Rat Liver Insulin Receptor Research Articles

A high-cholesterol diet increases the association between caveolae and insulin receptors in rat liver

Caveolin-1, a component of caveolae, regulates signaling pathway compartmentalization by interacting with tyrosine (Tyr) kinase receptors and their substrates. Perturbations in caveolae lipid composition have been shown in vitro to displace proteins from lipid microdomains, thereby altering their functionality and subsequent downstream signaling. The role of caveolin-1 in insulin receptor (IR) signaling has been widely investigated in vitro mainly in 3T3-L1 adipocyte cells. However, in vivo experiments investigating this connection in liver tissue have not been carried out. The objective of the present study was to investigate the effects of a high-cholesterol diet on caveolin-1 expression and IR localization and activity in the rat liver. Compared with a standard diet, rats fed with diet rich in cholesterol significantly altered liver caveolae by increasing both caveolin-1 (66%, P < 0.05) and caveolin-2 (55%, P < 0.05) expression while caveolin-1 mRNA levels were reduced. Concomitantly, a 25% increase in localization of the caveolae-resident signaling protein IR was observed. The distribution of caveolar and noncaveolar phosphorylated IR was unaffected but insulin-induced IR activation was significantly enhanced following consumption of the high-cholesterol diet (120%, P < 0.001). However, the downstream molecules IRS-1 and Akt have shown impaired activity in cholesterol-fed rats suggesting insulin resistance condition. Insulin stimulation failed to induce Tyr phosphorylation of caveolin-1 in cholesterol-fed rats. These findings suggest a mechanism by which a high-cholesterol diet altered caveolin-1 expression in vivo accompanied by altered IR localization and activity.

Natural vanadium-containing Mt. Fuji ground water improves hypo-activity of liver insulin receptor in Goto-Kakisaki rats

After daily treatments with Mt. Fuji ground water containing natural vanadium (approximately 65 microg/l) at doses of 0.53 microg/kg/day for 12 weeks, blood glucose (BG), serum hemoglobin A1C (HbA1C) levels and insulin secretion from the pancreas of Goto-Kakisaki (GK) rats, a genetic model of Type 2 diabetes, were improved. In GK rat liver insulin receptors, the binding properties of [125I] insulin, and the activities of insulin receptor beta subunit and primary insulin-like growth factor-1beta all recovered to normal levels of those found in Wistar rats. These results suggest that daily treatment with small concentrations of natural vanadium improves hyperglycemia by ameliorating liver insulin receptor activity.

Isolation and characterization of muscovy (Cairna moschata) duck insulin

Ducks (Anatidae Family, Anseriform order) are divided in two genera: Pekin duck (Anas platyrhynchos genus) and Muscovy duck (Cairina moschata genus) and differ for their number of liver insulin receptors (despite rather similar plasma insulin levels). The possibility that the presence of different endogenous insulins account for the difference in insulin receptor number between the two duck species led us to purify, sequence and characterize the binding properties of Muscovy duck insulin. The sequence of Muscovy duck insulin (measured mass: 5729. 11) was indentical to that described in two other species from the Anseriforme order: Pekin duck or goose. The binding affinity of Muscovy duck insulin for rat liver insulin receptors (either membrane bound or solubilized receptors) was lower than that of porcine insulin (0.3), which most likely accounts for the low biological potency of Pekin duck insulin previously described. In contrast, liver receptors from chicken and both duck species exhibited the same affinity for duck and porcine insulin suggesting the presence of specific changes in the structure of binding sites of bird liver insulin receptors. The decrease in the number of insulin receptors in Muscovy duck liver is not therefore the consequence of a change at the level of the insulin molecule itself. As discussed, among bird insulins, the hypoactive “duck type” insulin would have appeared after the hyperactive “chicken type” insulin during the evolution of Aves.

