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Degradation and intracellular accumulation of a residualizing hyaluronan derivative by liver endothelial cells
Author(s) -
McGary Carl T.,
YannarielloBrown Judith,
Kim Dennis W.,
Stinson Tami C.,
Weige Paul H.
Publication year - 1993
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.1840180628
Subject(s) - cetylpyridinium chloride , chemistry , intracellular , hyaluronic acid , chromatography , biochemistry , size exclusion chromatography , extracellular , degradation (telecommunications) , biology , pulmonary surfactant , telecommunications , genetics , computer science , enzyme
The release and intracellular accumulation of 125 I‐hyaluronan degradation products was studied in cultured liver endothelial cells with hyaluronan oligosaccharides (relative molecular mass = approximately 44,000) uniquely modified and radiolabeled at the terminal reducing sugar. Two methods were combined to measure 125 I‐hyaluronan degradation by liver endothelial cells. (a) Cetylpyridinium chloride precipitation of hyaluronan oligosaccharides was used as a rapid, convenient assay to monitor the appearance of hyaluronan degradation products. Hyaluronan oligosaccharides less than 54 to 60 monosaccharides in length were not precipitated with cetylpyridinium chloride and thus were assessed as degraded. (b) Gel filtration chromatography was used to estimate the size range of oligosaccharides produced by liver endothelial cells. After internalization of 125 I‐hyaluronan, liver endothelial cells released radioactive degradation products into the culture media after a lag period of 2.5 to 3.0 hr. The intracellular accumulation of degraded 125 I‐hyaluronan was linear for at least 2 hr even though no degradation products were released. The long lag before release of degraded 125 I‐hyaluronan is likely caused by the modified chemical structure at the reducing end of the hyaluronan derivative; the derivative acts like a residualizing label. After this lag the release of degraded 125 I‐hyaluronan proceeded linearly for up to 12 hr. The extracellular 125 I‐hyaluronan degradation products eluted with a distribution coefficient of 1.3 on a gel filtration column. The major intracellular 125 I‐labeled degradation product showed the same retardation (distribution coefficient = 1.3). This retention may be caused by the hydrophobic aromatic and alkyl modifications to the former reducing sugar, also characteristics of a residualizing label. In addition, at least two larger minor intermediates were observed intracellularly. The rate of intracellular 125 I‐hyaluronan degradation was dependent on hyaluronan concentration and reached a maximal rate (159 molecules/cell/sec) at 2 × 10 −7 mol/L. This was about half the maximal rate of endocytosis (285 molecules/cell/sec) at a hyaluronan concentration of 1.3 × 10 −7 mol/L. The apparent ligand concentration that gives half‐maximal responses for endocytosis and intracellular degradation was 0.6 × 10 −7 and 1.0 × 10 −7 mol/L, respectively. (HEPATOLOGY 1993;18:1465–1476.)