ATTEMPTS TO PURIFY A 2ND CELLULAR RECEPTOR FOR A COXSACKIEVIRUS-B3 VARIANT, CB3-RD FROM HELA-CELLS

A coxsackievirus B3 variant, CB3-RD, isolated on rhabdomyosarcoma (RD) cells is known to bind HeLa cells at two different receptor protein s ites, HR1 and HR2. Since HR2 occurs in almost 50 fold excess of HR1 in HeLa cells, purification of HR2 was attempted, to obtain its partial N-terminal amino acid sequence and its further characterization. This study describes the purification of HR2 from octylthioglucoside solubi lized HeLa cell membranes (HeLa-OTG) by preparative isoelectric focusi ng (IEF) followed by either preparative sodium dodecyl sulfate polyacr ylamide gel electrophoresis (SDS-PAGE) or affinity chromatography on i mmobilized receptor monoclonal antibody, RmcA (RmcA-agarose). IEF of H eLa-OTG showed that both HR2 and HR1 could be well separated by this t echnique and focused with peak maxima around pH 3.7 and 6.7, respectiv ely. Both RmcA and CB3-RD recognized HR2 as doublet bands (60 kD major polypeptide and a minor 55 kD polypeptide) on electroblots under non- reducing conditions. Preparative SDS-PAGE of the pool of IEF fractions containing HR2 (IEF pool) and simultaneous elution of polypeptides fr om the bottom of the gel during electrophoresis, is shown to be a usef ul technique in purifying HR2 with only one contaminating polypeptide (65 kD). However, affinity chromatography of the IEF pool on RmcA-agar ose yielded HR2 without any detectable contaminating polypeptide. A qu antitative chemiluminescence assay was developed to estimate the amoun t of HR2 on HeLa cells and in solution, when dot blotted on polyvinyli dene difluoride (PVDF) membranes and probed with RmcA. Assays revealed that about 1.2% of the total HR2 present on HeLa cells could be obtai ned by IEF followed by affinity chromatography. Efforts are continuing to obtain sufficient quantities of purified HR2 for partial N-termina l amino acid sequencing.