DIFFERENTIAL DETERGENT FRACTIONATION OF ISOLATED HEPATOCYTES - BIOCHEMICAL, IMMUNOCHEMICAL AND 2-DIMENSIONAL GEL-ELECTROPHORESIS CHARACTERIZATION OF CYTOSKELETAL AND NONCYTOSKELETAL COMPARTMENTS

Two-dimensional (2-D) gel electrophoresis is often used in toxicologic and metabolic studies to assess treatment- or stage-specific changes in protein synthesis, degradation or posttranslational modification. W hen combined with cell fractionation studies the detectability of low abundance proteins is enhanced, and changes in subcellular distributio n of proteins can also be monitored. Detergent fractionation is a simp ler alternative to differential pelleting, which partitions cellular c onstituents into functionally distinct populations while preserving cy toskeletal integrity. We defined and characterized a differential dete rgent fractionation (DDF) protocol to enable protein dynamics in cytos keletal and noncytoskeletal compartments of isolated hepatocytes to be monitored simultaneously. Rat hepatocytes were maintained in suspensi on culture and fractionated by sequential extraction with detergent-co ntaining buffers (digitonin/EDTA, Triton/EDTA, Tween/deoxycholate). DD F reproducibly yielded four electrophoretically distinct fractions enr iched in cytosolic, membrane-organelle, nuclear membrane and cytoskele tal-matrix markers, respectively. Immunoblotting with over 20 differen t antibodies corroborated the selectivity of fractionation and was use d to characterize the distribution profiles of cytoskeletal (actin, tu bulins, cytokeratins, vinculin, myosin, desmoplakins, fodrin, nuclear lamins) and noncytoskeletal proteins (heat-shock 70 proteins, glutathi one-S-transferase, calpains, carbamoyl phosphate synthetase, etc.), as well as to identify spots in 2-D gels. Detergent buffers were compati ble with equilibrium or nonequilibrium 2-D gel electrophoretic analysi s. Extensive 2-D maps of acidic and basic proteins in each fraction we re generated along with a tabular listing of M(r) and pI. Thus, DDF re producibly partitions hepatocytic proteins into functionally distinct cytoskeletal and noncytoskeletal compartments that are readily analyze d by 2-D gel electrophoresis. DDF is simple, applicable to use with ot her cell types or culture systems and is especially useful when biomat erial is limited (i.e., clinical studies).