The activity of dopamine-stimulated adenylate cyclase from rat brain striatum is modulated by temperature and the bilayer-fluidizing agent, benzyl alcohol

Benzyl alcohol achieved a marked activation of the adenylate cyclase activity in a partially purified membrane preparation from rat brain striata, although inhibition resulted at high concentrations. The degree of activation observed depended on the ligand used to stimulate the enzyme, with that observed in the presence of guanosine 5′-[β,γ-imido]triphosphate (p[NH]ppG) (5.8-fold)>dopamine+p[NH]ppG (5-fold)> GTP (3-fold)>dopamine+high GTP (2.25-fold)>dopamine (+low GTP)=basal (+low GTP) (1.7-fold). The differences in the concentration-dependence of both the activation and inhibition of dopamine-stimulated and basal activities of the enzyme meant that increasing benzyl alcohol concentrations caused a net elevation in the fold-stimulation of the basal activity by dopamine. Arrhenius plots of p[NH]ppG-, GTP-, fluoride-, dopamine-plus-high GTP- and dopamine-plus-p[NH]ppG-stimulated activities all exhibited a single break occurring at around 22°C. This break point was decreased to around 13°C when 50mm-benzyl alcohol was added to the assays. In the presence of dopamine (+low GTP), Arrhenius plots exhibited two distinct breaks, one at around 21°C and the other at around 11°C. When benzyl alcohol (50mm) was added to these assays of dopamine (+low GTP)-stimulated activity, a single break at around 14°C was observed. For the basal activity the Arrhenius plot exhibited a single break at around 15°C both in the presence and in the absence of 50mm-benzyl alcohol. It is suggested that the enzyme is activated by productive collisions between independent mobile entities and that the activity of the enzyme may be regulated by changes in membrane fluidity. The breaks in the Arrhenius plots of all of the ligand-stimulated activities, but not the basal activity, are attributed to lipid-phase separations occurring in either the inner or the outer halves of the bilayer.

Download Full-text

The activity of glucagon-stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its lipid environment

Biochemical Journal ◽

10.1042/bj1740179 ◽

1978 ◽

Vol 174 (1) ◽

pp. 179-190 ◽

Cited By ~ 114

Author(s):

I Dipple ◽

M D Houslay

Keyword(s):

Adenylate Cyclase ◽

Benzyl Alcohol ◽

Plasma Membranes ◽

Break Point ◽

Alcohol Concentration ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Lipid Phase ◽

Glucagon Receptor ◽

Arrhenius Plots

1. The local anaesthetic benzyl alcohol progressively activated glucagon-stimulated adenylate cyclase activity up to a maximum at 50 mM-benzyl alcohol. Further increases in benzyl alcohol concentration inhibited the activity. The fluoride-stimulated adenylate cyclase activity was similarly affected except for an inhibition of activity occurring at low benzyl alcohol concentrations (approx. 10 mM. 2. The fluoride-stimulated adenylate cyclase activity of a solubilized enzyme preparation was unaffected by any of the benzyl alcohol concentrations tested. 3. Increases in 3-phenylpropan-1-ol and 5-phenylpentan-1-ol concentrations progressively activated both the fluoride- and glucagon-stimulated adenylate cyclase activities up to a maximum, above which further increases in alcohol concentration inhibited the activities. 4. The ‘break’ points in Arrhenius plots of glucagon-stimulated adenylate cyclase activity in native plasma membranes, and in plasma membranes fused with synthetic dimyristoyl phosphatidylcholine so as to constitute 60% of the total lipid pool, were decreased by approx. 6 degrees C by addition of 40 mM-benzyl alcohol. This was accompanied by a fall in the associated activation energies. 6. Arrhenius plots of fluoride-stimulated adenylate cyclase activity in the presence and absence of 40 mM-benzyl alcohol were linear, although addition of benzyl alcohol caused a dramatic decrease in the associated activation energy of the reaction. 7. 5′-Nucleotidase activity was stimulated by benzyl alcohol, and the ‘break’ point in the Arrhenius plot of its activity was decreased by about 6 degrees C by addition of 40 mM-benzyl alcohol to the assay. 8. It is suggested that benzyl alcohol effects a fluidization of the bilayer, which is clearly demonstrated by its ability to lower the temperature of a lipid phase separation occurring at 28 degrees C in the outer half of the bilayer to around 22 degrees C. The increase in bilayer fluidity relieves a physical constraint on the membrane-bound adenylate cyclase, activating the enzyme. 9. The various inhibition phenomena are discussed in detail, together with the suggestion that the interaction between the uncoupled catalytic unit of adenylate cyclase and the lipids of the bilayer is altered on its physical coupling to the glucagon receptor.

