scholarly journals A comparison of lactogenic receptors from rat liver and Nb2 rat lymphoma cells by using cross-linking techniques

1988 ◽  
Vol 250 (1) ◽  
pp. 215-219 ◽  
Author(s):  
C F Webb ◽  
M Wallis
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Growth ◽  
Cell Receptor ◽  
Receptor Protein ◽  
Cross Linking ◽  
Pregnant Rats ◽  
Reducing Conditions ◽  
Nb2 Cells ◽  
Liver Membranes

Lactogenic receptors were analysed with the use of the cross-linking agent disuccinimidyl suberate to attach covalently 125I-labelled ovine prolactin or human growth hormone to binding sites from (1) liver from pregnant rats and (2) the rat-derived Nb2 lymphoma cell line. Analysis by SDS/polyacrylamide-gel electrophoresis of the proteins cross-linked to labelled hormone in rat liver indicated a major specifically-labelled complex with an Mr of 68,000-72,000, when run under reducing or non-reducing conditions. With Nb2 cells a major specifically-labelled complex with an Mr of 97,000-110,000 was identified, but only when electrophoresis was run using reducing conditions. Assuming one hormone molecule (Mr 22,000-24,000) per hormone-receptor complex, then the receptor proteins have an Mr of 44,000-50,000 for rat liver and 73,000-88,000 for the Nb2 cells. For both cell types the receptors were of lactogenic specificity; lactogenic hormones competed for binding whereas somatogenic hormones did not. These studies suggest that the lactogenic receptors in rat liver membranes and Nb2 cells differ in two respects. Firstly, the Mr of the labelled receptor protein in Nb2 cells is greater than that of the corresponding receptor protein in rat liver membranes; secondly, the Nb2 cell receptor appears to exist as a disulphide-linked oligomer whereas the receptor in rat liver membranes does not.

Pituitary regulation of human growth hormone binding sites in rat liver membranes

Metabolism ◽  
1976 ◽  
Vol 25 (3) ◽  
pp. 341-353 ◽  
Author(s):  
A.C. Herington ◽  
L.S. Phillips ◽  
W.H. Daughaday
Keyword(s):  
Growth Hormone ◽  
Rat Liver ◽  
Human Growth Hormone ◽  
Binding Sites ◽  
Human Growth ◽  
Hormone Binding ◽  
Liver Membranes

scholarly journals Lactogenic and somatogenic binding sites in intact and detergent-solubilized membrane preparations of female rat liver

1988 ◽  
Vol 252 (2) ◽  
pp. 509-514 ◽  
Author(s):  
L A Haldosén ◽  
J A Gustafsson
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Growth ◽  
Microsomal Membrane ◽  
Gel Chromatography ◽  
Female Rat ◽  
Microsomal Membranes ◽  
Triton X

The presence of lactogenic and somatogenic binding sites in intact microsomal membranes and in detergent-solubilized microsomal membrane preparations of female rat liver has been studied by affinity cross-linking-SDS/polyacrylamide-gel electrophoresis. In microsomal membrane preparations an Mr 40,000 lactogenic binder is present which is not disulphide-linked to another protein. Triton X-100 solubilization of membranes results in the appearance of three lactogenic 125I-human growth hormone (125I-hGH) binders with Mr values of 87,000, 40,000 and 35,000, and one somatogenic 125I-hGH binder with Mr 32,000. Treatment of rats with oestrogen increased the amount of lactogenic and somatogenic binding species in liver. The lactogenic binding sites are present as one entity in Triton X-100-solubilized preparations, clearly separated from the somatogenic binder as analysed by gel chromatography. Furthermore, 125I-hGH interacts with an Mr 95,000 somatogenic binder in membrane preparations to which the hormone can be cross-linked only following Triton X-100 solubilization.

BINDING OF HUMAN GROWTH HORMONE TO HEPATIC LACTOGENIC BINDING SITES: REGULATION BY OESTROGENS AND ANDROGENS

1976 ◽  
Vol 70 (3) ◽  
pp. 473-484 ◽  
Author(s):  
A. C. HERINGTON ◽  
H. G. BURGER ◽  
NOELLE M. VEITH
Keyword(s):  
Growth Hormone ◽  
Effective Dose ◽  
Rat Liver ◽  
Sex Steroids ◽  
Binding Sites ◽  
Human Growth ◽  
Female Rats ◽  
Male Rats ◽  
Minimum Effective Dose ◽  
Liver Membranes

