Isolation and characterization of two adenosine 3',5'-monophosphate-dependent protein kinases from bovine adrenal cortex.

Bovine adrenocortical cytosol contains two cAMP-dependent protein kinases separable by diethylamino-ethyl-cellulose chromatography. The kinases exhibit behavior characteristic of type I and type II enzymes i.e. the enzyme eluting at 80 mM NaCl is activated by NaCl and histone, whereas the enzyme eluting at 140 mM NaCl is not. In addition, a third cAMP-binding protein eluting from DEAE at 120 mM NaCl has been isolated and tentatively identified as type I regulatory subunit. A method is described for the isolation of the catalytic and regulatory subunits of both enzymes from the same starting material using cGMP to dissociate the enzyme subunits and elute the regulatory subunits from N6-aminoethyl-cAMP-Sepharose 4B. The isolated proteins were homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis. Using this technique, molecular weight values of 44,000 for the catalytic subunits, 51,000 for the cAMP-binding protein and type I regulatory subunit, and 56,000 for the type II regulatory subunit were obtained. Molecular weights obtained by sucrose density gradient centrifugation on the cGMP-dissociated holoenzymes were 95,000 and 101,000 for regulatory subunits from type I and II enzymes and 34,000 and 33,000 for their respective catalytic subunits. The apparent Km and Vmax values for catalytic subunits I and II were similar when histone, casein, or ATP was used as substrate. The Vmax for protamine phosphorylation was 3-fold higher with catalytic subunit II. Phosvitin was not a substrate for either subunit. The cAMP-binding protein and regulatory subunit I were able to recombine with, i.e. inhibit, either catalytic subunit. At a molar ratio of 4:1, inhibition was total. The type II regulatory subunit was considerably less effective. Preference for a particular catalytic subunit was not evident in the case of inhibition by either regulatory subunit or the cAMP-binding protein.

Ascorbic acid transport by isolated bovine adrenal cortical cells.

Viable, ACTH-sensitive, bovine adrenal cortical cells have the ability to accumulate [1-14C]ascorbic acid. Transport of the vitamin displays saturation kinetics (Km = 16.6 microM), requires Ca2+, and is inhibited maximally by ACTH at the same concentrations of the hormone necessary for maximal stimulation of steroidogenesis. Despite the transport of quantities of radioactive ascorbate amounting to approximately 25% of the total intracellular pool, no change in the ascorbate concentration in the cells can be detected by chemical methods. Transport of the vitamin is apparently a membrane carrier-mediated exchange process, since although intracellular ascorbate concentrations are 60- to 90-fold higher than those in the incubation medium, accumulated label can only be displaced when the cells are incubated in the presence of unlabeled ascorbic acid or metabolic inhibitors. Furthermore, ACTH fails to deplete ascorbic acid from [1-14C]ascorbate-loaded cells. It is hypothesized that the vitamin exists in two pools only one of which can be depleted by ACTH. This ACTH-depletable pool is no longer present in the isolated cell. In fact, as much as 66% of the ascorbic acid is lost from rat adrenals incubated for 15 min post adrenalectomy. The remaining pool of adrenal ascorbate is not depleted by ACTH and is also maintained against a concentration gradient.

Identification of a protein in adrenal particulates that binds adrenocorticotropin specifically and with high affinity.