Regulation of an hepatic low-Mr membrane-associated protein-tyrosine phosphatase

Protein-tyrosine phosphatases (PTPases), active against autophosphorylated insulin and epidermal growth factor (EGF) receptors in rat liver, are predominantly membrane associated. Fasting of rats for 48 h decreased hepatic particulate PTPase activity by 15.0-26.9%. This reduction in particulate PTPase activity was due to a rather specific decrease in activity of > 85% of a single species of PTPase, termed PTPase I. Disappearance of PTPase I activity from the particulate fraction was not accounted for by its translocation to the cytosol. PTPase I displayed the highest activity against autophosphorylated insulin and EGF receptors, relative to activity against a 32P-labelled peptide substrate, of three PTPases resolved from the liver particulate fraction. The M(r) value of PTPase I, as determined by gel filtration on a Superose 12 column was approx. 42,000, indicating that PTPase I belongs to the low-M(r) class of PTPases. An antibody raised against PTPase 1B, the prototype of this class of PTPases, did not react with PTPase I in Western blots. The potential importance of the novel change in activity of PTPase I in the regulation of insulin-receptor signal transduction is discussed.

Intermolecular Phosphorylation of Insulin Receptor as Possible Mechanism for Amplification of Binding Signal

Clustering of cell-surface insulin receptors has led to the speculation that intermolecular phosphorylation of unoccupied receptors catalyzed by ligand-occupied receptors within the cluster could be a mechanism by which the insulin-binding signal is amplified. We examined whether insulin receptors can be phosphorylated by an intermolecular mechanism. In this study, we used highly purified insulin receptors isolated from rat liver plasma membranes and human placental membranes. Rat liver insulin receptors were "activated" by incubation with 10 nM insulin in the presence of ATP. Subsequent to removal of insulin by immunodepletion, these receptors were used as an enzyme source to study phosphorylation of unphosphorylated "substrate" human receptors. Initially, we found no evidence that the addition of activated rat receptors increased phosphorylation of human receptors, when assessed by immunoprecipitation with a human-specific monoclonal antibody. To examine the possibility that these negative results were due to insufficient receptor concentration, activated human receptors were mixed with unphosphorylated substrate receptors at concentrations up to 60 micrograms/ml. In this study, we found that addition of activated receptors resulted in increased phosphorylation of the substrate receptors at the highest concentrations employed. These are the first data indicating that insulin receptors per se are capable of intermolecular phosphorylation. In vivo, this could be the initial step in amplifying the insulin-binding signal.

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

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.

Long-term maternal-fetal exposure to high-low insulin concentrations alter liver but not brain insulin receptors

We investigated the effects of long-term (5 to 6 days) in vivo exposure to hyperinsulinemia and hypoinsulinemia on maternal and fetal rat (18 to 20 days' gestation; term approximately 21 days) brain and liver insulin receptors. Further we studied the in vitro effects of long-term insulin exposure on cultured rabbit neuronal cell insulin receptors. Long-term glucose infusions to maternal rats resulted in maternal and fetal hyperglycemia and mild hyperinsulinemia, which decreases the liver insulin receptor abundance of the mother but increased that of the fetus. Streptozotocin-induced maternal diabetes resulted in maternal and fetal hyperglycemia with hypoinsulinemia (or near-normal insulin levels) and increased maternal and fetal liver insulin receptors. The maternal and fetal brain insulin receptor abundance failed to change at high or low insulin concentrations. Similarly long-term insulin (5 micrograms/ml) exposure failed to alter cultured neuronal cell insulin receptors as well. We conclude that perturbations in maternal and fetal circulating insulin concentrations fail to affect the brain insulin receptors. This protective phenomenon may be critical in maintaining the normal neuronal energy balance and functioning during certain disease states such as diabetes mellitus and hyperinsulinism.

