Exchange of partners in glucagon receptor-adenylate cyclase complexes. Physical evidence for the independent,mobile receptor model

The apparent target sizes of the glucagon receptor and the catalytic unit of adenylate cyclase in rat liver plasma membranes have been measured by the technique of radiation inactivation in an electron beam. When irradiated in the uncoupled state, the apparent target size for the catalytic unit assayed by fluoride-stimulated activity was 160 000, and for the receptor assayed by specific ¹²⁵I-labelled glucagon binding was 217 000. The corresponding target size estimated from glucagon-stimulated activity after irradiation in the uncoupled state was 389 000. When the complexes were irradiated in the coupled state in the presence of glucagon, the apparent target sizes from ¹²⁵I-labelled glucagon binding, and fluoride- or glucagon-stimulated activities had similar values of 310 000, 380 000 and 421 000, respectively. However, if the complexes were allowed to uncouple by removing glucagon after irradiation and activity was then assayed after readdition of glucagon, the apparent target size from the glucagon-stimulated activity increases from 421 000 to 811 000.The pattern of apparent target sizes obtained under these different conditions has been tested against the pattern predicted for simple models of the coupling mechanism. The only simple model that is consistent with the pattern of target sizes requires the receptors and catalytic units to be present in approximately equal numbers. On binding glucagon, the receptor forms a locking interaction with the catalytic units, so that the complex and its components are inactivated as a single target with an apparent size of about 380 000 ($\text{± 15\%}$). After the removal and readdition of glucagon to complexes that were irradiated in the coupled state, the new population of complexes must contain hybrids of active and inactive partners obtained by exchange between active and inactivated complexes, to account for the doubling in apparent target size to 811 000 for glucagon-stimulated activity. This hybridization of catalytic units and receptors is the essential feature of the model that distinguishes it from others in which permanently associated complexes of the two components are activated by lateral dimersation on binding glucagon. Simple models of this type are shown to be physically improbable. It is emphasized that the models described are based only on the relationships between the apparent target sizes of components that are defined by their functions, and the apparent target sizes do not necessarily relate solely to the components that can be defined structurally as the receptor or catalytic unit.     

Transference of the high molecular weight carbohydrate sequences of fetuin to the surface of carrot embryo protoplasts

A method is described for the removal of the carbohydrate sequences of glycoproteins, and their covalent attachment to hydrocarbon chains. These synthetic membrane components may then be incorporated into liposome and cell membranes. Pronase-liberated glycopeptides derived from fetuin were linked by a reduced Schiff's base linkage to tetradecyl aldehyde. The resulting glycolipid was incorporated by external addition, into phosphatidylcholine liposomes. Glycolipid transfer to these liposomes rendered them suseptible to agglutination by wheat germ lectin, which binds N-acetylneuraminic acid, the terminal carbohydrate of the high molecular weight fetuin sugar sequence. Sequential removal of the terminal sugars, and subsequent agglutination behaviour towards various lectins, suggests that the carbohydrate sequence had been transfered intact. The glycolipid was incorporated into plant protoplast membranes by incubation with glycolipid-containing liposomes for 2 h at 37°C. These synthetic glycolipids may find a use in the study of carbohydrate-based recognition systems in animal and plant membranes. In addition they may prove useful in the development of cell and membrane tagging and handling techniques, by the insertion of sugar groups not normally present in these membranes.     

Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells

We have utilized epithelial rat kidney cells and their Kirsten viral transformant (442) to examine the role of actin-binding proteins in cellular morphogenesis. Normal rat kidney cells are well spread while the transformed cells are more spherical, poorly adherent, and lack actin stress fibers (Rubin, R.W., Warren, R.H., Lukeman, D.S. and Clements, E. (1978) J. Cell Biol. 78, 28-35). By immunofluorescence, antitropomyosin prominently stains normal rat kidney cell stress fibers while only a weak, nonspecific fluorescence is observed in 442 cells. Using two-dimensional gel electrophoresis, tropomyosin can be detected in normal rat kidney cells homogenates. The tropomyosin subunits are enriched in Triton-extracted filamentous normal rat kidney cell models, and in extracts of normal rat kidney cell homogenate produced by using a rapid myosin affinity technique to isolate actin and actin-associated proteins. The identity of the tropomyosin subunits has been confirmed by electrophoretic mobility, lack of proline, and the peptide map generated by limited proteolysis. None of these techniques have detected tropomyosin in the corresponding 442 preparations. Our results suggest that the transformation of normal rat kidney cells has led to an overall reduction in tropomyosin content. This may be related to the inability of 442 cells to organize filamentous actin stress fibers.     

The Aedes aegypti genome: complexity and organization.

We describe the use of DNA reassociation kinetics to determine the total genome size and complexity together with the individual complexity and copy number of the single copy, middle repetitive and highly repeated DNA fractions of cell line and larval DNA from the mosquito, Aedes aegypti. The genome of Ae. aegypti is both large and complex, being one third the size of the human genome, and exhibits a short period interspersed repeat pattern. The implications of patterns of sequence arrangement and genome complexities for experiments aimed at isolating specific classes of DNA sequences, such as mobile genetic elements, are discussed.