Clinical studies of "big ACTH": its physico-chemical characteristics.

The big ACTH fractions available from human plasma and pituitary glands and from porcine pituitary glands were physico-chemically characterized by gel filtration, disc electrophoresis and isoelectric separation. In the case of healthy human subjects, big ACTH fractions were isolated by gel filtration from plasma samples taken during states of acute ACTH hypersecretion such as the lysine-8-vasopressin, insulin or metopyrone tests though none of these fractions were isolated from plasma sampled under normal conditions. Even with no stimulation of ACTH secretion, patients with Cushing's disease gave plasma samples that contained an isolable big ACTH fraction, but such a fraction was hardly isolated from plasma taken from patient with Addison's disease. Both human pituitaries and porcine pituitaries contained an isolable big ACTH fraction. By a gel filtration analysis the molecular weight of the big ACTH was estimated to be higher than 20 000. Disc electrophoresis with an acrylamide gel indicated that big ACTH is strongly basic while small ACTH is more acidic than pH 8.3. Isoelectric separation revealed that the isoelectric point of human big ACTH is higher than pH 10.0 while that of small ACTH is about pH 6.8.     

STUDIES OF THE NATURE OF PLASMA THYROID STIMULATING ACTIVITY IN HYPERTHYROIDISM

Previous reports from this and other laboratories indicate the importance of the activity of abnormal plasma long-acting thyroid stimulator (LATS) characteristic of thyrotoxicosis in relation to the various clinical features of the disease. Studies on the nature of this agent have now been carried out by comparison of its thyroid stimulating activity in the ¹³¹I-labelled mouse with that of bovine pituitary thyrotropic stimulating hormone (TSH) and plasma from hypothyroid patients in which an excess of pituitary TSH has been demonstrated, as well as by chemical studies. A short-acting response with a maximum at three hours is characteristic of pituitary TSH. A long-acting response with maximum at eight hours or longer is characteristic of LATS. However, in the present study a long-acting response with a maximum at eight hours has been demonstrated after multiple injections of bovine TSH. A long-acting response can also be produced by injecting bovine TSH after it has been mixed with an appropriate concentration of rabbit antiserum to bovine TSH. These findings indicate that the pattern of response is not necessarily a distinguishing feature differentiating LATS and TSH. In contrast to the activity of TSH, LATS activity is unaffected by an antiserum to bovine TSH prepared in the rabbit. However, LATS activity is diminished by mixing it with an antiserum to human 7S gamma globulin prepared in the sheep, which in turn fails to affect the thyroid stimulating activity of bovine TSH on plasma from hypothyroid patients. These findings indicate that there is a distinct immunological difference between LATS and TSH. Large plasma samples from three thyrotoxic patients have been subjected to gel filtration (with the use of a “Sephadex” G-200 apparatus). LATS activity was mainly confined to the 7S gamma globulin peak; although there was a degree of overlap into the macroglobulin and albumin peaks, starch gel electrophoresis revealed 7S gamma globulin was invariably a component of these fractions. Further purification was achieved by DEAE- “Sephadex” chromatography. This produced three fractions—the first two were found to have LATS activity and to contain 7S gamma globulins on starch gel electrophoresis, while the third contained beta globulins and albumin but was devoid of 7S gamma globulin and was inactive in the mouse assay. These findings are consistent with the findings of other laboratories, in that they clearly indicate that LATS belongs to the 7S gamma globulins and is therefore probably an antibody. These data strongly support clinical evidence indicating the importance of immunological mechanisms in the pathogenesis of hyperthyroidism. The possibility of hyperthyroidism being an autoimmune disorder is discussed.     

Insulin receptor isolation studies

The observation that N^α,B1-biotinylinsulin binds firmly to resins in which succinoylavidin is covalently attached to AH Sepharose 4B and can be retrived 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^α,B1-biotinylinsulin is noncovalently attached to AH Sepharose 4B-immobilized-succinoylavidin. 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.     

Caracterisation partielle d'un facteur hypothalamique de liberation des hormones gonadotropes chez la Carpe (Cyprinus carpio L.) etude in vitro

The influence of different factors on in vitro secretion of carp pituitary gonadotrophin has been studied in a specific incubation medium. After gel filtration of acid extracts of hypothalamus and neurohypophysis on Sephadex G 25, a “releasing factor” type activity was found in fractions having a molecular weight less than 5000. This activity is different from that of oxytocin, vasopressin and the neuroamines, adrenalin, noradrenalin, serotinine and dopamine which do not stimulate gonadotrophin release from carp pituitary in vitro.Hypothalamic secretion in vitro of “releasing factor” is not increased when dopamine is added.These results confirm the existence of a hypothalamic factor which stimulates pituitary gonadotrophin secretion. The possible similarity between this factor and mammalian $\text{LH}\text{FSH-RH}$ is discussed.     