The Rat Insulin Receptor: Primary Structure and Conservation of Tissue-Specific Alternative Messenger RNA Splicing

To investigate whether alterations in the polypeptide structure of the insulin receptor might explain the heterogeneity observed in its properties between species and in different tissues, we obtained the complete primary structure of the rat liver insulin receptor precursor by cDNA cloning and sequencing. The rat proreceptor contains 1357 amino acids and has 95.2% identity with deduced polypeptide sequences reported for the human insulin receptor precursor. In addition, the rat liver insulin receptor cDNA was similar to the form of the human insulin receptor mRNA that contains Exon 11 in its coding region, which undergoes tissue-specific alternative splicing. Using the polymerase chain reaction to amplify a cDNA segment corresponding to this region in several rat tissues, the splicing pattern of sequences homologous to Exon 11 was found to be highly conserved, providing further evidence that these two forms of the insulin receptor may serve important functional or regulatory roles. These studies have also identified subtle variations in the primary structure of the insulin receptor which may influence its properties between species.

An Extracellular Domain of the Insulin Receptor β-Subunit with Regulatory Function on Protein-Tyrosine Kinase

Abstract Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.

Intrinsic Kinase Activity of the Insulin Receptor: The intact (α2β2) insulin receptor from rat liver contains a kinase domain with greater intrinsic activity than the intact insulin receptor from human placenta

We are interested in developing methods to rigorously characterize the intrinsic enzymatic activity of the insulin receptor. We have previously shown that the intact, kinase active form of the receptor can be separated from inactive forms isolated from human placenta. Therefore, the determination of kinase activity, when normalized to the number of receptors based on binding, is not complicated by the presence of insulin receptor forms which bind insulin normally, but are kinase inactive. We now have extended this separation technique to insulin receptor preparations from rat liver. Thus, the determination and comparison of the intrinsic kinase activity of insulin receptor from human placenta and rat liver was performed. When normalized to the same number of insulin receptors which are autophosphorylated to the same degree, the rat liver insulin receptor catalyzes the transfer of phosphate from ATP to three different substrates, on average, 2.8-fold quicker than receptor from human placenta. This probably represents an inherent difference in the intrinsic kinase activity (Vmax), since the values for KM of the substrates are essentially identical, for insulin receptors from both sources. Intrinsic kinase differences may reflect different biological roles and/or differential regulation by exogenous factors. We are now examining this hypothesis in light of reports that demonstrate regulation of intrinsic kinase activity of the insulin receptor in certain physiological and pathological states.

Proteolytic activity in the insulin receptor

When a highly purified preparation of rat liver insulin receptor is incubated with trypsin, the receptor develops hydrolytic activity towards N alpha-benzoyl-L-arginine ethyl ester, N alpha-p-tosyl-L-arginine methyl ester, and N alpha-benzoyl-DL-arginine-p-nitroanilide, (compounds which are synthetic substrates of trypsin). The activity toward N alpha-benzoyl-DL-arginine-p-nitroanilide is inhibited by soybean trypsin inhibitor but not by N alpha-p-tosyl-L-lysil chloromethyl ketone. These data are consistent with the presence of proteolytic activity in the insulin receptor specific for the bonds whose carbonyl functions are provided by arginine but not by lysine. Furthermore we found that the esterase activity toward N alpha-benzoyl-L-arginine ethyl ester in the presence of trypsin is enhanced by insulin, and that the concentration of insulin that produced the half maximum stimulation is of the same magnitude as the dissociation constant for the soluble receptor. These data suggest that the insulin receptor is a zymogen, activated by trypsin, and based on its specific activity, may be the protease which releases a peptide chemical mediator of intracellular action of insulin.

