scholarly journals A radio-ligand receptor assay for the long-acting thyroid stimulator. Inhibition by the long-acting thyroid stimulator of the binding of radioiodinated thyroid-stimulating hormone to human thyroid membranes

1975 ◽  
Vol 145 (1) ◽  
pp. 105-111 ◽  
Author(s):  
S Q Mehdi ◽  
S S Nussey
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Thyroid Stimulating Hormone ◽  
Human Thyroid ◽  
Experimental Conditions ◽  
Sucrose Density Gradient Centrifugation ◽  
Low Concentrations ◽  
Chloramine T ◽  
Long Acting ◽  
Stimulating Hormone

Highly purified bovine TSH (thyroid-stimulating hormone) was labelled with 125I by using very low concentrations of chloramine-T. Human thyroid membranes prepared by discontinuous sucrose-density-gradient centrifugation were homogeneous on examination by electron microscopy. Incubation of radioiodinated TSH with the membranes showed that radioactivity could be bound to the membranes. Under the experimental conditions described here, binding was dependent on time and temperature and was a saturable phenomenon. Preincubation of the membranes with unlabelled hormone inhibited the subsequent binding of 125I-labelled TSH. Similarly, inhibition by the long-acting thyroid stimulator also showed a saturation behaviour. A rapid and sensitive method for the detection of the long-acting thyroid stimulator is described.

scholarly journals Peptide-chain initiation in duodenal mucosa of rachitic chicks after 1α-,25-dihydroxycholecalciferol administration

1984 ◽  
Vol 219 (1) ◽  
pp. 99-106 ◽  
Author(s):  
G Mezzetti ◽  
M Moruzzi ◽  
B Barbiroli
Keyword(s):  
Complex Formation ◽  
Gradient Centrifugation ◽  
Active Form ◽  
Ribosomal Subunit ◽  
Sucrose Density Gradient ◽  
Duodenal Mucosa ◽  
Binding Activity ◽  
Initiation Factor ◽  
Experimental Conditions ◽  
Sucrose Density Gradient Centrifugation

The post-ribosomal fraction of chick duodenal mucosa contains Met-tRNAMetf-binding protein(s) that behaves like the eukaryotic initiation factor (eIF-2) in protein synthesis. The binding activity of cytosol protein can be measured by retention of the radioactive complex formed on a nitrocellulose membrane. Complex-formation requires Met-tRNAMetf and GTP or guanosine [beta, gamma-methylene] triphosphate, and is inhibited by aurintricarboxylic acid. The ternary initiation complex thus formed can bind to ribosomal particles from chick intestine. By sucrose-density-gradient centrifugation, [35S]Met-tRNAMetf was found to bind exclusively to 40S and not to 60S ribosomal subunit particles. In the duodenal mucosa of rachitic chicks the ability of the cytosol proteins to promote the binding of Met-tRNAMetf to ribosomal particles via ternary-complex formation is detectably increased by 3 h after injection of 1 alpha,25-dihydroxycholecalciferol, the active form of vitamin D. Cholecalciferol and ergocalciferol under the same experimental conditions failed to stimulate Met-tRNAMetf-binding activity.

A COMPARISON OF THE HEAT STABILITY OF LONG-ACTING THYROID STIMULATOR AND HUMAN THYROID-STIMULATING HORMONE

1965 ◽  
Vol 32 (1) ◽  
pp. 29-33 ◽  
Author(s):  
A. R. McGIVEN ◽  
D. D. ADAMS ◽  
H. D. PURVES
Keyword(s):  
Heat Stability ◽  
Thyroid Stimulating Hormone ◽  
Human Thyroid ◽  
Human Antibody ◽  
Initial Value ◽  
Long Acting ◽  
Stimulating Hormone

SUMMARY Studies of the heat stability of a human antibody against thyroglobulin showed that after heating at 70° for 10 min. its potency was reduced to 2% of the initial value. Similarly, the potency of a preparation of long-acting thyroid stimulator (LATS) was reduced to significantly less than 10% by this treatment, whereas the potency of a human thyroid-stimulating hormone (TSH) preparation was reduced only to 45%, with a lower fiducial limit of 26% for P = 0·05. It is concluded that LATS is less stable to heat than is TSH. LATS did not differ from the antibody in heat stability.

scholarly journals The preferential loss of the polylysine- or polyornithine-stimulated activity of Clostridium perfringens polynucleotide phosphorylase during proteolysis

1969 ◽  
Vol 112 (4) ◽  
pp. 489-495 ◽  
Author(s):  
P. S. Fitt ◽  
Helga Wille
Keyword(s):  
Clostridium Perfringens ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Polynucleotide Phosphorylase ◽  
Sucrose Density Gradient Centrifugation ◽  
Charge Effects ◽  
Low Concentrations ◽  
Preferential Loss ◽  
Single Enzyme

1. An improved method for the purification of Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate–polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is described. The product was stable and was highly stimulated by polylysine or polyornithine. 2. It migrated as a single enzyme during sucrose-density-gradient centrifugation, and no separation of polymerization and phosphorolytic activities was observed. 3. Trypsin digestion caused a rapid, preferential loss of the polylysine- or polyornithine-stimulated activity, which was prevented by low concentrations of polyornithine. 4. The protection by polyornithine was not specific. 5. It is concluded that charge effects on the clostridial polynucleotide phosphorylase itself are primarily responsible for the stimulation of this enzyme by polylysine or polyornithine.

