Immunological reactivity of insulin to sepharose coupled with insulin-antibody—Its use for the extraction of insulin from serum

Akanuma, Yasuo; Kuzuya, Tsunehiko; Hayashi, Masaki; Ide, Takehiko; Kuzuya, Nobusada

doi:10.1016/0006-291x(70)90813-2

Cited by 59 publications

(15 citation statements)

References 3 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, the progress made thus far indicates that their complete isolation is well within reach. Immunoabsorption methods, which have proven so successful in the rapid purifica tion of specific proteins from complex mixtures [32,33], should prove quite helpful when adequate supplies of monospecific antibodies to the PAPPs become available. Preliminary experiments on PAPP-C in our laboratory has indicated its feasibility.…”

Section: Discussionmentioning

confidence: 99%

Three Pregnancy-Associated Human Plasma Proteins: Purification, Monospecific Antisera and Immunological Identification

Lin

Halbert

Kiefer

et al. 1974

Int Arch Allergy Immunol

View full text Add to dashboard Cite

Three human pregnancy-associated plasma proteins (PAPPs), which were detected only during pregnancy with appropriately absorbed antisera to pregnancy plasma, were purified from third trimester plasma. The authors employed combinations of sequential DEAE-cellulose and hydroxylapatite chromatography; Sephadex G-100, G-200 and Sepharose 6B gel filtration; isoelectric focusing; and/or ammonium sulfate salting out. Each final product was shown to contain only one PAPP, to be immunologically free of most normal plasma proteins, and to have specific activities 100- to 145-fold greater than in the original pregnancy plasma. Rabbits immunized with these preparations responded with antisera which became mono-specific to their respective PAPP after appropriate absorption. PAPP-D, the one with low molecular weight (20,000), was shown immunologically to represent human placental lactogen. Another, PAPP-C, has been found to be identical to the pregnancy-specific β₁-glycoprotein described by Bohn (designated by him as SP₁ protein). The other, PAPP-A, of molecular weight 750,000, is distinct from the other two pregnancy-associated proteins of Bohn (designated SP₂ and SP₃), as well as the α₂-pregnoglobulin of Berne. Since the latter and SP₃ proved to be identical immunologically, and since it has been reported that SP₃, Xh factor, pregnancy-associated globulin (PAG or Pal), and the pregnancyzone proteins are identical to each other, PAPP-A may represent a new pregnancy-specific protein. None of the PAPPs were detected in sera from 102 patients with various malignancies.

show abstract

Section: Discussionmentioning

confidence: 99%

Three Pregnancy-Associated Human Plasma Proteins: Purification, Monospecific Antisera and Immunological Identification

Lin

Halbert

Kiefer

et al. 1974

Int Arch Allergy Immunol

View full text Add to dashboard Cite

show abstract

“…This procedure gives identical recovery to acid-ethanol extraction according to the method of Davoren [18] as shown previously [10,11]. A 0.5 ml aliquot of the supernatant was then placed on 0.9 x 2.0 cm columns of CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) covalently linked to anti-insulin antibodies [12,[19][20][21] and equilibrated with 0.2 mol/1 glycine buffer 2.5 g/1 HSA pH 8.8. The antibodies were purified prior to the linking reaction by ammonium sulphate precipitation from guinea-pig anti-porcine insulin antiserum kindly provided by Dr. P.H.…”

Section: Methodsmentioning

confidence: 99%

Glucose stimulated proinsulin biosynthesis

1978

View full text Add to dashboard Cite

Summary. Proinsulin biosynthesis was stimulated progressively to a plateau level at 45 min by 5.5 mmol/l glucose. The rate of biosynthesis promptly decreased following cessation of either 15 or 60 min exposure to this concentration of glucose. Actinomycin D had no effect on the rate of decline. In contrast, exposure to 20 mmol/l glucose caused a prolonged increase in proinsulin biosynthesis which was still apparent at 75 rain. After removal of the stimulus there was an initial decrease in the rate of proinsulin biosynthesis followed by persistently elevated rates. When proinsulin biosynthesis was stimulated by 20 mmol/1 glucose in the presence of actinomycin D, there was a rapid switch off of biosynthesis and no persistent effect. Thus, two controis for regulation of proinsulin biosynthesis can be characterised: a prompt, rapidly reversible stimulation in response to low or high glucose concentrations and a persistent stimulation in response to high glucose concentrations. These two effects may represent the translational and transcriptional effects of glucose respectively.