Role of divalent metals in the kinetic mechanism of insulin receptor tyrosine kinase

The kinetic and thermodynamic interrelationships of peptide substrate (Val 5-angiotensin 11), metal-ATP, and divalent metal cations with rat liver insulin receptor tyrosine kinase (IRTK) were investigated. Results of the initial rate studies with varying peptide and MnATP substrates indicates that the kinetic mechanism for IRTK is of the sequential type and therefore rules out a ping pong Bi Bi pathway. Hence, peptide substrate and metal-ATP bind to the kinase prior to the release of products. MnADP was a linear competitive inhibitor of MnATP and a noncompetitive inhibitor of peptide substrate. A synthetic tyrosine-containing pentapeptide, GluGluPheTyrPhe (EEFYF), was a linear competitive inhibitor of peptide substrate and a noncompetitive inhibitor of MnATP. Accordingly, the data show that phosphorylation of peptide substrate occurs via a rapid random equilibrium Bi Bi mechanism in which the kinase has the potential to react initially with either of the two substrates. In contrast, divalent metal cations and metal-ATP were found to interact with the kinase in a mutually inclusive manner, with metal binding to the kinase prior to MnATP. It was also found that divalent metals increase the affinity of the kinase for metal-ATP but do not affect the affinity of IRTK for metal-ADP product. Hence, divalent metals, during the reaction of association of enzyme with one of its substrates to form the binary complex, increase the relative concentration of E-ATP complex versus E-peptide complex, thus introducing a thermodynamic-dependent ordering for the interaction of substrates with the enzyme. To investigate the thermodynamics of this system, we assumed that under initial conditions the kinetic data we obtained reflected the association constants of reactants with the enzyme.

Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations.

Insulin stimulates autophosphorylation of the beta subunit of its receptor and activates the associated tyrosine kinase. This kinase, in turn, phosphorylates a number of specific protein substrates; however, the functional and structural identity of these substrates is largely unknown. In this study, we demonstrate that insulin also stimulates the phosphorylation of calmodulin by rat hepatocyte insulin receptors partially purified by wheat germ agglutinin affinity chromatography. Phosphorylation occurred predominantly on tyrosine residues and had an absolute requirement for insulin receptors, divalent cations, and certain basic proteins. Maximal 32P incorporation was observed at an insulin concentration of 5 X 10(-9) M, and the K0.5 for insulin was approximately 4 X 10(-10) M. Phosphorylation of calmodulin was dependent upon ATP, saturating at 100 microM ATP with a K0.5 of 30 microM. Insulin-stimulated phosphorylation of calmodulin was also dependent upon Mg2+ or Mn2+, but was approximately 12-fold greater in the presence of Mg2+. Maximal phosphorylation was observed in the absence of Ca2+ and was inhibited at Ca2+:EGTA ratios greater than 0.8 (0.16 microM free Ca2+). Certain basic proteins, such as polylysine, histone Hf2b, and protamine sulfate, were necessary to observe insulin-stimulated phosphorylation of calmodulin. The relative amount of insulin-stimulated phosphorylation of calmodulin observed in the presence of each of these proteins differed. Maximal insulin-stimulated phosphorylation was observed in the presence of polylysine. These data suggest that both Ca2+ and calmodulin may participate in the early post-receptor events in the cellular mechanism of insulin action in hepatocytes.

Ligand-induced changes in the subcellular distribution of insulin receptors in rat liver: Effects of colchicine

The in vivo effects of colchicine on the subcellular distribution of insulin receptors have been studied in insulin-injected rats and in control animals. Colchicine (0.1 mg/100 g or 10 mg/100 g body weight i.v.) did not affect the ability of plasma membranes and Golgi fractions of control rats to bind insulin. As previously reported (Desbuquois et al., 1982), the injection of native insulin (8 nmol i.v.) caused a 50% decrease in the insulin binding activity of plasma membranes and a concomitant 50% increase in insulin binding to Golgi fractions. These changes occurred at 4 and 40 min after insulin injection but were no longer detectable at 3 h. Colchicine treatment did not affect the initial changes in the distribution of insulin receptors induced by insulin; however, in rats treated with the low dose of colchicine, insulin binding to plasma membranes at 3 h was not fully restored. Colchicine treatment did not alter the amount of acid-extractable insulin associated with Golgi fractions of insulin-injected rats. The time course of uptake of 125I-insulin was similar in plasma membranes, microsomal fraction and Golgi fractions of colchicine-treated (0.1 mg/100 g) and of untreated rats. These results suggest that colchicine does not interfere with the endocytosis of insulin receptors induced by their ligand and has little effect, if any, on the reinsertion of internalized receptors in the plasma membrane.

