B. Richard Martin

Activation of rat liver plasma membrane adenylate cyclase by a cytoplasmic protein factor.

Studies of adenylate cyclase have tended to concentrate upon the mechanism of action of polypeptide hormones. These act by binding to a protein receptor on the outer surface of the plasma membrane. The hormone-receptor complex which is formed interacts with a catalytic unit of adenylate cyclase on the inner surface of the plasma membrane and modulates its activity [l]. There has been less interest in the possibility that cytoplasmic factors acting on the inner surface of the plasma membrane may also be important in regulating the activity of adenylate cyclase but there have been a number of observations which suggest that this may be the case. GTP and its analogue GMP-PNP have been shown to affect the activity of adenylate cyclase and to modulate the response of the enzyme to hormones [2] and it is likely that GTP exerts its effect on the inner surface of the cell membrane. The precise physiological function of this response is obscure since cytoplasmic concentrations of GTP are well in excess of the concentrations needed to produce a maximal response of adenylate cyclase activity. Ho et al. [3] have shown that fat cells produce a factor in response to adrenaline that act as an antagonist of the hormone’s lipolytic effect and have suggested that this factor is produced inside the cell and acts as a feed back inhibitor of adenylate cyclase. Low an Werner [4] have suggested that adenylate cyclase activity may be modulated by the redox state of the cell. Gill and King [5] have suggested that a cytoplasmic factor may be involved in the action of cholera toxin in stimulating adenylate cyclase.

Inhibition of PI-kinase in rat liver membranes by F−

In a number of membrane preparations GTP or its non-hydrolysable analogues stimulate the breakdown of PIP2 generating the second messengers, inositol triphosphate and diacylglycerol. The G-protein which couples the PIP2-specific phospholipase C with the receptors can also be activated by F-. However, the level of PIP2 is dependent upon the activity of a number of enzymes in the PI-pathway. Besides stimulating the breakdown of PIP2, we report that in rat liver membranes F- also decreases the labelling of the polyphosphoinositides through inhibition of the PI-kinase.

The Analysis of Interactions between Hormone Receptors and Adenylate Cyclase by Target Size Determinations Using Irradiation Inactivation

Publisher Summary The irradiation inactivation is a method of protein size determination in which a protein molecule being hit will depend upon its size and, accordingly, upon the molecular weight. There is no requirement for the protein to be pure or for the protein to be in solution. All that is required is a measure of the activity of the protein under study. Thus, the method offers the opportunity to examine the size of proteins in intact biological membranes. Where multisubunit enzymes are examined by irradiation inactivation the target size sometimes corresponds to the whole enzyme, and sometimes corresponds to the monomer molecular weight. There are two types of approach to the study of protein-protein interaction by target size analysis. In the first case, the preparation is irradiated in the absence of any effectors or in the basal state. The activity under study, for example, the catalytic activity of adenylate cyclase, is then determined in the usual way, and the target size for the basal state can be calculated. The activity can also be determined in the presence of an activator such as a hormone. In the second type of general approach, the membranes are preincubated with the effector prior to irradiation. In this case, the preincubations should, so far as possible, be identical to the conditions which are normally employed in the assay of the activity which is under study, although minor modifications may be necessary.

Insulin alters the target size of the peripheral cyclic AMP phosphodiesterase but not the integral cyclic GMP-stimulated cyclic AMP phosphodiesterase in liver plasma membranes

Radiation inactivation of the two high affinity cyclic AMP phosphodiesterases (PDE) found in liver plasma membranes afforded an estimation of their molecular target sizes in situ. The activity of the peripheral plasma membrane PDE decayed as a single exponential with a target size corresponding to a monomer of circa 54 kDa. The integral, cyclic GMP-stimulated PDE decayed as a dimer of circa 125 kDa. Preincubation of plasma membranes with insulin (10nM), prior to irradiation, caused the target size of only the peripheral plasma membrane PDE to increase. We suggest that insulin addition causes the peripheral plasma membrane PDE to alter its coupling to an integral plasma membrane protein with a target size of circa 90 kDa.

Effect of Insulin on the Membrane Potentials of Rat White Epididymal Fat Cells

Insulin produces a number of rapid changes in the membrane transport processes of fat cells. Examples are the stimulation of the transport of glucose,(1) amino acids,(2) potassium,(3,4) calcium,(5) and phosphate.(6) It is possible that the changes in the ionic balance caused by insulin may play a role in mediating or maintaining some of the effects of insulin. An understanding of ionic transport by mechanisms that are electrogenic or electrophoretic requires a knowledge of the electrical potential across the plasma membrane. We have therefore studied the electrical potentials across the plasma membrane as well as the organelle membranes of fat cells stimulated by insulin.