show abstract

“…Coupling of anti-insulin globulin to cyanogen bromideactivated Sepharose-4B was accomplished by a technique similar to that of Akanuma et al (10). One hundred twenty milliliters of a solution consisting of 1 vol of serum and 2 vol of buffer (0.113 M boric acid, pH 8.0) was passed over 2.0 ml of anti-insulin globulin-coupled agarose (binding capacity, 1.5 units/ml) in a 10 X 150 mm siliconized column at a rate of 15 ml/hr at room temperature.…”

Section: Case Reportmentioning

confidence: 99%

Insulin resistance due to a defect distal to the insulin receptor: Demonstration in a patient with leprechaunism

Kobayashi¹,

Olefsky²,

Elders³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We have studied a 2-year-old girl with acanthosis nigricans, glucose intolerance, marked hyperinsulinemia, and somatic features characteristic of the leprechaunism syndrome. Circulating plasma insulin levels were increased up to 50-fold and the patient showed a blunted hypoglycemic response to an injection of exogenous insulin (0.2 units/kg), indicating the presence of severe insulin resistance. Insulin purified from the patient's plasma was normal on the basis of chromatographic, electrophoretic, and immunologic criteria. Furthermore, the purified insulin competed effectively with 125I-labeled insulin for binding to insulin receptors on cultured IM-9 lymphocytes and rat fat cells and also exhibited normal biological potency when tested on rat fat cells. Anti-insulin receptor and anti-insulin antibodies were not detected in the patient's plasma, and plasma levels of glucagon, growth hormone, and cortisol were normal. Insulin binding to the patient's circulating mononuclear leukocytes was only slightly depressed into the low normal range and could not account for the severe insulin resistance. Studies on the patient's fibroblasts revealed normal levels of insulin receptors but a total absence of insulin's ability to accelerate glucose transport. Because rates of glucose transport and metabolism were normal in the basal state in the absence of insulin, we conclude that this patient's insulin resistance is due to an inherited cellular defect in the coupling mechanism between occupied insulin receptors and the plasma membrane glucose transport system. Leprechaunism is a rare, inherited disease characterized by an unusual facial appearance, hirsutism, cliteromegaly, and sparse subcutaneous fat stores (1-3). We have performed detailed metabolic studies on a 2-year-old girl with this syndrome who also presented with acanthosis nigricans and severe insulin resistance. Theoretically, insulin resistance can be due to abnormal beta cell secretory products, circulating insulin antagonists, or a cellular defect in insulin action (4). To elucidate the etiology of this patient's insulin resistance, each of these possibilities was examined in detail. Thus, we studied the integrity of her circulating insulin, assayed for potential insulin antagonists, measured cellular insulin receptors, and studied the insulin responsiveness of the patient's cultured fibroblasts in vitro. The results indicate that this patient's insulin resistance is due to a defect in insulin action distal to the insulin receptor. CASE REPORTThe patient, an 18-month-old white girl, had been born after a full-term (41-week gestation) uncomplicated pregnancy and delivery to a gravida I para 1 28-year-old mother who had takenThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3469 no medications during her pregnancy; birth weight was 2600 g. At birth she was noted to have bilateral ingui...

show abstract

Immunological reactivity of insulin to sepharose coupled with insulin-antibody—Its use for the extraction of insulin from serum

Cited by 59 publications

References 3 publications

Three Pregnancy-Associated Human Plasma Proteins: Purification, Monospecific Antisera and Immunological Identification

Three Pregnancy-Associated Human Plasma Proteins: Purification, Monospecific Antisera and Immunological Identification

Glucose stimulated proinsulin biosynthesis

Insulin resistance due to a defect distal to the insulin receptor: Demonstration in a patient with leprechaunism

Contact Info

Product

Resources

About