Pineal factors other than melatonin.

Some sheep pineal factors other than melatonin are described.A “nonmelatonin” antigonadotropic activity has been detected by application of the inhibition of compensatory ovarian hypertrophy (COH) in unilaterally ovariectomized adult Charles River CD-1 mice. The factor has been extracted from sheep pineals under rather simple and mild experimental conditions. This antigonadotropic activity has been partially purified by gel filtration and ultrafiltration and was localized on paper chromatograms and paper electropherograms.Inhibitory and stimulatory activities on the hypothalamic-hypophyseal system in vitro have been detected in sheep pineal fractions obtained after gel filtration and ultrafiltration.A substance with distinct fluorescence characteristics, which could not be detected in sheep cerebral cortex extracts and which differs from melatonin, has been isolated from a low molecular Sephadex G-25 fraction of an extract of sheep pineals.     

Characterization of mouse ACTH in plasma and in extracts of pituitary and of adrenotropic pituitary tumor.

The predominant component of immunoreactive ACTH in the plasma of adrenalectomized normal mice and of mice bearing the adrenotropic mouse pituitary tumor, AtT-20, and in extracts of the normal mouse pituitary and pituitary tumor, has an elution volume on Sephadex G-50 gel filtration approximately midway between the void volume and the elution volume of human ACTH (1-39 peptide). The tumor extracts are shown to contain, in addition to this intermediate ACTH, 2 other components of immunoreactive ACTH, one which coelutes with 131I-labeled albumin (big ACTH) and the other with [125I]hACTH (little ACTH). Big and intermediate ACTH are urea-stable. Controlled tryptic digestion of mouse-tumor big ACTH results within 10 seconds in conversion to an intermediate component followed by continued loss of immunoreactivity. Under the same conditions of tryptic digestion of intermediate ACTH, there is only continuous loss of immuno-reactivity with no change of hormonal form. These findings strengthen the hypothesis that mouse intermediate ACTH is not a precursor for little ACTH.     

Immunoreactive gastrin components in human serum

The apparent molecular size and charge of immunoreactive gastrin components were studied in sera from patients with pernicious anaemia or gastrinomas (the Zollinger-Ellison syndrome) by Sephadex gel filtration and aminoethylcellulose chromatography. The following serum components were distinguished: (1) a monophasic component I similar in size to proinsulin which was converted into ;little' gastrin I by trypsin digestion; (2) a biphasic component II, corresponding to ;big' gastrins I and II (Gregory and Tracy); (3) a biphasic component III corresponding to ;little' gastrins I and II (Gregory and Tracy); and (4) a biphasic component IV, corresponding to ;minigastrins' I and II (Gregory and Tracy). ;Big, big' gastrin, a plasma component found in the void volume of the Sephadex G-50 column by Yalow and Berson (1972) was undetectable in the sera investigated. A component in gastrinoma and antral mucosa extracts corresponding in size to ;big big' gastrin was detectable by the assay; the ;big big' gastrin fraction from gastrinoma tissue was heterogenous, with components of apparent MW 30 000-100 000. It is concluded that serum gastrin circulates in the form of at least four components, of which the three smaller ones are in pairs.     

Characterization of circulating insulin in insulin autoimmune syndrome

We examined the forms of circulating insulin in three patients with the insulin autoimmune syndrome by a method combining gel filtration and reverse phase high performance liquid chromatography (RP-HPLC). Insulin bound to circulating antibody was dissociated by molecular sieve chromatography at acid pH. The free insulin peak eluted from a Sephadex G-50 column was subsequently chromatographed on a Bio-Gel P-30 column. In all three patients, insulin coeluted with normal human insulin. However, when the partially purified insulins, obtained by gel filtration, were applied to RP-HPLC, an abnormally migrating insulin was found in two of three patients. The insulins were more hydrophobic than normal human, porcine, or bovine insulin, but were different from each other. A third patient had only a single insulin peak on RP-HPLC which corresponded to normal insulin. In contrast, the insulin from insulin-treated diabetic patients with antibodies to exogenous insulin corresponded to either porcine or bovine and normal human insulin. The antibodies in the circulation of these patients with the autoimmune syndrome were of the immunoglobulin G type and contained kappa and lambda-chains in the same proportions as antibodies in insulin-treated patients. Autoantibodies could not be distinguished from those secondary to exogenous insulin treatment on the basis of displacement of binding by human, beef, or pork insulin. These results suggest that in certain patients with the insulin autoimmune syndrome, there may be a molecular abnormality of circulating insulin. Whether this comprises a cause for the syndrome or is a result of posttranslational processing of insulin remains to be determined.     