Insulin-related changes in the subcellular distribution of insulin receptors in intact rat liver: effects of acute hypoinsulinemia induced by diazoxide, somatostatin, and xylazine.

The effects of inhibitors of insulin secretion on the concentration of plasma insulin and on the subcellular distribution of hepatic insulin receptors have been examined in rats. Liver plasma membranes, Golgi fractions, and a total particulate fraction were prepared various times after the injection of diazoxide (10 mg), xylazine (0.5 mg), and somatostatin (20 micrograms as a bolus followed by a 20 micrograms/15-min infusion), solubilized by Triton X-100, and examined for specific insulin binding. Injection of each of these drugs caused a 4- to 8-fold decrease in plasma insulin concentration, and concomitantly, a 25-35% decrease in insulin binding to Golgi fractions; a 10% increase in insulin binding to plasma membranes also occurred in diazoxide-treated rats. Insulin binding to the total particulate fraction was unaffected. These changes achieved maximum by 10-30 min and underwent complete reversal in 1 h. The decrease in insulin binding to Golgi fractions resulted exclusively from a change in receptor number and was accompanied by a 4- to 6-fold decrease in the content of extractable insulin in these fractions; the latter observation suggests that receptor internalization, rather than receptor synthesis, is diminished. The effects of diazoxide on plasma insulin concentration, insulin binding to Golgi fractions, and insulin content of Golgi fractions were fully prevented by tolbutamide, a stimulant of insulin secretion. These results show that acute hypoinsulinemias in rats are accompanied by changes in the subcellular distribution of hepatic insulin receptors that are opposite to those previously observed in acute hyperinsulinemias. Furthermore, both in acute hyperinsulinemic and hypoinsulinemic models, a significant correlation is observed between plasma insulin concentration on the one hand, and insulin binding to Golgi fractions as well as insulin content of Golgi fractions on the other. It is concluded that the extent to which hepatic insulin receptors are internalized is a function of their occupancy by endogenous hormone, and that, at least under acute conditions, receptor internalization is one major mechanism involved in receptor regulation.

Disulfide bonds within the α-subunit of insulin receptors in rat liver and brain membranes

We have characterized inter- and intrasubunit disulfide bonds of insulin receptors using reductant-treated rat liver and brain membranes. In autoradiograms of 125I-insulin cross-linked to both membranes pretreated with dithiothreitol, the intensity of affinity-labeled bands of the αβ-heterodimer and α-subunit was increased. Interestingly, labeled 120 and 110 kDa bands considered to be the α-subunit in partially reduced liver and brain membranes moved to 130 and 120 kDa bands under further reduced conditions, respectively. Double electrophoresis of each partially reduced band in the presence of reductants clearly demonstrates that the α-subunit of insulin receptors contains intrasubunit disulfide bonds.

Insulin receptors of chicken liver and brain. Characterization of alpha and beta subunit properties.

