Janet M. Stein

The effect of insulin on 32P1 incorporation into rat fat cell phospholipids

Abstract 1. 1. The effect of insulin on the incorporation of 32Pi into the phospholipids of rat fat cells incubated in glucose-free medium has been investigated. 2. 2. The incorporation of 32Pi into fat cell phospholipid increased with time, and incubation of fat cells with insulin resulted in increased incorporation of 32Pi into phospholipid compared with controls. 3. 3. Fat cells incubated with insulin for I h showed increased specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and cardiolipin compared with controls. 4. 4. ATP concentrations in fat cells were determined by the luciferase assay. Insulin incubated for 1 h with fat cells produced no significant change in ATP concentrations compared with controls. Glucose incubated for I h with fat cells either with insulin or without insulin did not result in any change in ATP concentration compared with incubated controls. 5. 5. The effect of insulin on the incorporation of 32Pi into ATP was determined and the specific radioactivity of ATP calculated. Incubation of fat cells with insulin resulted in an increase in the specific radioactivity of ATP compared with ATP in control cells, and the increase was time-dependent.

Effects of GTP,GDP[βS] and glucose on adenylate cyclase activity of E. coli B

Adenylate cyclase activity was measured in suspensions of E. coli B, rendered permeable with toluene. The enzyme was activated in a dose‐dependent manner by GTP and by its non‐hydrolysable analogue, GTP[γS]. In contrast, incubation with GDP[βS], a non‐phosphorylatable analogue of GDP, caused a dose‐related inhibition of adenylate cyclase; this was partially overcome by addition of GTP. GTP did not relieve, and GDP[βS] augmented, the non‐competitive and dose‐related inhibition of E.coli adenylate cyclase by glucose.

The effect of carbacyclin, a prostaglandin analogue, on adenylate cyclase activity in platelet membranes

The effect of carbacyclin, a chemically stable analogue of prostacyclin, on the activity of adenylate cyclase in platelet membrane was measured, and compared with the effect of PGE1. When GTP was added in concentrations up to 10 μM the activation of adenylate cyclase by carbacyclin was increased, whereas higher concentrations of GTP were inhibitory. The addition of a non‐hydrolysable analogue of GDP, guanosine 5′‐[β‐thio]diphosphate (GDP[βS]) resulted in a dose‐dependent inhibition of adenylate cyclase activation by carbacyclin; this inhibition was relieved by adding increased amounts of GTP.

The effect of adrenaline on synthesis of phosphatidylcholine in fat cells

Abstract (1) Isolated fat cells from rat epididymal fat pad when incubated in vitro in medium containing 32 P i , showed a time-dependent increase in specific radioactivity of microsomal phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol; incubation of fat cells in the presence of adrenaline resulted in an increased specific radioactivity only of phosphatidylcholine compared with phospholipids in incubated control cells. (2) Investigation of the incorporation of 32 P i into the water-soluble precursors of phosphatidylcholine showed that the increase in radioactivity of phosphorylcholine was time-dependent, and was similar in both control and adrenaline-stimulated fat cells. Incorporation of 32 P i into CDP-choline was less than into phosphorylcholine, and was markedly increased in fat cells incubated with adrenaline when compared with controls. (3) The concentration of phosphorylcholine in fat cells has been determined. (4) The fractional turnover rate of phosphatidylcholine has been calculated, and was found to be increased in adrenaline-stimulated cells when compared with incubated controls.

L-antigen and active potassium transport in HK and LK red cells of barbary sheep (Ammotragus lervia)

1. The potassium concentration in red cells of 21 Barbary sheep showed a bimodal distribution, with five animals of LK type (K+ conc. 30-45 mM) and 16 of HK type (K+ conc. 80-95 mM). 2. Evidence is presented that both Lp and Ll antigens are present on LK Barbary sheep red cells. 3. Active K+ transport in LK Barbary sheep red cells was stimulated 3-5 fold by sheep and goat anti-L. 4. Active K+ transport in HK Barbary sheep red cells was higher than in LK red cells. Five out of six HK animals tested showed no stimulation of active K+ transport with anti-L. One HK animal (2BA2) showed some stimulation of active K+ transport, and also absorbed some anti-L from antisera, suggesting that Lp antigen is present on these red cells. 5. Ouabain-sensitive ATPase in membranes from HK and LK Barbary sheep red cells showed kinetics characteristic of HK and LK membranes of domestic goats and sheep; the ATPase of LK Barbary sheep membranes sensitized with anti-L was stimulated 2-fold due to an alteration in the internal sodium and potassium affinities in favour of sodium.