scholarly journals Demonstration of inhibitory guanine nucleotide regulatory protein (Gi) function in liver and hepatocyte membranes from streptozotocin-treated rats

1992 ◽  
Vol 284 (2) ◽  
pp. 301-304 ◽  
Author(s):  
D M Kirkham ◽  
G J Murphy ◽  
P Young
Keyword(s):  
Plasma Membrane ◽  
Regulatory Protein ◽  
Diabetic Animal ◽  
Membrane Preparation ◽  
Pathological State ◽  
Guanine Nucleotide ◽  
Functional Inhibition ◽  
Inhibitory G Protein ◽  
Guanine Nucleotide Regulatory Protein ◽  
Plasma Membrane Preparation

By using a defined plasma-membrane preparation, functional inhibition of adenylate cyclase activity by the inhibitory G-protein (Gi) was observed in liver and hepatocyte membranes from rats made diabetic by streptozotocin. These observations contrast with previous reports which have shown a defect in Gi in this diabetic animal model. These results suggest that Gi function is not impaired in the livers of streptozotocin-treated rats and that plasma-membrane preparation procedures should be clearly defined before ascribing Gi defects to a pathological state such as diabetes.

scholarly journals The action of islet activating protein (pertussis toxin) on insulin's ability to inhibit adenylate cyclase and activate cyclic AMP phosphodiesterases in hepatocytes

1986 ◽  
Vol 235 (1) ◽  
pp. 145-149 ◽  
Author(s):  
C M Heyworth ◽  
A M Grey ◽  
S R Wilson ◽  
E Hanski ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pertussis Toxin ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Guanine Nucleotide ◽  
Cyclic Amp Phosphodiesterase ◽  
Peripheral Plasma ◽  
Guanine Nucleotide Regulatory Protein

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the ‘dense-vesicle’ cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and ‘dense-vesicle’ cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes.

scholarly journals A rapid immunological procedure for the isolation of hormonally sensitive rat fat-cell plasma membrane

1976 ◽  
Vol 154 (1) ◽  
pp. 11-21 ◽  
Author(s):  
J P Luzio ◽  
A C Newby ◽  
C N Hales
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Membrane Preparation ◽  
Fat Cells ◽  
Fat Cell ◽  
Cell Plasma ◽  
Rat Fat Cells ◽  
Plasma Membrane Preparation ◽  
Isolated Rat

1. A rapid method for the isolation of hormonally sensitive rat fat-cell plasma membranes was developed by using immunological techniques. 2. Rabbit anti-(rat erythrocyte) sera were raised and shown to cross-react with isolated rat fat-cells. 3. Isolated rat fat-cells were coated with rabbit anti-(rat erythrocyte) antibodies, homogenized and the homogenate made to react with an immunoadsorbent prepared by covalently coupling donkey anti-(rabbit globulin) antibodies to aminocellulose. Uptake of plasma membrane on to the immunoadsorbent was monitored by assaying the enzymes adenylate cyclase and 5′-nucleotidase and an immunological marker consisting of a 125I-labelled anti-(immunoglobulin G)-anti-cell antibody complex bound to the cells before fractionation. Contamination of the plasma-membrane preparation by other subcellular fractions was also investigated. 4. By using this technique, a method was developed allowing 25-40% recovery of plasma membrane from fat-cell homogenates within 30 min of homogenization. 5. Adenylate cyclase in the isolated plasma-membrane preparation was stimulated by 5 μm-adrenaline.

Effects of cations on binding of human choriogonadotropin

1988 ◽  
Vol 66 (12) ◽  
pp. 1258-1264
Author(s):  
Patrick J. McIlroy
Keyword(s):  
Binding Sites ◽  
Regulatory Protein ◽  
Guanine Nucleotides ◽  
Guanine Nucleotide ◽  
Human Choriogonadotropin ◽  
Receptor Sites ◽  
Number Of Binding Sites ◽  
Receptor Number ◽  
Guanine Nucleotide Regulatory Protein ◽  
Low Ionic Strength

The effect of various salts on the binding of human choriogonadotropin to rat luteal membranes has been examined. Increasing salt concentrations had biphasic effects, initially increasing binding, then decreasing it. With NaCl, these effects were on both the affinity and the number of receptor sites. The affinity increased with increasing NaCl concentrations, to a maximum at 40 mM, and then decreased. Above 40 mM NaCl, the number of binding sites increased. NaCl also altered the effects of Mg2+ and guanyl nucleotides. At low ionic strength, Mg2+ was necessary to observe binding. Guanine nucleotides modulated this binding by decreasing the affinity. At 40 mM NaCl, Mg2+ increased receptor number without altering affinity. Guanyl nucleotides modulated this binding by reducing the number of sites to that observed in the absence of Mg2+. At 150 mM NaCl, Mg2+ and guanine nucleotides had no effect. The results suggest the presence of two pools of human choriogonadotropin receptor in rat corpus luteum, one coupled to the guanine nucleotide regulatory protein (Ns) and being Mg2+ dependent and guanine nucleotide sensitive, and the other not coupled to Ns and being Mg2+ independent and guanine nucleotide insensitive.

scholarly journals Desensitization of tumour Leydig cells by lutropin: evidence for uncoupling of the lutropin receptor from the guanine nucleotide-binding protein

1982 ◽  
Vol 202 (3) ◽  
pp. 739-745 ◽  
Author(s):  
Clive J. Dix ◽  
Matthias Schumacher ◽  
Brian A. Cooke
Keyword(s):  
Cyclic Amp ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Linear Increase ◽  
Guanine Nucleotide ◽  
Production Rates ◽  
Guanine Nucleotide Binding Protein ◽  
Intact Cells ◽  
Guanine Nucleotide Binding ◽  
Guanine Nucleotide Regulatory Protein

Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED50 (dose that produces a response that is 50% of the maximum response) 60±5.7ng/ml and 8±1.8ng/ml (mean±s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/106 cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific 125I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4°C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5′-[β,γ-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50–60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- and the p(NH)ppG-stimulated cyclic AMP production rates were unchanged but no effect of lutropin could be demonstrated with or without added p(NH)ppG. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.

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