This paper is concerned with the identification and isolation in cross-linked form of a protein of bovine adrenal cortical particulates that binds the ACTH probe 125I-[Phe2,Nle4,DTBct25]ACTH-(1-25) amide specifically, reversibly, and with high affinity. This protein may well represent the long sought, adenylate cyclase-linked, low affinity ACTH receptor or a portion thereof. Evaluation of the binding data by Scatchard analysis afforded a linear plot corresponding to a dissociation constant of 2.7 X 10(-9) M with a single class of binding sites. Competitive binding studies using nonradioactive ACTH analogs served to establish the specificity of the binding. ACTH-(1-24), was the most active competitor, followed by [Gln5]ACTH-(1-20) amide, [Gln5,Phe9] ACTH-(1-24), and ACTH-(11-20) amide, the weakest binder of the group. These findings correlate well with the ability of the peptides to stimulate cAMP formation in bovine adrenal cortical cells, i.e. ACTH-(1-24) greater than [Gln5]ACTH-1-20) amide greater than [Gln5,Phe9]ACTH-(1-24). ACTH-(11-20) amide is biologically inactive but inhibits ACTH-(1-24)-stimulated adenylate cyclase with a 50% inhibition ratio of 400:1. Nonspecific binding was suppressed by inclusion in the incubates of the protease inhibitors pepstatin, bacitracin, and benzamidine. The binding protein was cross-linked to the radioactive probe with disuccinimidyl suberate with a high cross-linking yield. The cross-linked material was solubilized with sodium dodecyl sulfate (SDS), and the 100,000 X g supernatant was subjected to SDS-polyacrylamide gel electrophoresis, followed by a autoradiography. The gel showed the presence of a band corresponding to an apparent mol wt of 43,000 (assuming a molecule of ligand bound). This band was absent when cross-linking was performed in the presence of unlabeled ACTH-(1-24). Similar results were obtained when cross-linking was performed with dithiobis (succinimidyl)propionate or ethyleneglycolbis (succinimidyl)succinate. The soluble cross-linked material bound to a column of succinoylavidin Sepharose and could be eluted with guanidinium chloride at pH 1.5. SDS-polyacrylamide gel electrophoresis and autoradiography of the affinity-purified material afforded the same pattern as the unpurified material; however, considerably more radioactivity was present in the high mol wt region of the gels.

Ampholyte displacement and high pressure liquid chromatographic separation of the estrogen-responsive neurophysin from human plasma.

Estrogen-stimulated neurophysin (ESN) or oxytocin (OT)-neurophysin (Np) was measured in plasma of seven men before and after oral administration of 25 mg diethylstilbestrol (DES). Pre-DES levels of ESN averaged 0.93 +/- 0.3 (+/- SEM) ng/ml and increased to 29.8 +/- 6.5 and 25.4 +/- 5.1 ng/ml 24 and 48 h after DES treatment, respectively. To compare the estrogen-responsive Np in plasma with human OT-Np which is present in the posterior pituitary gland, the Np fraction of post-DES plasma was concentrated by double precipitation with ammonium sulfate and applied to ampholyte displacement and Sephadex G-75 columns. The Np fraction of this plasma extract contained ESN immunoreactivity (IR) but no nicotine-stimulated neurophysin-IR. ESN-IR of plasma and of an extract of human posterior pituitary eluted identically from a Sephadex G-75 column, indicating similar mol wt. The plasma extract containing ESN-IR eluted from the ampholyte displacement column at pH 4.3-4.2. No nicotine-stimulated Np (arginine vasopressin-Np)-IR was found in the plasma samples. ESN-IR in an extract of human posterior pituitary gland eluted from the ampholyte displacement column at the same pH as that of the ESN extracted from plasma. Peak ESN-IR-containing fractions from the ampholyte displacement were pooled, dialyzed, lyophilized, and reconstituted in appropriate carrier buffer for reverse phase high pressure liquid chromatography. The ESN-IR was resolved into two distinct ESN-IR peaks by high pressure liquid chromatography. Plasma and posterior pituitary gave identical pairs of peaks. Thus, the Np that is increased in human plasma in response to estrogen is identical to pituitary OT-Np, providing strong evidence that estrogen stimulates the human neurohypophysis.

A novel oxytocin-like and vasotocin-like peptide in human plasma after administration of estrogen.

