RESUMEN
OBJECTIVE: To evaluate the frequency of malignant ulcers in patients presenting with leg ulcers. DESIGN: A descriptive study from data collected between July 1988 and June 1995 from 981 patients (2448 ulcers) attending a leg ulcer clinic. SETTING: A specialised leg ulcer clinic at a tertiary teaching hospital. SUBJECTS: 43 patients with 55 malignant skin lesions. OUTCOME MEASURES: Tissue biopsies in ulcerated lesions that suggested malignancy or were not responding to appropriate treatment. RESULTS: Forty-three patients were found to have malignant lesions on the legs, giving a frequency of malignant ulcers of 4.4 per 100 leg ulcer patients, or 2.2 per 100 leg ulcers. Seventy-five per cent of the malignant ulcers were basal cell carcinoma and 25% were squamous cell carcinoma. CONCLUSIONS: Malignant skin changes are common in chronic leg ulcers. A biopsy should be taken from all suspicious ulcers or ulcers that do not respond to appropriate treatment.
Asunto(s)
Úlcera de la Pierna/epidemiología , Neoplasias Cutáneas/epidemiología , Anciano , Anciano de 80 o más Años , Biopsia , Carcinoma Basocelular/epidemiología , Carcinoma Basocelular/patología , Carcinoma de Células Escamosas/epidemiología , Carcinoma de Células Escamosas/patología , Enfermedad Crónica , Femenino , Úlcera del Pie/epidemiología , Úlcera del Pie/patología , Tejido de Granulación/patología , Hospitales de Enseñanza , Humanos , Pierna/irrigación sanguínea , Úlcera de la Pierna/etiología , Úlcera de la Pierna/patología , Masculino , Persona de Mediana Edad , Piel/patología , Neoplasias Cutáneas/patología , Enfermedades Vasculares/complicaciones , Australia Occidental/epidemiologíaRESUMEN
Classical insulin and insulin-like growth factor I (IGF-I) receptors exist as well defined alpha 2 beta 2 heterotetrameric complexes that are assembled from two identical alpha beta heterodimeric half-receptor precursors. Recent evidence suggests that insulin and IGF-I half-receptors can heterologously assemble to form alpha 2 beta 2 insulin/IGF-I hybrid receptor complexes in vivo and in vitro. We have utilized hybrid receptor complexes to examine ligand-stimulated transmembrane signaling of wild-type insulin (alpha beta INS.WT) or IGF-I (alpha beta IGF.WT) half-receptors assembled with a kinase-defective insulin half-receptor mutant (alpha beta INS.A/K). In vitro assembly of either (alpha beta)IGF.WT/(alpha beta)INS.A/K or (alpha beta)INS.WT/(alpha beta)INS.A/K hybrid receptors resulted in decreased substrate protein kinase activity. The degree of protein kinase inactivation directly correlated with the amount of immunologically cross-reactive hybrid receptors formed. In contrast to substrate kinase activity, insulin-stimulated autophosphorylation of the (alpha beta)INS.WT/(alpha beta)INS.A/K hybrid receptor complex was completely unaffected in comparison to the wild-type (alpha beta)INS.WT/(alpha beta)INS.WT receptor. To assess a molecular basis for this difference, autophosphorylation of a hybrid receptor composed of a truncated beta-subunit insulin half-receptor with the kinase-defective half-receptor, (alpha beta)INS. delta CT/(alpha beta)INS.A/K, demonstrated the exclusive autophosphorylation of the (alpha beta)INS.A/K half-receptor beta subunit. These results demonstrate that ligand-dependent substrate phosphorylation by insulin and IGF-I holoreceptors requires interactions between two functional beta subunits within the alpha 2 beta 2 heterotetrameric complex and occurs through an intramolecular trans-phosphorylation reaction.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptor de Insulina/metabolismo , Receptores de Superficie Celular/metabolismo , Membrana Celular/metabolismo , Femenino , Humanos , Insulina/farmacología , Cinética , Placenta/metabolismo , Embarazo , Multimerización de Proteína , Receptores de SomatomedinaRESUMEN
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.