Download Full-text

Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma-membrane enzymes occurring on hibernation

Biochemical Journal ◽

10.1042/bj1740909 ◽

1978 ◽

Vol 174 (3) ◽

pp. 909-919 ◽

Cited By ~ 76

Author(s):

M D Houslay ◽

R W Palmer

Keyword(s):

Adenylate Cyclase ◽

Adenosine Triphosphatase ◽

Plasma Membranes ◽

Lipid Phase ◽

Arrhenius Plots ◽

Liver Plasma Membranes ◽

Break Points ◽

Lipid Phase Separation ◽

Outer Half ◽

Single Break

1. Arrhenius plots of the glucagon-stimulated adenylate cyclase, 5′-nucleotidase, (Na+ + K+)-stimulated adenosine triphosphatase and Mg2+-dependent adenosine triphosphatase activities of control hamster liver plasma membranes exhibited two break points at around 25 and 13 degrees C, whereas Arrhenius plots of their activities in hibernating hamster liver plasma membranes exhibited two break points at around 25 and 4 degrees C. 2. A single break occurring between 25 and 26 degrees C was observed in Arrhenius plots of the activities of fluoride-stimulated adenylate cyclase, basal adenylate cyclase and cyclic AMP phosphodiesterase of liver plasma membranes from both control and hibernating animals. 3. Arrhenius plots of phosphodiesterase I activity showed a single break at 13 degrees C for membranes from control animals, and a single break at around 4 degrees C for liver plasma membranes from hibernating animals. 4. The temperature at which break points occurred in Arrhenius plots of glucagon- and fluoride-stimulated adenylate cyclase activity were decreased by about 7–8 degrees C by addition of 40 mm-benzyl alcohol to the assays. 5. Discontinuities in the Arrhenius plots of 4-anilinonaphthalene-1-sulphonic acid fluorescence occurred at around 24 and 13 degrees C for liver plasma membranes from control animals, and at around 25 and 4 degrees C for membranes from hibernating animals. 6. We suggest that in hamster liver plasma membranes from control animals a lipid phase separation occurs at around 25 degrees C in the inner half of the bilayer and at around 13 degrees C in the outer half of the bilayer. On hibernation a change in bilayer asymmetry occurs, which is expressed by a decrease in the temperature at which the lipid phase separation occurs in the outer half of the bilayer to around 4 degrees C. The assumption made is that enzymes expressing both lipid phase separations penetrate both halves of the bilayer, whereas those experiencing a single break penetrate one half of the bilayer only.

Download Full-text

Phenobarbital selectively modulates the glucagon-stimulated activity of adenylate cyclase by depressing the lipid phase separation occurring in the outer half of the bilayer of liver plasma membranes

Biochemical Journal ◽

10.1042/bj1970675 ◽

1981 ◽

Vol 197 (3) ◽

pp. 675-681 ◽

Cited By ~ 48

Author(s):

M D Houslay ◽

I Dipple ◽

L M Gordon

Keyword(s):

