Insulin processing and action in adipocytes: evidence for generation of insulin-containing vesicles by leupeptin and monensin

AbstractIncubation of adipocytes with 125I-insulin plus leupeptin or monensin, but not chloroquine, resulted in the appearance of a novel peak of 125I-insulin (modal density about 1.20 gml) on density gradient centrifugation; the appearance of the peak depended on the presence of specific insulin receptors on the cell surface. The fractions comprising this peak contained vesicles, probably originating from the Golgi apparatus, and dit not appear to be contaminated with lysosomes, mitochondria or plasma membrane. Entrapment of insulin in these vesicles per se did not prevent the activation of glucose transport, acetyl-CoA carboxylase or pyruvate dehydrogenase by insulin

Degradation of nuclear proteins: studies on transplanted B82 cell karyoplast proteins

AbstractKaryoplasts were prepared from B82 cells (thymidine kinase deficient mouse L cells) by cytochalasin B mediated enucleation. Morphological measurements show that the nucleus constitutes 89% of a karyoplast by volume. Homokaryons were obtained by Sendai virus mediated karyoplast-B82 cell fusion. Transplanted nuclei were not destroyed catastrophically but were maintained intracellularly for at least 140 h. Transplanted nuclear proteins were degraded with an average half-life of 84 ± 7 h by processes partially sensitive to inhibition by NH4Cl (50%) and leupeptin (30%). The data therefore suggest that some nuclear proteins are translocated to the cytoplasm for lysosomal degradation

Ubiquitin—protein conjugates and αB crystallin are selectively present in cells undergoing major cytomorphological reorganisation in early chicken embryos

AbstractUbiquitin—protein conjugates and αB crystallin are detected immunohistochemically in cells undergoing extensive morphological reorganisation in early chicken embryos. Cytoplasmic ubiquitinated proteins and αB crystallin are coordinately found in cells of the lens, notochord and myotome. The antigens appear in the myotome cells precisely, at the point at which the cells begin to migrate from the dorsomedial lip of the dermamyotome. The findings indicate that ubiquitin and αB crystallin may have a coordinate role in the extensive architectural remodeling which occurs in these developing tissues in the early chick embryo, Some form of functional association between protein ubiquitination and αB crystallin in cells may explain why αB crystallin is found with ubiquitin—protein deposits in some neurodegenerative diseases