Asunto(s)
Membrana Eritrocítica/química , Hígado/química , Receptor de Insulina/química , Animales , Membrana Celular/química , Centrifugación por Gradiente de Densidad , Reactivos de Enlaces Cruzados , Etilmaleimida , Humanos , Insulina/metabolismo , Insulina/farmacología , Yodoacetamida , Sustancias Macromoleculares , Peso Molecular , Fosforilación , Placenta/química , Proteínas Quinasas/metabolismo , Ratas , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/metabolismo , Pavos/sangreRESUMEN
Examination of 125I-IGF-1 affinity cross-linking and beta-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated alpha beta heterodimeric IGF-1 receptors into an alpha 2 beta 2 heterotetrameric state, in a similar manner to that observed for the insulin receptor [Morrison, B.D., Swanson, M.L., Sweet, L.J., & Pessin, J.E. (1988) J. Biol. Chem. 263, 7806-7813]. The formation of the alpha 2 beta 2 heterotetrameric IGF-1 receptor complex from the partially purified alpha beta heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulate the protein kinase activity of the purified alpha beta heterodimeric insulin receptor complex. Incubation of the alpha 2 beta 2 heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter 125I-IGF-1 binding of IGF-1 stimulation of protein kinase activity. In addition, IAN did not affect the Mn/MgATP-dependent noncovalent association of IGF-1 receptor alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state. However, IAN treatment of the alpha beta heterodimeric IGF-1 receptors inhibited the IGF-1-dependent covalent formation of the disulfide-linked alpha 2 beta 2 heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated alpha beta heterodimeric IGF-1 receptor complexes into a disulfide-linked alpha 2 beta 2 heterotetrameric state whereas Mn/MgATP induces a noncovalent association.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Factor I del Crecimiento Similar a la Insulina , Receptor de Insulina , Somatomedinas , Reactivos de Enlaces Cruzados/metabolismo , Humanos , Fosforilación , Conformación Proteica , Proteínas Quinasas/metabolismo , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/metabolismo , Especificidad por SustratoRESUMEN
Insulin and Mn/MgATP treatment of immunoaffinity-purified alpha beta heterodimeric insulin receptors induced the formation of an alpha 2 beta 2 heterotetrameric insulin receptor complex. In contrast, insulin-like growth factor-1 (IGF-1) treatment was completely ineffective in inducing the association of alpha beta heterodimeric insulin receptors. Similarly, IGF-1 or Mn/MgATP, but not insulin, treatment of immunoaffinity-purified alpha beta heterodimeric IGF-1 receptors induced the formation of an alpha 2 beta 2 heterotetrameric IGF-1 receptor complex. A monoclonal antibody specific for the insulin receptor (MA5) completely immunoprecipitated all the insulin binding activity from both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes but did not immunoprecipitate IGF-1 receptors. Conversely, the IGF-1 receptor-specific monoclonal antibody (alpha IR-3) immunoprecipitated all the IGF-1 binding activity, but not insulin receptors. The simultaneous treatment of pooled equal amounts of alpha beta heterodimeric insulin and IGF-1 receptors with a combination of insulin and IGF-1 resulted in the formation of alpha 2 beta 2 heterotetrameric insulin and IGF-1 receptor complexes. However, in the mixed alpha 2 beta 2 heterotetrameric receptor fraction MA5 immunoprecipitated 94% of the insulin binding in addition to 27% of the IGF-1 binding activity whereas alpha IR-3 immunoprecipitated 97% of the IGF-1 binding in addition to 38% of the insulin binding activity. Treatment of the mixed alpha beta heterodimeric insulin and IGF-1 receptors with Mn/MgATP also resulted in the formation of cross-immunoreactive (42-46%) alpha 2 beta 2 heterotetrameric receptors. These data directly demonstrate the formation of insulin/IGF-1 hybrid receptors by both a combination of insulin plus IGF-1 or Mn/MgATP treatment of purified human placenta alpha beta heterodimeric insulin and IGF-1 half-receptors in vitro.
Asunto(s)
Receptor de Insulina/metabolismo , Receptores de Superficie Celular/metabolismo , Adenosina Trifosfato/farmacología , Membrana Celular/metabolismo , Cromatografía en Gel , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Sustancias Macromoleculares , Magnesio/farmacología , Manganeso/farmacología , Placenta/metabolismo , Embarazo , Multimerización de Proteína , Receptor de Insulina/aislamiento & purificación , Receptores de Superficie Celular/aislamiento & purificación , Receptores de SomatomedinaRESUMEN
We have extended these observations to examine the role of polylysine on the divalent metal ion requirement for ligand-stimulated protein kinase activity and the transmembrane signaling mechanism of both the human placenta insulin and insulin-like growth factor 1 (IGF-1) receptors. Polylysine (0.2-1 microM) was found to activate maximally the alpha 2 beta 2 heterotetrameric insulin receptor autophosphorylation and exogenous substrate protein kinase activity 25-50-fold in the presence of insulin without significantly affecting the basal protein kinase activity in the absence of insulin. The polylysine-dependent insulin stimulation of protein kinase activity required the presence of both magnesium and manganese but at relatively low divalent metal ion concentrations (0.1 mM) compared to the typical 2-10 mM Mg/Mn used in the standard in vitro kinase assays. The stimulation of the insulin receptor kinase by insulin in the presence of polylysine occurred primarily due to an increase in Vmax with no significant effect on the Km for ATP. In addition, autophosphorylated insulin receptors which are protein kinase-active and insulin-independent at high metal ion concentrations still displayed the polylysine-dependent insulin stimulation of protein kinase activity to the same extent as nonphosphorylated insulin receptors at low Mg/Mn (0.1 mM) concentrations. Surprisingly, polylysine was completely unable to stimulate the IGF-1-dependent protein kinase activity of the homologous human placenta IGF-1 receptor. These data suggest that the insulin receptor tyrosine-specific protein kinase activity may be regulated by unique endogenous basic proteins that are distinct from those which modify the IGF-1 receptor.