Biochemical properties of big renin extracted from human plasma.

The properties of big renin, a relatively inactive form of renin isolated from human plasma, were examined following partial purification by gel filtration. Exposure of big renin to pH 3.0-3.6, or brief incubation with trypsin or pepsin, resulted in a ten-fold increase in enzymatic activity. Activation was not effected by 4M NaCl, 6M urea, or incubation with neuraminidase. Both before and after inactivation, big renin eluted from Sephadex gel more rapidly than normal plasma renin. During polyacrylamide gel disc electrophoresis, inactive big renin migrated more slowly than either normal renin or big renin previously activated. Using sheep substrate, the enzyme kinetics of normal renin and previously activated big renin were identical, while inactive big renin possessed a higher Michaelis constant. These data indicate that big renin is closely related biochemically to normal plasma renin. As the activation of big renin results in the formation of the substance even more similar to normal renin, the possibility exists that big renin may prove to be a precursor form of normal renin.     

Evidence for a gonadotropin-releasing hormone binding protein in goldfish (Carassius auratus) serum

The binding of salmon gonadotropin-releasing hormone (sGnRH) and its superactive analog, [D-Arg6, Pro9-NEt]-sGnRH, to a macromolecular component in goldfish serum was studied, using 125I-[D-Arg6, Pro9-NEt]-sGnRH and 125I-sGnRH as labeled ligands. Bound was separated from free labeled ligand by gel filtration with Sephadex G-50. The binding of labeled ligand to goldfish serum was dose-dependent. The results indicate a single class of binding site having low affinity and high capacity. The existence of a GnRH binding protein in serum may, in part, contribute to the long-lasting pharmacological action of GnRHs in goldfish.     

In vivo specific uptake of labeled insulin by liver,adipose tissue,pituitary, and adrenals in the turtle Chrysemys dorbigni

Insulin labeled with ¹²⁵I was injected into turtles (Chrysemys dorbigni) to study its specific uptake by tissues. The maximum specific uptake of radioactivity by turtle tissues was obtained 1 hr after administration of [¹²⁵I]iodoinsulin. Besides liver and adipose tissue, specific uptake of labeled insulin was detected in some endocrine glands, such as pituitary and adrenals. Both glands were as active in concentrating labeled insulin as liver and adipose tissue. A significant reduction of the uptake was observed when unlabeled insulin was injected together with the labeled hormone. This reduction was dose dependent, and the concentration of unlabeled insulin that prevented 50% of the tissue uptake of [¹²⁵I]iodoinsulin was of 1 to 10 μg/kg body weight. These doses were able to induce blood glucose decrease in the turtle. Prolactin, growth hormone, or glucagon were unable to displace labeled insulin uptake. The major proportion of the radioactive material extracted from liver and pituitary 1 hr after [¹²⁵I]iodoinsulin injection into turtle coeluted with [¹²⁵I]iodoinsulin in Sephadex G-50 column. The presence of radioactive degradation products are consistent with the intracellular receptor mediated degradation hypothesis. These findings suggest the presence of specific insulin binding sites in liver, adipose tissue, pituitary, and adrenal glands from turtles.     

Effects of insulin at two dose levels on gluconeogenesis from alanine in fasting man

We have determined the effect of insulin infused at 1 and 5 mU/kg/min on gluconeogenesis from alanine in 48-hr fasted men. The conversion of alanine to glucose was measured by the arterial-hepatic venous catheterization technique combined with the infusion of ¹⁴C-alanine. During insulin infusion, euglycemia was maintained by variable glucose infusion. When insulin was infused at 1 mU/kg/min the net splanchnic production of ¹⁴C-glucose was suppressed by 80% but glucagon infused at the end of the study resulted in substantial release of ¹⁴C-glucose from the liver suggesting marked accumulation of labeled glucose in glycogen. When insulin was infused at 5 mU/kg/min the splanchnic release of ¹⁴C-glucose was also markedly suppressed but in contrast to the lower insulin dose very little labeled glucose accumulated in glycogen. Neither the high nor the low dose insulin infusion had any effect on net splanchnic alanine uptake and plasma glucagon levels fell by 35% in both protocols. These data demonstrate that in 48-hr fasted man, (1) a small increment in insulin concentration will suppress glucose production but mostly by diverting the newly formed glucose into glycogen; (2) at higher concentrations, insulin will inhibit glucose production mainly by suppressing gluconeogenesis; and (3) this insulin-induced suppression of gluconeogenesis is due to an intrahepatic effect rather than an effect on the splanchnic extraction of alanine.     