Using three antisera to oxytocin (OT Pitt Ab-1, OT Pitt Ab-2, and TOR OT Ab), we found comparable levels of OT in response to infant suckling and during infusion of synthetic OT, and identical standard curves with biological and synthetic standards of OT. Pitt Ab-1, but not Pitt Ab-2 or TOR OT Ab, measured increased OT in response to estrogen. Using an arginine vasotocin RIA (TOR AVT Ab), we found an increase in AVT immunoreactivity after estrogen treatment. Mean basal OT levels measured with OT Pitt Ab-2 in plasma of men [0.75 +/- 0.06 (+/- SEM) microU/ml] and women (0.8 +/- 0.09 microU/ml) were lower than OT measured with Pitt Ab-1 (1.7 +/- 0.09 microU/ml in men and 1.7 +/- 0.07 microU/al in women; P less than 0.001). Mean OT measured with Pitt Ab-2 in the plasma of women given estrogen chronically (0.8 +/- 0.04 microU/ml) and acutely (0.6 +/- 0.15 microU/ml) were not significantly different from basal levels. OT levels measured with Pitt Ab-1 in the same samples were 4.6 +/- 0.5 and 4.3 +/- 0.5 microU/ml, respectively, both significantly increased from basal levels (P less than 0.001) and significantly higher than OT measured with Pitt Ab-2 (P less than 0.001). Mean OT measured with Pitt Ab-1 in the plasma of pregnant women was 8.6 +/- 1.02 microU/ml, significantly higher than OT measured with Pitt Ab-2 (1.0 +/- 0.3 microU/ml; P less than 0.001). Men given 25 mg diethylstilbestrol had significant increases in OT measured with Pitt Ab-1 and in AVT measured with TOR AVT (P less than 0.01), but not in OT measured with Pitt Ab-2. Plasma from a man given diethylstilbestrol was prepared for high performance liquid chromatography and applied to a C18 muBondapak reverse phase column. The plasma contained two peaks of immunoreactivity detected as OT with Pitt Ab-1 and as AVT using TOR AVT Ab. The material was not detected by Pitt Ab-2 or TOR OT Ab and did not coelute with standards of OT, AVT, or AVP. Pregnancy plasma, thioglycolic acid, chymotrypsin, and trypsin reduced Pitt Ab-1, Pitt Ab-2, and TOR OT immunoreactivity of synthetic OT. The percent recovery of OT immunoreactivity was not significantly different with Pitt Ab-1 vs. Pitt Ab-2. A novel peptide, which is increased in response to administered estrogen, is present in human plasma and is detected by some antisera to OT and AVT. The observation explains the wide variability in OT levels in the estrogen-primed state and provides a new mechanism to study estrogen-related physiology and pathophysiology.

High pressure liquid chromatographic separation of an oxytocin/arginine vasotocin-like peptide from the plasma of patients with chronic renal failure.

Levels of a novel oxytocin (OT)- and arginine vasotocin (AVT)-like peptide detected by one antiserum to OT (Pitt Ab-1) and one antiserum to AVT (Tor AVT) were recently found to rise in human plasma in response to administration of estrogen. The novel peptide rose in parallel with the estrogen-stimulated neurophysin (ESN). The mean level (+/- SEM) of ESN in plasma of 11 individuals with altered renal function (nondialyzed) was significantly higher than the level in individuals with normal renal function (4.2 +/- 0.9 vs. 1.1 +/- 0.04 ng/ml; P less than 0.01). In patients treated with hemo- or peritoneal dialysis, mean (+/- SEM) levels of ESN were 18.1 +/- 3.2 and 16.8 +/- 3.7 ng/ml, respectively. Levels of estradiol and estrone were not elevated and did not correlate with high levels of ESN. Levels of OT Pitt Ab-1, AVT, and ESN immunoreactivity were measured in plasma form nine patients undergoing hemodialysis and eight patients undergoing peritoneal dialysis. Mean (+/- SEM) levels of all three peptides were elevated (12.9 +/- 1.5 microU/ml, 32.1 +/- 6.7 pg/ml, and 13.5 +/- 4.0 ng/ml, respectively). ESN was significantly correlated with OT Pitt Ab-1 and AVT (R2 = 0.80; P less than 0.001). Plasma samples from the same patients were pooled, treated, and separated by reverse phase HPLC. The plasma contained a peak of immunoreactivity detected by Pitt Ab-1 and Tor AVT Ab. The position of the material was distinct from that of synthetic OT, AVT, or AVP and corresponded to the position of the novel OT-like peptide found in plasma of individuals given estrogen. The findings support parallel secretion of the OT-like peptide with ESN and represent the first disease state characterized by high levels of this OT- and AVT-like peptide.

