPL may provide a structural basis for the multiple fates of LPL on the surface of adipocytes, i.e. internalization, degradation, or recycling to the cell surface and translocation into the medium.
Misra
K B
KB
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.
Kim
K C
KC
Cho
S
S
Low
M G
MG
Bensadoun
A
A
D017667:Adipocytes / Q000737:chemistry*; D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D017261:Glycosylphosphatidylinositols; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000302:isolation & purification* / Q000378:metabolism; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008970:Molecular Weight; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011509:Proteoglycans / Q000302:isolation & purification* / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D008071:Lipoprotein Lipase; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases; D011509:Proteoglycans