NOVEL LIGAND BLOT ASSAY DETECTS DIFFERENT HYALURONAN-BINDING PROTEINSIN RAT-LIVER HEPATOCYTES AND SINUSOIDAL ENDOTHELIAL-CELLS

We have developed a sensitive ligand blot assay to detect hyaluronan ( HA) binding proteins in cell extracts using I-125-HA. Samples to be te sted are electrophoresed using standard, nonreducing SDS-PAGE conditio ns, electro-transferred to nitrocellulose then blocked in buffer conta ining Tween 20. After incubation with I-125-HA the nitrocellulose is w ashed and HA-binding proteins are detected by autoradiography. This me thod was used to detect different HA-binding proteins in isolated rat liver cell preparations. Two HA-binding bands of 175 kD and 350 kD wer e detected in sinusoidal endothelial cells. Both bands were competed v irtually 100% with nonlabeled HA. Using rat hepatocytes, the assay det ected major bands at 85 kD and 180 kD. In addition, histones present i n both cell types were readily detected in the low MW region. Thus, tw o different liver cell types show different HA-binding patterns. The b locking procedure is critical for successful renaturation of HA-bindin g activity, since substitution of BSA for Tween 20 did not result in d etectable I-125-HA-binding. This ligand blot assay will be a powerful tool to detect HA-binding proteins in various other tissues and cell t ypes. (C) 1996 Academic Press, Inc.