Insulin receptor isolation studies.

The observation that N alpha,B1-biotinylinsulin binds firmly to resins in which succinoylavidin is covalently attached to AH Sepharose 4B and can be retrieved by exposure of the resins to 20 mM biotin provided the basis for the present investigations. Solubilized, partially purified insulin receptor from human placenta binds to affinity resins in which N alpha,B1-biotinylinsulin is noncovalently attached to AH Sepharose 4B-immobilized-succinolylavidin. Exposure of the receptor loaded resin to 20 mM biotin results in liberation of a high molecular weight material containing bound 125I-biotinylinsulin, which precipitates with polyethyleneglycol and cross reacts with human insulin receptor antibodies. The technique is biospecific and appears to be applicable to the purification of insulin receptors on a preparative scale. Crude solubilized insulin receptor from human placenta is contaminated with "insulinase" which is inhibited by N-ethylmaleimide. HPLC provides a tool to assess "insulinase" activity that is more sensitive than the TCA precipitation method.

The plasma of pregnant women contains a novel oxytocin-vasotocin-like peptide.

Basal levels of immunoreactive oxytocin (OT) were measured in plasma of healthy pregnant women using two antisera to OT, Pitt Ab-1 and Pitt Ab-2, and an antiserum to arginine vasotocin, Tor AVT Ab. The mean (+/- SEM) level of immunoreactive OT Pitt Ab-1 was significantly higher, 7.7 +/- 0.9 microU/mL, than immunoreactive OT Pitt Ab-2, 0.9 +/- 0.2 microU/mL, P less than .001 measured in the same samples. AVT immunoreactivity in plasma of nonpregnant individuals was 0.8 +/- 0.16 pg/mL and in plasma of women in late pregnancy was 5.0 +/- 0.4 pg/mL. In four pregnant women receiving an infusion of synthetic OT (Pitocin, Parke-Davis, Morris Plains, NJ) a linear correlation was found between the dose of OT infused and the concentration of OT in plasma in samples measured with Pitt Ab-2, but no correlation was found in the same samples measured with Pitt Ab-1. Immunoreactive OT Pitt Ab-1 in plasma was not destroyed by a 60-minute incubation with pregnancy plasma. Pooled plasma from pregnant women was separated by reverse phase high pressure liquid chromatography (HPLC). OT Pitt Ab-1 and Tor AVT immunoreactivities in pregnancy plasma eluted in a position separate from synthetic OT. The differences found in levels of immunoreactive OT in the same samples of plasma measured with two antisera to OT illustrate an important reason why levels of OT in pregnant women may be reported to be variable among laboratories.

Oxytocin and vasopressin are present in human and rat pancreas.

Immunoreactive oxytocin and vasopressin were found in human and rat pancreatic extracts. The pancreatic oxytocin and vasopressin eluted on Sephadex G-75 gel filtration chromatography and on reverse phase high pressure liquid chromatography in the same positions as their respective reference preparations. The immunoreactive oxytocin was biologically active in the rat milk ejection assay. The presence of oxytocin and vasopressin in human and rat pancreatic extracts suggests the possibility of local synthesis of both hormones. The neurohypophysial hormones are known to be endocrine mediators of insulin and glucagon release. The finding of oxytocin and vasopressin in the pancreas raises the possibility, although yet unproven, of local synthesis and perhaps a paracrine function for the neurohypophysial peptides upon pancreatic hormone release or for a local function upon the liver.

Development of affinity resins for isolation of angiotensin receptors.

The development of affinity resins for the isolation of angiotensin II receptors from adrenal fasciculata cells is described. The approach is based on the avidin-biotin interaction. The advantages of the technique are delineated.

ATP sensitizes the insulin receptor to insulin.