IODINATION OF THYROID-STIMULATING HORMONE FOR RECEPTOR-BINDING STUDIES WITH HUMAN THYROID MEMBRANES: EFFECTS OF SPECIFIC ACTIVITY AND METHOD OF IODINATION

1980 ◽  
Vol 84 (3) ◽  
pp. 439-447 ◽  
Author(s):  
J. C. KERMODE ◽  
B. D. THOMPSON
Keyword(s):  
Receptor Binding ◽  
Specific Activity ◽  
Thyroid Stimulating Hormone ◽  
Human Thyroid ◽  
Receptor Interaction ◽  
Binding Studies ◽  
Ph Optimum ◽  
Chloramine T ◽  
Dramatic Shift ◽  
Stimulating Hormone

Three different methods were compared for 125I-labelling of thyroid-stimulating hormone (TSH) for use in receptor-binding studies with human thyroid membranes: these were the chloramine-T, Bolton–Hunter and lactoperoxidase methods. Chloramine-T proved to be an inferior method to the other two. Iodinations to different specific activity (0·7–9·4 Bq/pg) were also compared: too high a specific activity led to reduced binding and a dramatic shift in the pH optimum for the TSH–receptor interaction. A specific activity of 2·5 Bq/pg should not be exceeded if binding of 125I-labelled TSH is to be representative of the binding of the natural hormone. Under these conditions, pH 7·5 was optimal for binding of TSH to its receptor. Repurification of the labelled TSH by receptor adsorption also proved to be essential.

EFFECTS OF HUMAN THYROID-STIMULATING HORMONE AND IMMUNOGLOBULINS ON ADENYLATE CYCLASE ACTIVITY AND THE ACCUMULATION OF CYCLIC AMP IN HUMAN THYROID MEMBRANES AND SLICES

1978 ◽  
Vol 79 (1) ◽  
pp. 121-130 ◽  
Author(s):  
S. D. HOLMES ◽  
SUSAN M. DIRMIKIS ◽  
T. J. MARTIN ◽  
D. S. MUNRO
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Thyroid Stimulating Hormone ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Maximum Response ◽  
Human Thyroid ◽  
Tissue Slices ◽  
Long Acting ◽  
Stimulating Hormone

The activation of adenylate cyclase and the accumulation of cyclic AMP resulting from the action of human thyroid-stimulating hormone (TSH), long-acting thyroid stimulator (LATS) or LATS-protector (LATS-P) have been investigated in preparations of human thyroid membranes and slices. Human TSH significantly increased adenylate cyclase activity in membranes from non-toxic goitres whereas LATS and LATS-P had no consistent effect. However, pre-incubation of goitrous membranes with LATS–immunoglobulin G inhibited the effect of TSH on adenylate cyclase. When thyroid membranes were prepared from the glands of patients with Graves's disease neither TSH nor thyroid-stimulating immunoglobulins (TSIg) stimulated adenylate cyclase significantly. Whether from non-toxic goitres or thyrotoxic tissue, the concentration of TSH needed to induce half of the maximum response was lower in thyroid slices than in membranes. Both LATS and LATS-P significantly stimulated the accumulation of cyclic AMP in slices of goitrous tissue but thyrotoxic tissue slices did not respond. In goitrous slices, submaximum concentrations of TSH and TSIg caused additive responses in the accumulation of cyclic AMP but TSIg did not increase the maximum response to TSH.

scholarly journals Polyamines and nucleic acid metabolism in chick embryo. Incorporation of labelled precursors into nucleic acids of subcellular fractions and polyribosomal patterns

1968 ◽  
Vol 107 (5) ◽  
pp. 609-613 ◽  
Author(s):  
G. Moruzzi ◽  
B Barbiroli ◽  
C. M. Caldarera
Keyword(s):  
Chick Embryo ◽  
Nucleic Acids ◽  
Orotic Acid ◽  
Nuclear Dna ◽  
Amine Oxidase ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Nucleic Acid Metabolism ◽  
Experimental Conditions ◽  
Sucrose Density Gradient Centrifugation

1. An increase in polyamine concentration, caused by inhibiting the amine oxidase activities with iproniazid, increased the incorporation of [3H]orotic acid into chick-embryo RNA and DNA. On the other hand, a decrease in polyamine concentration, obtained by causing an increase in amine oxidase activities, decreased [3H]orotic acid incorporation into nucleic acids. This was particularly evident for nuclear DNA and ribosomal RNA. 2. Polyribosomal patterns obtained by sucrose-density-gradient centrifugation showed highest radioactivity in the regions of 259s and 280s aggregates in those embryos in which the polyamine contents were enhanced, whereas a decrease in the radioactivity was observed when the polyamine concentrations were decreased. 3. The activity of DNA-dependent RNA polymerase, assayed in the same experimental conditions, also varied in the same fashion with changes in polyamine concentration.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

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