EMPLOYMENT OF A TURBIDIMETRIC ASSAY SYSTEM TO STUDY THE BIOCHEMICAL ROLE OF HSP70 IN HEAT-INDUCED PROTEIN AGGREGATION

1. The biochemical role of the 70 kDa constitutive heat shock protein (cHSP70) in heat-induced protein aggregation was studied by a turbidimetric assay system. 2. When rhodanese (2.5 mg/ml) was diluted 100-fold into heated cytoplasmic proteins (500 mug/ml), turbidity caused by protein rhodanese aggregation rapidly increased and reached maximum within 5 min. 3. The amount of protein aggregates was reduced 2-fold by adding cHSP70 (20 mug/ml), but not bovine serum albumin, during beating at 45.5-degrees-C. Similar results were observed when cHSP70 was added to cytoplasmic proteins from cells heated at 45.5-degrees-C for 10 min. 4. The reduction of protein aggregation by treatment with HSP70 was dependent on ATP content. The aggregation was also significantly reduced when cytoplasmic proteins from thermotolerant cells that contain high level of HSP70 were used. The reduction effects were markedly decreased by adding anti-HSP70 antibody (0.1 mg/ml) to cytoplasms before heating at 45.5-degrees-C for 20 min. 5. Thus, data from the turbidimetric assay system indicate that HSP70 plays an important role in reduction of protein aggregation, and perhaps dissociation of protein aggregates in the presence of ATP.