Insulin receptor with high insulin binding and tyrosine kinase activities has been prepared from human placenta. Based on a molecular mass of 306 kDa for the receptor (the value obtained from the sum of the amino acid residues), this preparation is capable of binding 1.48 mol of insulin per mol of receptor. The receptor is free from phosphatase and ATPase activity and is not stimulated by sodium vanadate. Autophosphorylation is linear with respect to receptor concentration, and the 32P incorporated is stable even in the presence of a 100-fold excess of unlabeled ATP. The Km for ATP is 208 microM. N-Ethylmaleimide inhibits autophosphorylation. Alkylation with 3H-labeled N-ethylmaleimide results in the incorporation of 1.13 +/- 0.37 mol of N-ethylmaleimide per mol of insulin binding activity exclusively into the beta subunit of the receptor. The nonhydrolyzable ATP analog adenosine 5'-[beta,gamma-imido]triphosphate stimulates autophosphorylation of the receptor, an effect that is evident at ATP concentrations below 1 mM. The stimulatory effect of adenosine 5'-[beta,gamma-imido]triphosphate is the result of increasing the binding of insulin to the alpha subunit, and this reflects itself in a shift to the left of the insulin dose-response curve for autophosphorylation. The same is true for ATP. As a consequence, it is now possible to reconcile the concentration of insulin necessary for stimulating the autophosphorylation reaction with physiological levels and with the levels of insulin required for its classical biological effects.

Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.

The ligand N alpha, B1-(6-biotinylamido)hexanoyl-insulin was attached noncovalently to Sepharose 4B immobilized succinoylavidin to form an insulin-affinity resin. This resin was used to isolate highly purified insulin receptor from human placental tissue by a four step process involving (i) preparation of a crude membrane fraction, (ii) solubilization with Triton X-100, (iii) wheat germ agglutinin purification, and (iv) insulin-affinity chromatography. NaDodSO4/PAGE of the purified 125I-labeled receptor under nonreducing conditions showed the presence of a major component with an approximate molecular weight of 350,000 and a minor component with a molecular weight of approximately equal to 166,000. Based on the assumption that the degree of labeling is comparable in both components, the material corresponding to the Mr 350,000 peak represents approximately equal to 94% of the receptor preparation as determined by scanning the autoradiograms. The specific insulin binding capacity of the preparation is 18 +/- 6 micrograms of 125I-labeled insulin per mg of protein as determined by the polyethylene glycol assay and analyzed by Scatchard plot. Insulin binding activity was stable at 4 degrees C and pH 7.6 for at least 12 weeks but was destroyed by freezing and thawing. The availability of highly purified receptor afforded the opportunity to explore its precipitability by polyethylene glycol under assay conditions. Whereas trichloroacetic acid precipitated 95% of the 125I-labeled receptor, polyethylene glycol precipitated only 30%. If the specific activity of the receptor is corrected for incomplete precipitability by polyethylene glycol, the apparent specific binding would be 3.5 +/- 1.2 mol of insulin per mol of receptor. These results are in disagreement with the current receptor model, which postulates that 1 mol of receptor (Mr, 350,000) binds 2 mol of insulin. Clearly, the problems associated with the method available for determining insulin binding are sufficiently serious to preclude their use in determining receptor valence.

Ligands for insulin receptor isolation.

Biotinylated insulins are bivalent molecules having the ability to bind to insulin receptors on the one hand and to "avidins" on the other. In order to be useful as ligands for insulin receptor isolation, biotinylated insulins must be developed that have the capacity to bind simultaneously to both and insulin receptor. The present investigation addresses this problem. A series of biotinylated and dethiobiotinylated insulins has been prepared in which the distance between the biotin carboxyl group and the insulin varies from 7 to 20 atoms. These compounds form complexes with succinoylavidin. The dissociation rates (K-1) of these complexes have been determined from the [14C]biotin exchange assay. The dissociation kinetics of most of these complexes are biphasic, and the kinetic constants reported are those corresponding to the slow rate. Ligands containing dethiobiotin dissociate more rapidly than the corresponding biotin derivatives. The interposition of a spacer arm substantially decreases the rate of dissociation. The [14C]biotin exchange assay could not be used with streptavidin complexes of the above ligand since biotin dissociates more rapidly from streptavidin than from succinoylavidin. However, the relative dissociation rates of a series of ligands could be determined and were as follows: 6-(dethiobiotinylamido)-hexanoic acid greater than dethiobiotinyl-A1-insulin greater than biotinylinsulin greater than biotinyl-A1-insulin greater than biotinyl-A2-insulin. Dethiobiotin and its amide failed to form complexes with streptavidin. The affinity of the ligands for insulin receptors was determined by measuring their ability to stimulate 14CO2 formation from [1-14C]glucose in rat epididymal adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Labile disulfide bonds in human placental insulin receptor.