Asunto(s)
Insulina/farmacología , Polilisina/farmacología , Proteínas Tirosina Quinasas/metabolismo , Membrana Celular/metabolismo , Activación Enzimática , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Cinética , Magnesio/farmacología , Manganeso/farmacología , Fosforilación , Placenta/enzimología , Placenta/metabolismo , Embarazo , Receptor de Insulina/aislamiento & purificación , Receptor de Insulina/metabolismo , Receptores de Superficie Celular/aislamiento & purificación , Receptores de Superficie Celular/metabolismo , Receptores de SomatomedinaRESUMEN
The purified human placental alpha 2 beta 2 heterotetrameric insulin-receptor complex was reduced and dissociated into functional alpha beta heterodimers by a combination of alkaline pH and dithiothreitol treatment. Wheat germ agglutinin (WGA) was observed to stimulate the beta-subunit autophosphorylation of both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes in the absence of insulin. However, WGA was without effect on the insulin stimulation of beta-subunit autophosphorylation in these insulin-receptor complexes. In contrast, monomeric WGA was unable to stimulate the basal exogenous substrate protein kinase activity in either the alpha 2 beta 2 heterotetrameric or alpha beta heterodimeric insulin receptor complexes. The stimulatory effect of WGA was biphasic, increasing the basal insulin receptor beta-subunit autophosphorylation at low concentrations (250 ng/ml to 2.5 micrograms/ml) and inhibiting at high concentrations (greater than 25 micrograms/ml). Similarly, equilibrium tracer insulin binding was not significantly altered by low concentrations of WGA (less than 1 microgram/ml) but was dramatically reduced at high WGA concentrations (greater than 2.5 micrograms/ml). In contrast to the insulin-induced covalent association of the alpha beta heterodimeric insulin receptors to form a disulfide-linked alpha 2 beta 2 heterotetrameric complex, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the WGA stimulation of beta-subunit autophosphorylation in the alpha beta heterodimer preparation occurred in the absence of covalent association. Nondenaturing Bio-Gel A-1.5m gel filtration chromatography and [125I]insulin affinity cross-linking demonstrated that WGA treatment of the alpha beta heterodimeric insulin receptors induced a noncovalent association into an alpha 2 beta 2 heterotetrameric state. These data support the hypothesis that the insulin receptor protein kinase activity, although dependent upon alpha beta heterodimeric subunit interactions, does not necessarily require covalent disulfide bond formation between the individual alpha beta heterodimeric species.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Receptor de Insulina/metabolismo , Aglutininas del Germen de Trigo/farmacología , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Insulina/metabolismo , Sustancias Macromoleculares , Peso Molecular , Fosforilación , Placenta/metabolismo , Embarazo , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/aislamiento & purificaciónRESUMEN
The purified human placenta alpha 2 beta 2 heterotetrameric insulin receptor was reduced and dissociated into a functional alpha beta heterodimeric complex by a combination of alkaline pH and dithiothreitol treatment. In the presence of Mn/MgATP, insulin binding to the isolated alpha beta heterodimeric insulin receptor was found to induce the formation of a covalent disulfide-linked alpha 2 beta 2 heterotetrameric complex. In the absence of insulin, a noncovalent association of the alpha beta heterodimeric insulin receptor complex into an alpha 2 beta 2 heterotetrameric state required the continuous presence of both a divalent metal ion (Mn or Mg) and an adenine nucleotide (ATP, ADP, or AMPPCP). Thus, Mn/MgATP binding and not insulin receptor autophosphorylation was responsible for the noncovalent association into the alpha 2 beta 2 heterotetrameric state. However, the divalent metal ions or NaATP separately was ineffective in inducing the noncovalent association between the alpha beta heterodimers. The specific sulfhydryl agent iodoacetamide (IAN) was observed to inhibit the insulin-dependent covalent association of the alpha beta heterodimers without affecting the Mn/MgATP-induced noncovalent association into the alpha 2 beta 2 heterotetrameric state. Insulin treatment of the isolated alpha beta heterodimeric complex in the presence of IAN demonstrated that the Mn/MgATP-induce noncovalent association into the alpha 2 beta 2 heterotetrameric state was sufficient for insulin stimulation of beta-subunit autophosphorylation and exogenous substrate protein kinase activity. These data indicate that although interaction between the individual insulin receptor alpha beta heterodimers is necessary for insulin stimulation of protein kinase activity it does not require covalent disulfide bond formation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Placenta/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptor de Insulina/metabolismo , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Insulina/metabolismo , Sustancias Macromoleculares , Peso Molecular , Fosforilación , Embarazo , Receptor de Insulina/aislamiento & purificaciónRESUMEN
The purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex was reduced and dissociated into functional alpha beta heterodimers by a combination of alkaline pH and dithiothreitol treatment. Insulin treatment of the isolated alpha beta heterodimeric complex was observed to induce the complete reassociation to an alpha 2 beta 2 heterotetrameric state when analyzed by nondenaturing Bio-Gel A-1.5m gel filtration chromatography. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-insulin affinity cross-linked and 32P-autophosphorylated alpha beta heterodimers demonstrated that the insulin-dependent reassociation to the alpha 2 beta 2 heterotetrameric state occurred both covalently and noncovalently under these conditions. Comparison by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the insulin-dependent covalent reassociation to an alpha 2 beta 2 heterotetrameric complex was due to the formation of a disulfide linkage(s) between the alpha beta heterodimers. beta subunit autophosphorylation of the control alpha 2 beta 2 heterotetrameric insulin receptor preparation was maximally stimulated within 5 min of insulin preincubation and occurred exclusively in the Mr = 400,000 alpha 2 beta 2 complex. Similarly, maximal insulin-stimulated beta subunit autophosphorylation of the alpha beta heterodimeric preparation occurred within 5 min of insulin pretreatment in the Mr = 210,000 alpha beta complex. However, 4 h of insulin pretreatment of the alpha beta heterodimer preparation induced the formation (6-fold) of a covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Maximal stimulation of substrate phosphorylation for the alpha 2 beta 2 heterotetrameric complex was also observed to occur within 5 min of insulin treatment, whereas maximal insulin-stimulated substrate phosphorylation of the alpha beta heterodimeric complex required greater than 4 h. These data demonstrate that (i) insulin treatment can induce the reassociation of the alpha beta heterodimeric complex into a covalent alpha 2 beta 2 heterotetrameric state, and (ii) insulin-dependent protein kinase activation of the alpha beta heterodimeric insulin receptor correlates with the covalent reassociation into a disulfide-linked alpha 2 beta 2 heterotetrameric complex.
Asunto(s)
Disulfuros/metabolismo , Insulina/metabolismo , Receptor de Insulina/metabolismo , Alquilación , Cromatografía en Gel , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Humanos , Concentración de Iones de Hidrógeno , Sustancias Macromoleculares , Peso Molecular , Placenta/análisis , PolímerosRESUMEN
The dissociation of the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex into an alpha beta heterodimeric state was found to occur in a pH- and dithiothreitol (DTT)-dependent manner. Formation of the alpha beta heterodimeric complex, under conditions which preserved tracer insulin binding and protein kinase activities (pH 8.75 for 25 min followed by 2.0 mM DTT for 5 min) occurred with an approximate 50% efficiency. The resulting nondissociated alpha 2 beta 2 heterotetrameric complexes could then be separated effectively by Bio-Gel A-1.5m gel filtration chromatography at neutral pH. The isolated DTT-treated but nondissociated alpha 2 beta 2 heterotetrameric complex was resistant to any further dissociation by a second round of DTT and alkaline pH treatment, whereas the isolated alpha beta heterodimeric complex was stable to spontaneous reassociation for at least 72 h at pH 7.60. Kinetic analyses of the insulin receptor protein kinase activity demonstrated that the insulin stimulation of glutamic acid:tyrosine (4:1) synthetic polymer phosphorylation for both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes occurred via an increase in Vmax without any significant change in Km. Examination of beta subunit autophosphorylation of the alpha beta heterodimeric complex, in the presence but not in the absence of insulin, demonstrated the appearance of the covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Further, the initial rate of insulin-stimulated beta subunit autophosphorylation in the isolated alpha beta heterodimeric complex occurred in a dilution-dependent (intermolecular) manner. These data demonstrate that the isolated alpha beta heterodimeric insulin receptor complex is fully capable of expressing insulin-dependent activation of the beta subunit protein kinase domain with the covalent reassociation of the alpha beta heterodimeric complex into an alpha 2 beta 2 heterotetrameric disulfide-linked state.