Adenylate Cyclase ◽

Spin Probe ◽

Plasma Membranes ◽

Chemical Constituents ◽

Lipid Phase ◽

Lipid Phase Transition ◽

Arrhenius Plots ◽

Lipid Environment ◽

Lipid Phase Separation ◽

Outer Half

The glucagon-stimulated (coupled) activity of rat liver plasma-membrane adenylate cyclase could be selectively modulated by the anionic drug phenobarbital, whereas the fluoride-stimulated (uncoupled) activity remained unaffected. It is suggested that the cationic drug phenobarbital preferentially interacts with the external half of the bilayer, as the negatively charged phospholipids are found at the cytosol-facing side. This results in a selective fluidization of the external half of the bilayer, leading to a depression in the high-temperature onset of the lipid phase transition (from 28 degree to 16 degree C) occurring there. This was detected both by e.s.r. analysis, using a fatty acid spin probe, and also by Arrhenius plots of glucagon-stimulated activity, where the enzyme forms a transmembrane complex with the receptor and is sensitive to the lipid environment of both halves of the bilayer. However, in the absence of hormone, adenylate cyclase only senses the lipid environment of the inner (cytosol) half of the bilayer. Thus its fluoride stimulated activity and Arrhenius plots of this activity remained unaffected by the presence of phenobarbital (less than 12 mM) in the assay. These results support the view that independent modulation of the fluidity or chemical constituents of each half of the bilayer can selectively affect the receptor-coupled and uncoupled activities of adenylate cyclase.

Download Full-text

The role of Gi and the membrane-fluidizing agent benzyl alcohol in modulating the hysteretic activation of human platelet adenylate cyclase by guanylyl 5′-imidodiphosphate

Biochemical Journal ◽

10.1042/bj2910945 ◽

1993 ◽

Vol 291 (3) ◽

pp. 945-949 ◽

Cited By ~ 6

Author(s):

S Spence ◽

M D Houslay

Keyword(s):

Steady State ◽

Adenylate Cyclase ◽

Benzyl Alcohol ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Steady State Rate ◽

Fluidizing Agent ◽

State Rate ◽

Lag Period ◽

Pre Treatment

The non-hydrolysable GTP analogue guanylyl 5′-imidodiphosphate (p[NH]ppG) elicited a profound increase in the adenylate cyclase activity of human platelets. This occurred after a well-defined lag period of around 6 min, whereupon an enhanced steady-state rate was evident. The duration of the lag period was unchanged over a range of concentrations of p[NH]ppG which gave very different steady-state rates of adenylate cyclase activity. Prior activation of the stimulatory G-protein Gs by cholera-toxin pre-treatment abolished the lag period and elicited a small increase in the steady-state rate. Manipulating function of the inhibitory G-protein Gi also led to profound changes in the lag periods. Thus marked decreases in the lag were seen (approximately 70-81%) when Gi function was ablated through pre-treatment of platelet membranes with pertussis toxin, or by using elevated (25 mM) Mg2+ levels in the assay, or when Mg2+ was replaced by 5 mM Mn2+ in the assay. In contrast with this, potentiation of Gi function led to an increase in the lag period, as seen under conditions of agonist occupancy of inhibitory alpha 2-adrenoceptors (increase approximately 74%) or with the addition of 100 mM NaCl to the assays (increase approximately 44%). The local anaesthetic and membrane-fluidizing agent benzyl alcohol elicited both a profound decrease (around 70% at 80 mM) in the p[NH]ppG-induced lag period and a marked augmentation (around 5-fold) in the steady-state adenylate cyclase activity. When adenylate cyclase assays were done at 35 degrees C instead of 25 degrees C, then the lag period for activation by p[NH]ppG was decreased by around 33% and the steady-state rate increased by around 3-fold. At 35 degrees C, the addition of benzyl alcohol led to the apparent abolition of the lag period for p[NH]ppG activation of adenylate cyclase and amplified the steady-state rate by only around 2.2-fold. It is shown that Gi plays a fundamental role in determining the rate of activation of Gs. The proposal is formulated that such an action may be mediated through the release of beta gamma-subunits. Thus beta gamma-subunit dissociation is proposed as providing the rate-limiting step in Gi activation.

Download Full-text