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Preparations of β-adrenergic receptor and Gs from turkey erythrocytes were delipidated by previously developed procedures. Three synthetic phospholipids, dioteoylglycerophosphoethanolamine, dioleoylglycerophosphocholine and dioleoylglycerophosphoserine plus an unphosphorylated lipid, were all required to restore receptor-mediated activation of Gs by GTP[γS]. The same lipids were necessary for the reconstitution of the isoproterenol-enhanced GTPase. The requirement for the unphosphorylated lipid could be fulfilled by l-monooleoyl glycerol, α-tocopherol or oleic acid. Cholesterol hemisuccinate further enhanced the receptor-mediated activity of the relipidated system when present in addition to the lipids specified above. Cholesterol hemisuccinate had no effect on the basal rate of Gs activation and depressed the basal GTPase. It is therefore suggested that cholesterol hemisuccinate affects the receptor or the coupling of the receptor to Gs.
In the system relipidated with the three dioleoyl phospholipids, plus α-tocopherol and cholesterol hemisuccinate, the initial rate of Gs activation per mole receptor appeared to be considerably higher than in the native turkey erythrocyte membrane. [ABSTRACT FROM AUTHOR]

Detergent-solubilized preparations of the β-adrenergic receptor (R) and of the guanyl nucleotide binding proteins (Gs) were extensively treated to remove phospholipids and cholesterol. Reconstitution of an R-Gs system was subsequently performed in the presence of a mixture of natural phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine or the synthetic dioleoyl derivatives of the same phospholipids. In both cases, an additional lipid was required for the agonist-dependent activation of Gs. The requirement could be fulfilled by α-tocopherol, or by unsaturated fatty acids such as oleic acid. Inclusion of this non-phosphorylated lipid in the reconstituted system enhanced the isoproterenol-dependent activation of Gs by guanosine 5'-O-[γ-thio]triphosphate 16-33-fold. The rate of activation was largely dependent on the addition of the agonist. Efficient functional reconstitution of R-Gs was thus achieved in a totally defined lipid system. Additional studies of the reconstituted system and of the native membrane led to the notion that the non-phosphorylated lipid plays a role in the function of the hormone-R complex. [ABSTRACT FROM AUTHOR]

Rat parotid gland slices showed a very slow rate of glucose utilization and lactic acid production, both in the presence and in the absence of oxygen. Epinephrine, which caused rapid enzyme secretion by the slices did not affect the low rate of glycolysis. A rough calculation indicates that in the parotid gland ATP is produced by oxidative phosphorylation 50 times faster than by glycolysis. The mitochondria, when isolated and tested in presence of EDTA or EGTA showed respiratory activity and respiratory control comparable to those of liver mitochondria. Adenosine 3':5'-cyclic phosphate, which is an intermediate in induction of enzyme secretion, showed no clear-cut effect on the functions of the parotid mitochondria. Isolation of the mitochondria in the absence of the above chelators yielded a preparation which was almost depleted of NAD, respired feebly on NAD linked substrates and showed no respiratory control. Addition of NAD+ restored respiration. The damage caused to these mitochondria during isolation was attributed to the high calcium concentrations found in the gland. Addition of small amounts of free calcium to functional parotid mitochondria indeed caused the loss of NAD and the decline of respiration. Tested in parallel experiments, liver mitochondria were much more resistant to added calcium. Their initial calcium content was one tenth that of the parotid mitochondria. The control of calcium distribution in the parotid gland and the possible functions of calcium are discussed. [ABSTRACT FROM AUTHOR]