Antibody to Transcobalamin II in Patients Treated with Long Acting Vitamin B12 Preparations

⁵⁷Co-labelled vitamin B₁₂ bound to transcobalamin I, transcobalamin II or non-protein bound, has been mixed with serum from patients with pernicious anaemia treated with long acting vitmain B₁₂ preparations. By paper electrophoresis, immunoelectrophoresis and Sephadex G-200 gel filtration, an antibody of the γG-globulin type reacting with transcobalamin II was demonstrated. By immunoelectrophoresis the antibody was demonstrated in 8 of 19 patients. Gel filtration experiments showed that the affinity of the antibody to TC II is very sensitive to changes in pH, ionic strength and temperature.     

The purification of a high-molecular-weight, enzymatically inactive renin precursor from human kidney

A high-molecular-weight enzymatically inactive form of renin has been purified to homogeneity from human kidney. It has an Mr of 48 000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and an Mr of 51 000 by gel filtration on Sephadex G100. It is activated by treatment with trypsin and reversibly activated by exposure to acid. We conclude that this material represents a human prorenin.     

Electrophoretic characterization of circulating human proinsulin and insulin

Summary The serum proinsulin and insulin components of normal weight subjects treated with tolbutamide, or glucose and tolbutamide, were shown to be homogeneous on both Sephadex filtration and polyacrylamide gel electrophoresis. Serum of obese subjects treated with glucose and tolbutamide contained, in addition to proinsulin and insulin, two intermediate species when electrophoresed on polyacrylamide gel, i.e., desdipeptide proinsulin and diarginyl insulin. It is suggested that, 1. the conversion of proinsulin to insulin proceeds via these intermediates under physiological conditions, and 2. intermediates may appear in the circulation with excessive elevations of plasma immunoreactive insulin materials.Supported by VA Funds and NIH Grant # AM 11578.     

HUMAN INSULIN1

Two methods of synthesis of human insulin have been developed to the stage of commercial production. One entails synthesis of human insulin by bacteria (“biosynthetic” human insulin) and one entails the conversion of pork insulin into human insulin by an amino acid substitution (“semisynthetic” human insulin). Both forms of human insulin have been shown to be safe, and to have similar efficacy and pharmacokinetics to purified pork insulin. Human insulin given by subcutaneous injection has been shown to elicit antibody formation in man, although the extent of this may be slightly less than in the case of purified pork insulin, and is certainly less than in the case of beef insulin and beef-pork combinations. The clinical significance of these differences in immunogenicity is doubtful. However there are rare defined situations, such as allergy to pork insulin and antibody-mediated insulin resistance where human insulin has been shown to have advantages over purified pork insulin. Bacterial synthesis of insulin provides an assured supply for the world's future needs, and it is conceivable that further technological refinement may make bacterial synthesis cheaper than extraction and purification of animal insulins. However, current evidence provides no basis for recommending human rather than purified animal insulin for the routine management of insulin dependent diabetes.     

Purification and kinetic properties of the soluble Mn2+-dependent adenylyl cyclase of the rat testis

The soluble Mn²⁺-dependent adenylyl cyclase (AC) of the rat testis was purified 1500-fold with some 19% yield of the initial activity. These results were accomplished by conventional separation techniques including (NH₄)₂SO₄ precipitation of testis cytosol (106 000 × g), gel filtration (Sephadex G-200) and ion-exchange chromatography (Sephadex DEAE-A50) followed by Sephadex G-100 gel filtration and isoelectric focusing.Analysis by polyacrylamide-gel electrophoresis (PAGE) of aliquots from each purification step revealed the following. (a) The Mn²⁺-dependent AC migrated with a R_f value of 0.40 irrespective of the degree of purification. (2) The AC peak from the isoelectric focusing column separated into 2 major protein bands; however, only one band (R_f 0.40) had AC activity.The molecular weight emerging from the position of migration on the Sephadex G-200 and G-100 columns appeared to be consistent at 47 000–48 000 D, as estimated from the relationship log MW versus elution volume.The purified enzyme fulfilled the requirements for a simple Michaelis-Menten kinetics with an apparent Km for Mn²⁺ and MnATP^2? of 6.7 and 2.5 mM, respectively. Varying the concentrations of ATP or Mn²⁺ separately did not alter the apparent affinity (K_m^app) for the other parameter.These and previous data from our laboratory show that the physico-chemical and kinetic properties (molecular weight and K_mapp for Mn²⁺ and MnATP^2?) do not alter during purification. Furthermore, the additional step of affinity chromatography seems obligatory if a homogeneous AC preparation is to be obtained.     