Asunto(s)
Insulina/metabolismo , Receptor de Insulina/metabolismo , Ditiotreitol/farmacología , Humanos , Concentración de Iones de Hidrógeno , Cinética , Sustancias Macromoleculares , Placenta/análisis , Proteínas Tirosina Quinasas/metabolismoRESUMEN
To investigate the role of subunit communication in the insulin binding and tyrosine-specific protein kinase activities of the purified human placental insulin receptor, we have developed the methodology to isolate a functional alpha beta heterodimeric insulin receptor complex from the native alpha 2 beta 2 heterotetrameric disulfide-linked state. The dissociation of the alpha 2 beta 2 heterotetrameric insulin receptor into an alpha beta heterodimer was found to be approximately 50% efficient by treatment with alkaline pH (8.75) and dithiothreitol (2 mM). Removal of the dithiothreitol and pH neutralization (pH 7.60) by rapid Sephadex G-50 gel filtration resulted in the preservation of tracer insulin binding activity. The nondissociated alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes could then be effectively separated by Bio-Gel A-1.5m gel filtration. Scatchard analyses of insulin binding to the alpha 2 beta 2 heterotetrameric control or dithiothreitol-treated but nondissociated alpha 2 beta 2 heterotetrameric insulin receptor complexes demonstrated a curvilinear binding isotherm with a maximum of 1 mol of insulin bound/mol of alpha 2 beta 2 heterotetrameric complex. However, binding analyses performed on the isolated alpha beta heterodimeric complex yielded a nearly linear binding curve also, with 1 mol of insulin bound/mol of alpha beta heterodimeric complex at saturation. These data demonstrate that the insulin half-site binding reactivity observed in the alpha 2 beta 2 heterotetrameric insulin receptor complex results from either an asymmetric assembly of identical alpha beta heterodimers or from absolute negative cooperativity.
Asunto(s)
Insulina/metabolismo , Placenta/análisis , Receptor de Insulina/aislamiento & purificación , Cromatografía en Gel , Disulfuros , Ditiotreitol/farmacología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Sustancias Macromoleculares , Embarazo , Receptor de Insulina/metabolismoRESUMEN
The glutamic acid:tyrosine (Glu:Tyr) synthetic polymer was observed to inhibit the insulin receptor beta subunit autophosphorylation with an IC50 of 0.20 mg/ml in the absence and 0.15 mg/ml in the presence of insulin. Even though complete blockade of beta subunit autophosphorylation was observed at 4.0 mg/ml Glu:Tyr, insulin was still capable of stimulating the exogenous protein kinase activity of the insulin receptor toward Glu:Tyr. Histone H2B (1.3 mg/ml) was also observed to inhibit the beta subunit autophosphorylation by approximately 80% with an IC50 of 0.31 and 0.35 mg/ml in the absence and presence of insulin, respectively. Similar to the results with Glu:Tyr, insulin was found to stimulate histone H2B phosphorylation under these conditions. Comparisons between the time courses of beta subunit autophosphorylation with those of Glu:Tyr phosphorylation both in the presence and absence of insulin confirmed that insulin can stimulate the exogenous protein kinase activity of the insulin receptor in the complete absence of beta subunit autophosphorylation. Prephosphorylation of the insulin receptor (from 0 to 1.3 mol of phosphate/mol of insulin receptor) in the absence of insulin was found to have no significant effect on the exogenous protein kinase activity when assayed both in the presence and absence of insulin. Insulin was observed to stimulate the phosphorylation of Glu:Tyr approximately 3-fold independent of the extent of beta subunit autophosphorylation. In contrast, prephosphorylation of the insulin receptors in the presence of insulin was observed to enhance the exogenous protein kinase activity dependent on the extent of autophosphorylation, such that by 1.4 mol of phosphate incorporated per mol of insulin receptor, insulin was found to maximally stimulate the initial rate of Glu:Tyr phosphorylation (approximately 9-fold). These results demonstrate that the insulin-dependent autophosphorylation of the insulin receptor results in an amplification of the insulin stimulation of the exogenous protein kinase activity, whereas the insulin-independent autophosphorylation does not.