The disulfide crosslinking pattern of human placental insulin receptor was investigated using selective reduction with tributylphosphine followed by alkylation with N-[3H]ethylmaleimide. Insulin receptor contains a single sulfhydryl group in each beta subunit whose alkylation with N-[3H]ethylmaleimide inhibits receptor autophosphorylation. Alkylation is partially inhibited by ATP or the nonhydrolyzable substrate analog adenosine 5'-[beta,gamma-imido]triphosphate when the nucleotides are added as Mn2+ complexes. Neither insulin nor 6 M guanidinium chloride renders additional sulfhydryl groups accessible to alkylation. When the receptor is reduced under drastic conditions with tributylphosphine in guanidinium chloride, 32 of the 37 sulfhydryl groups in the receptor's alpha subunit can be alkylated with N-[3H]ethylmaleimide. Surprisingly only three of the 10 cysteines in the beta subunit become titratable under identical conditions. By using highly selective reducing conditions, we were able to determine quantitatively the maximum number of disulfide bridges that link the two alpha beta halves to form the tetrameric structure and those that couple the alpha to the beta subunits. Liberation of two sulfhydryl groups in the alpha and one in the beta subunit resulted in formation of alpha beta dimers. Free beta subunit was formed when an additional disulfide bond was reduced. It is remarkable that the tetrameric structure of this highly complex receptor molecule, which contains a large number of cysteine residues, is maintained by such a small number of disulfide bonds. Three models of the arrangement of the labile disulfide bonds, consistent with these findings, are proposed.

Angiotensin stimulation of adrenal fasciculata cells.

In this paper we provide evidence to show that the pathways by which adrenocorticotropic hormone (ACTH) and angiotensin II (AII) stimulate steroidogenesis in bovine fasciculata cells are only partially independent. Both hormones have the same intrinsic activity but a 500-fold higher dose of AII is required to achieve 50% stimulation of steroidogenesis. Whereas ACTH acts by way of cAMP, AII appears to operate through protein kinase C. The phorbol ester, 12-O-tetradecanoylphorbol-13 acetate (TPA), and the calcium ionophore, A23187, each stimulate steroidogenesis and, when added together, act synergistically. To test the relationship between the ACTH and AII pathways, we added the two hormones simultaneously and measured steroid production. When the hormones were present at submaximal concentrations, their effects were additive. At maximal doses, steroid production was 40% above that elicited by either hormone alone. In contrast to the action of AII in the glomerulosa cell where it inhibits ACTH-stimulated cAMP formation, AII causes no inhibition in the fasciculata. Cycloheximide inhibits steroidogenesis stimulated by AII or a mixture of TPA and A23187. Scatchard analysis of the binding of 125I-AII to particulates from adrenal cortical fasciculata indicates the presence of a single class of binding sites (Kd = 0.6 X 10(-8) M). Binding is not inhibited by ACTH. Biotin-containing AII analogs that bind specifically to the particulates have been evaluated as potential tools for avidin-biotin affinity chromatography of the receptor. One of these, [N epsilon-6-(biotinylamido)hexyllys1, Val5] AII, is a promising candidate for receptor isolation.

Hormonal properties of avidin-biotinylinsulin and avidin-biotinylcorticotropin complexes.