Regulation of insulin receptors in erythroid cells

Previous studies using inhibitors have suggested that protein synthesis is necessary for “down-regulation” of insulin receptors. We have tested this hypothesis without the use of inhibitors by studying the ability of cells of the erythroid series to down-regulate their insulin receptors in vitro. The cells tested include mature erythrocytes and reticulocytes from rabbits and Friend erythroleukemia cells (a model for the basophilic erythroblast, a primitive nucleated erythrocyte). All cells were maintained at 37°C for 18 hr ± insulin (10^?8M). Cultures were then incubated with phosphate buffered saline (pH 7.0) at 30°C for 40 min to remove bound insulin. Receptors were quantitated by computerized analysis of Scatchard plots of subsequent insulin binding studies. Cells fully capable of both mRNA synthesis and protein synthesis, such as the undifferentiated and differentiated Friend erythroleukemia cell, had reductions in insulin receptors of 60% and 43%, respectively. Reticulocytes, which were capable of protein synthesis but not mRNA synthesis, had decreases of 25%–30% in 8 separate experiments. Mature erythrocytes, capable of neither RNA nor protein synthesis had no significant change in receptor concentration. Since mature erythrocytes do not “down-regulate” their insulin receptor concentration, studies of these receptors in erythrocytes of patients should be interpreted with caution.     

Characterization of immunoreactive insulin in human saliva: evidence against production in situ

Summary The presence of insulin immunoreactivity in extrapancreatic tissues and fluids suggests multiple sites of insulin production. Immunoreactive insulin occurs in human saliva and concentrations increase after oral glucose ingestion. The goal of these experiments was to determine whether the presence of immunoreactive insulin in this extra-pancreatic site is independent of pancreatic production or merely represents the accumulation of circulating pancreatic insulin. The mean ±SEM concentration of extracted salivary immunoreactive insulin in five normal volunteers increased during an oral glucose tolerance test from basal values of 36±3.0 to 291±40pmol/l; however, the peak occurred 45–90 min later than in serum. On this basis, it was not possible to distinguish between the stimulation (by increased blood glucose concentrations) of insulin synthesis in the saliva glands from the accumulation of blood insulin. Therefore, we studied a group of five volunteers during intravenous infusion of insulin (1 and 10 mU·kg^–1·min^–1, sequentially) and glucose (euglycaemic clamp). Under these conditions, salivary immunoreactive insulin concentrations increased significantly from 254±100 to 1919±437 pmol/l (p<0.05), while simultaneous mean plasma C-peptide concentrations were unchanged. Thus, the concentration of salivary immunoreactive insulin was clearly related to the amount of insulin in the blood and not to the plasma glucose concentration. Physico-chemical and immunological characterization of salivary immunoreactive insulin by dilution in radioimmunoassay, gel filtration and polyacrylamide disc gel electrophoresis demonstrated that the majority of it was indistinguishable from insulin standards. We conclude that salivary insulin is accumulated from the blood and there is no reason to suggest that it is synthesized elsewhere than in the pancreas.     

Plasma clearance of intravenously administered pituitary human growth hormone: gel filtration studies of heterogeneous components

Both pituitary and plasma human GH (hGH) comprise heterogeneous components, exhibiting similar patterns when gel filtered on Sephadex G-100. To determine at what rate the components are cleared from the circulation, blood was obtained at specific intervals following a bolus injection of pituitary hGH in hypopituitary patients. Each sample was gel filtered to determine its component profile of RIA values, which, when plotted vs. the time interval it represented, yielded a means of monitoring its disappearance from the plasma. Total hGH was cleared with a t 1/2 of 21.5 min, the little component was cleared at 19.0 min, the big component was cleared at 26.5 min, and the pre-big component was cleared at 45 min. These data indicate that the larger the hGH component, the longer it takes to be cleared from the plasma.