SUMMARY The binding of 125I-labelled human growth hormone (HGH) to the 'lactogenic' binding sites of rat liver membranes has been shown to be highly dependent on the oestrogen and androgen status of the animal from which the membranes were prepared. Oestradiol treatment of either male or female rats induced a highly significant rise in HGH binding. The minimum effective dose used was 2–5 μg/day and the rise in HGH binding was apparent after 4 days of treatment. Following cessation of oestradiol treatment of male rats HGH binding declined with a half-time of approximately 9 days. In contrast to the stimulatory effect of oestrogen, treatment of female rats with testosterone propionate (minimum effective dose 100–200 μg/day) led to a marked reduction in HGH binding. The influence of both oestrogens and androgens was confirmed following the removal of endogenous sex steroids by adrenalectomy–ovariectomy of female rats and castration of male rats. Scatchard analysis showed that, with the possible exception of adrenalectomy–ovariectomy, all pharmacologically and physiologically induced changes in HGH specific binding reflected changes in binding site capacity; there were no changes in binding affinity. While earlier studies have indicated that the oestrogen effect is primarily indirect and is mediated by the pituitary gland, the mode of action of the androgens is currently unknown. The relatively slow response of HGH binding to hormonal changes would support an indirect action for both the sex steroids. The stimulatory effect of oestrogens and the inhibitory effect of androgens may provide an explanation for the marked sex difference in HGH binding to rat liver membranes.

scholarly journals The endogenous functional turkey erythrocyte and rat liver insulin receptor is an α2β2 heterotetrameric complex

1990 ◽  
Vol 271 (1) ◽  
pp. 99-105 ◽  
Author(s):  
J L Treadway ◽  
B D Morrison ◽  
J A Wemmie ◽  
I Frias ◽  
T O'Hare ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Rat Liver ◽  
Human Placenta ◽  
Alkylating Agents ◽  
Insulin Receptors ◽  
Cross Linking ◽  
Protein Kinase Activity ◽  
Liver Membranes ◽  
Alpha Beta ◽  
Beta 2

Previous studies have indicated that turkey erythrocyte and rat liver membranes contain endogenous alpha beta heterodimeric insulin receptors in addition to the disulphide-linked alpha 2 beta 2 heterotetrameric complexes characteristic of most cell types. We utilized 125I-insulin affinity cross-linking to examine the structural properties of insulin receptors from rat liver and turkey erythrocyte membranes prepared in the absence and presence of sulphydryl alkylating agents. Rat liver membranes prepared in the absence of sulphydryl alkylating agents displayed specific labelling of Mr 400,000 and 200,000 bands, corresponding to the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes respectively. In contrast, affinity cross-linking of membranes prepared with iodoacetamide (IAN) or N-ethylmaleimide identified predominantly the alpha 2 beta 2 heterotetrameric insulin receptor complex. Similarly, affinity cross-linking and solubilization of intact turkey erythrocytes in the presence of IAN resulted in exclusive labelling of the alpha 2 beta 2 heterotetrameric insulin receptor complex, whereas in the absence of IAN both alpha 2 beta 2 and alpha beta species were observed. Turkey erythrocyte alpha 2 beta 2 heterotetrameric insulin receptors from IAN-protected membranes displayed a 3-4-fold stimulation of beta subunit autophosphorylation and substrate phosphorylation by insulin, equivalent to that observed in intact human placenta insulin receptors. Turkey erythrocyte alpha beta heterodimeric insulin receptors, prepared by defined pH/dithiothreitol treatment of IAN-protected membranes, were also fully competent in insulin-stimulated protein kinase activity compared with alpha beta heterodimeric human placenta receptors. In contrast, endogenous turkey erythrocyte alpha beta heterodimeric insulin receptors displayed basal protein kinase activity which was insulin-insensitive. These data indicate that native turkey erythrocyte and rat liver insulin receptors are structurally and functionally similar to alpha 2 beta 2 heterotetrameric human placenta insulin receptors. The alpha beta heterodimeric insulin receptors previously identified in these tissues most likely resulted from disulphide bond reduction and denaturation of the alpha 2 beta 2 holoreceptor complexes during membrane preparation.

Hepatic Binding Sites for Angiotensin II in the Rat

1979 ◽  
Vol 56 (1) ◽  
pp. 33-40 ◽  
Author(s):  
J. J. Lafontaine ◽  
M. P. Nivez ◽  
R. Ardaillou
Keyword(s):  
Angiotensin Ii ◽  
Rat Liver ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Liver Membranes ◽  
Clear Increase

1. 125I-labelled (Asn1, Val5)-angiotensin II (125I-labelled AII) incubated with purified rat liver membranes was degraded with time, as estimated by three techniques: binding to an excess of specific antibody, polyacrylamide-gel electrophoresis and rebinding to fresh membranes. Degradation was inhibited in the presence of an excess of β1–24-corticotrophin but still very marked. 2. 125I-labelled AII became bound to purified rat liver membranes. Association and dissociation rates were slow. Binding was competitively inhibited by (Asn1, Val5)-AII, (Asp1, Ile5)-AII and (Des, Asn1, Ile5)-AII. Apparent KD was approximately 0·1 nmol/l. 3. Bound hormone was also partly degraded independently of time. 4. Angiotensinases inhibitors had different effects on 125I-labelled AII binding. A clear increase was observed in the presence of β1–24-corticotrophin and phenylmethylsulphonylfluoride whereas binding was decreased in the presence of EDTA or 8-hydroxyquinoline. 5. These results demonstrate the presence of high-affinity binding sites for AII and of angiotensinases in hepatic membranes.

Sign in / Sign up

Export Citation Format

Share Document