In connection with the development of affinity columns (based on the avidin-biotin interaction) for retrieval of peptide and protein hormone receptors, the hormonal properties of a number of avidin-biotinylinsulin and avidin-bioinylcorticotropin complexes were examined. Of particular interest was an evaluation of streptavidin as a ligand for the attachment of biotinylated hormones to solid supports and its possible advantage over SpHPP-avidin (S = succinoylated; pHPP = 3-(p-hydroxyphenyl)propionyl). As concerns binding kinetics using rat liver plasma membranes, streptavidin was found superior to avidin since it does not display apparently nonsaturable binding. Scatchard analyses of the binding of 125I-streptavidin, 125I-S-streptavidin and 125I-SpHPP-avidin to rat liver plasma membranes gave KD value of 6.7, 13.2, and 10.6 nM respectively. The binding was saturable and the unlabeled proteins competed with their labeled counterparts for the membrane binding sites. Biotinylinsulin, attached to either streptavidin or SpHPP-avidin was able to compete for 125I-insulin-binding sites on rat liver plasma membranes though somewhat larger concentrations of the complexes than of insulin were required to achieve comparable inhibition. The ID50 values for insulin and the biotinylinsulin complexes were 5 and 80 nM respectively. Biotinylcorticotropin was found to be a more effective activator of particulate rat adrenal adenylate cyclase when complexed with unmodified avidin than with streptavidin, S-streptavidin or SpHPP-avidin.

Synthetic tools for adrenocorticotropin receptor identification.

Biotinylated photoaffinity derivatives of adrenocorticotropin (ACTH) are potentially useful tools for the identification of ACTH receptors. The hormone can be attached covalently to its receptor by photoactivation, and the presence of biotin in the molecule facilitates isolation of the solubilized hormone-receptor complex on columns of immobilized succinoylavidin (Suc-avidin). Six photoprobes of ACTH1-24 have been prepared by reacting ACTH1-24, [25-biocytin]ACTH1-25 amide, and [25-dethiobiocytin]ACTH1-25 amide with either 4- or 5-azido-2-nitrophenylsulfenyl (4-NAPS and 5-NAPS, respectively) chlorides in acetic acid. The homogeneity of the photoprobes was carefully monitored by thin-layer chromatography and amino acid analyses of acid hydrolysates. The presence of underivatized starting material in the photoprobes was critically scrutinized by high-pressure liquid chromatography and was estimated to be less than 0.5%. Both the 4- and 5-NAPS derivatives stimulated maximal steroidogenesis (as compared with ACTH1-24) in calf adrenal cortical cells. However, the potencies of the two isomers differed significantly. The ED50 for steroidogenesis with 5-NAPS-ACTH1-24 was 100-fold greater than the standard (ACTH1-24) while that for 4-NAPS-ACTH1-24 was only approximately 7 times greater. Although 4-NAPS-ACTH1-24 was capable of stimulating maximal adenosine cyclic 3',5'-phosphate (cAMP) production, the 5-NAPS derivative was usually not. The level of stimulation with the 5-NAPS derivative varied considerably from cell preparation to cell preparation. ACTH1-24-induced cAMP production was inhibited by 5-NAPS-ACTH1-24 or 5-NAPS-[25-dethiobiocytin]ACTH1-25 amide.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding and autophosphorylating activity of human insulin analogs.

Insulin receptor binding and autophosphorylating activities of a number of synthetic analogs of human insulin have been examined using highly purified insulin receptor from human placenta. In general, autophosphorylation correlates well with the ability of the analogs to stimulate glucose oxidation and to inhibit lipolysis in adipocytes although their biological activities varied over a wide range. These findings support the hypothesis that autophosphorylation is an obligatory step in the pathways leading to glucose oxidation and inhibition of lipolysis. The relative biological potencies of the analogs in the autophosphorylation assay also correlated well with their receptor-binding affinities except for the peptides [endo-TyrB16a]insulin, in which an additional Tyr has been inserted between TyrB16 and LeuB17 and [ProA2]insulin. The relative receptor binding affinity of [endo-TyrB16a]insulin is significantly greater than its biological activity in the adipocyte or receptor autophosphorylation assays. The converse is true for [ProA2]insulin. These results demonstrate that the amino-acid residues involved in binding and receptor activation may not be identical.