CHANGES IN PROTEIN SYNTHESIS PROFILES OF MEGAKARYOCYTES (MK) DURING MATURATION

Abstract

Several key differentiation events occur within the recognizable MK compartment; however, little is known about the macromolecular changes responsible for these events. In this study, protein synthesis profiles of morphologically immature and mature guinea pig MK populations have been analyzed by twodimensional gel electrophoresis after in vivo labeling with 35S-methionine. MK were enriched by a bovine plasma aggregation enrichment procedure (Blood 69:173, 1987) and then fractionated into immature and mature populations based on differences in their respective buoyant densities (Brit. J. Haematol. 64:33, 1986). With this protocol, immature and mature MK populations were obtained in which MK constituted 95% of the cell mass. Ninety percent of the MK in the immature population had basophilic, immature morphology while ≥90% of those in the mature population had acidophilic, mature staining characteristics after Wright's staining. Protease inhibitors were used throughout the isolation procedure. The cells were solubilized and proteins subjected to two-dimensional electrophoresis according to O'Farrell (J. Biol. Chem. 250:4007, 1975). To examine basic proteins, proteins were electrophoresed in the first dimension under nonequilibrium conditions in a pH gradient as described by O'Farrell et al. (Cell 12:1133, 1977). Analyses of fluorograms revealed both qualitative and quantitative differences in synthesis profiles between these two MK populations. Among acidic proteins whose synthesis was readily detected in immature but not mature MK were ones whose MW and pi were respectively: 120K, 6.4; 7OK, 5.9; 70K, 6.9; 65K, 6.8; 55K, 6.2; 55K, 6.0; 53K, 5.8; 53K, 6.5; 52K, 6.7; 50K, 6.8; 41K, 5.5 and 33K, 6.7. Acidic and neutral proteins prominently synthesized in mature but not immature MK were found at MW and PI of: 110K, 5.7; 110K, 5.8 and 80K, 7.2. Basic proteins prominently synthesized in immature but not mature MK were found at MWs of: 110K; 70K; 52K; 48K; 39K and 18K. Basic proteins actively synthesized by mature but not immature MK had MWs of: 83K; 43K and 17K. These findings demonstrate that differences in protein synthesis patterns can be readily detected between immature and mature MK and provide baseline data with which to explore the role of these proteins in MK differentiation