Receptors on membranes of chicken liver and brain bound porcine 125I-insulin in a specific and temperature-dependent manner. Competition with unlabeled insulin derivatives exhibited typical insulin potency ratios, i.e. chicken greater than porcine insulin greater than human proinsulin (2.1/1/0.02). Apparent binding affinity was higher in brain with a 50% inhibition of tracer binding of 1.3 +/- 0.2 nM porcine insulin as compared to 2.8 +/- 0.3 nM in liver. The apparent molecular mass of the 125I-insulin cross-linked alpha subunit of the insulin receptor was 139 +/- 2 kDa for chicken liver and 127 +/- 2 kDa for chicken brain. These molecular masses were similar to those of rat liver and brain insulin receptors. Neuraminidase treatment of the cross-linked insulin receptor increased the mobility of the alpha subunit from liver but did not affect that from brain, suggesting a difference in the glycosylation of the chicken brain alpha subunit as previously described in the rat. Despite this change, both receptors could be purified on wheat germ agglutinin (WGA) chromatography after Triton solubilization. In the presence of CTP and vanadate (phosphatase inhibitors) insulin-stimulatable tyrosine-specific phosphorylation of exogenous substrates was demonstrated with chicken liver and brain receptors. The reaction was dependent on Mg2+ and Mn2+. As noted with other insulin receptors, the best artificial substrate for phosphorylation was poly(Glu,Tyr)4:1. In both chicken liver and brain the smallest effective insulin dose as well as maximal stimulation of phosphorylation of the substrate was similar to that seen with rat liver, and in all three tissues chicken insulin was more potent than porcine insulin. In chicken liver an active ATP hydrolytic activity copurified with the insulin receptors during WGA chromatography. Further purification using S-300 Sephacryl filtration or affinity (insulin-biotin-avidin) chromatography could dissociate the phosphorylation and the hydrolytic activities. Gel electrophoresis, under reducing conditions revealed beta subunits with apparent Mr of 97-99 kDa in chicken liver and brain, which were phosphorylated in the presence of insulin. Similar apparent molecular masses have been described for the beta subunit of rat liver receptors. These studies suggest that both chicken brain and liver insulin receptors exhibit coupling of alpha and beta subunits with fully active tyrosine kinase and that the structural difference of the brain insulin receptor is widespread and phylogenetically old.

Chronic treatment with phenobarbital decreases the expression of rat liver EGF and insulin receptors

In male F-344 rats fed phenobarbital 0.05% with chow the binding of EGF to microsomal and Golgi liver fractions decreased after 2 weeks, dropped by more than 60% after one month and remained low for the following five months of the experiment. The binding of insulin remained stable for 2 weeks after which it decreased by half. In both cases the receptors decreased in number without changes in their affinity. Autophosphorylation of the receptors showed changes parallel to those of the binding of the ligands.

The demonstration of insulin-like material in the honey bee, Apis mellifera

1.1. Partially purified extracts from bees (Apis mellifera) and their separated heads cross-reacted in a porcine insulin radioimmunoassay whereas the decapitated bodies were devoid of activity.2.2. The active extracts displaced porcine insulin from rat liver insulin receptors and showed insulin-like activity with rat adipocytes which could be abolished with bovine insulin antiserum.3.3. The presence of insulin in bee royal jelly was demonstrated.4.4. Thus an insulin of close similarity to mammalian is present in the head of the bee, indicating high evolutionary conservation of the molecule.

Radiation inactivation of membrane proteins: Molecular weight estimates in situ and after Triton X-100 solubilization

Target size analysis by radiation inactivation is widely used for molecular weight determination of membrane enzymes and receptors in situ without the need for prior solubilization or purification. However, since most molecular weight data available in the literature on membrane proteins involve the use of detergents for solubilization, the target sizes of membrane proteins in situ and after solubilization by detergent treatment have been compared. Using data from the literature and personal results, three different types of behavior of membrane proteins in presence of detergents were found: (i) uncoupling of subunits (electric eel acetylcholinesterase, placental steroid sulfatase, and human nonspecific β-glucosidase); (ii) coupling of protein molecules (mouse liver neuraminidase, and rat liver insulin receptor regulatory component); and (iii) no major change in quaternary structure (rat liver insulin receptor, kidney γ-glutamyltransferase, asialoglycoprotein receptor, insulin degrading enzyme, and human leucocyte neuraminidase). For all these proteins, there is a statistically significant increase in target size of about 24% over the value obtained in situ without detergent. A relatively large body of literature data involving a variety of membrane proteins, membrane types, and irradiation conditions (electron accelerators or 60Co sources, and proteins irradiated in lyophilized form or frozen solution) was examined, and it was concluded that target sizes of membrane proteins, irradiated in the presence of Triton X-100, should be diminished by a factor of about 24% to obtain the molecular weight value.