Thyrotropin interactions with human fat cell membrane preparations and the finding of soluble thyrotropin binding component.
Explore the source record for details and available documents.
Adipose Tissue↗
Biomedical subjects
L D Kohn
Publications and source records attributed to L D Kohn.
Use of thyrotropin and cholera toxin to probe the mechanism by which interferon initiates its antiviral activity.
Thyrotropin (10 muM) inhibited the antiviral activity of interferon. When added after interferon, thyrotropin (TSH) had no effect on antiviral activity. There was also no inhibition of interferon action in cells washed with medium between incubations with TSH and interferon. 125I-Labeled TSH and 125I-labeled cholera toxin could bind to preparations of mouse L-cell plasma membranes. The binding was specific in that it was prevented by unlabeled thyrotropin or cholera toxin, but not by insulin, glucagon, prolactin, growth hormone, human chorionic gonadotropin, or luteinizing hormone. Mouse interferon inhibited 125I-labeled TSH binding to L-cell plasma membranes. The effect of mouse interferon on 125I-labeled cholera toxon binding was more complex, inhibition occurring only after an initial enhancement at low interferon concentrations. A 10-fold higher concentration of interferon was required to inhibit 125I-labeled TSH binding. Mouse interferon was also able to displace bound 125I-labeled TSH, but not bound 125I-labeled cholera toxin. The interferon interaction with cell membranes was temperature-sensitive. Human interferon could induce changes in binding of 125I-labeled TSH and 125I-labeled cholera toxin to mouse L-cell plasma membranes similar to those induced by mouse interferon. Mouse interferon induced similar changes in plasma membranes of human KB-3 cells, which are insensitive to both human and mouse interferons. In view of these results, the species specificity of interferons does not appear to reside solely at the point of the initial interaction with their binding sites.
Bacterial Toxins↗
Relationship of gangliosides to the structure and function of thyrotropin receptors: their absence on plasma membranes of a thyroid tumor defective in thyrotropin receptor activity.
Plasma membranes derived from rat thyroid tumor (1-8R) which is unresponsive to thyrotropin but is responsive to dibutyryl adenosine 3':5'-cyclic monophosphate bind less than 20% of the [125I] thyrotropin which can be bound to plasma membranes from normal rat thyroids under conditions which optimize tumor membrane binding relative to normal thyroid membranes. In addition, the binding is different from thyrotropin binding to normal thyroid membranes both in its altered sensitivity to changes in hydrogen ion concentration and in a decreased sensitivity to competition by unlabeled thyrotropin. This reduced capacity to bind [125I] thyrotropin cannot be attributed to degradation of the hormone by membrane-associated proteases. Although the supernatant phase of the thyroid tumor homogenates contains a soluble component which inhibits [125I] thyrotropin binding to thyrotropin receptors on plasma membranes, its level is the same as in homogenates of normal thyroid tissue. Trypsin digestion does not expose thyrotropin receptors in a manner analogous to that seen in normal thyroid tissue. The major ganglioside in the tumor membranes is N-acetylneuraminylgalactosylglucosylceramide and the membranes lack the N-acetylgalactosaminyltransferase required for the synthesis of more complex gangliosides. In contrast, the normal rat thyroid membranes contain more complex gangliosides such as galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide and N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide as well as the glycosyltransferase activities required for their syntheses. Galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide can also be detected in normal membranes, but not in tumor membranes, by selective labeling with galactose oxidase (D-galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) and [3H] sodium borohydride. These results support the hypothesis that gangliosides are important structural or functional components of thyrotropin receptors on thyroid plasma membranes.
Animals↗
Thyrotropin-ganglioside interactions and their relationship to the structure and function of thyrotropin receptors.
Gangliosides inhibit 125I-labeled thyrotropin binding to the thyrotropin receptors on bovine thyroid plasma membranes, on guinea pig retro-orbital tissue plasma membranes, and on human adipocyte membranes. This inhibition by gangliosides is critically altered by the number and location of the sialic acid residues within the ganglioside structure, the efficacy of inhibition having the following order: GD1b greater than GT1 greater than GM1 greater than GM2 = GM3 greater than GD1a. The inhibition results from the interaction of thyrotropin and gangliosides, rather than the interaction of membrane and gangliosides. Fluorescence studies show that the inhibition is associated with a distinct conformational change of the thyrotropin molecule and that the progression from a "noninhibitory conformation" to an "inhibitory conformation" parallels exactly the order of effectiveness in inhibiting 125I-labeled thyrotropin binding. The ganglioside inhibition of 125I-labeled thyrotropin binding appears to be hormonally specific in that it is not affected by albumin, glucagon, insulin, prolactin, follicle-stimulating hormone, growth hormone, or corticotropin. The possibility that a ganglioside or ganglioside-like structure is a component of the thyrotropin receptor is suggested by the finding that gangliosides more complex than N-acetylneuraminylgalactosylglucosylceramide are present in bovine thyroid membranes in much higher quantities than have been previously found in extraneural tissue. The finding that the B component of cholera toxin, which also interacts with gangliosides, has a peptide sequence in common with the beta subunit of thyrotropin, suggests that thyrotropin and cholera toxin may be analogous in their mode of action on the membrane.
Adipose Tissue↗
Cholera toxin interactions with thyrotropin receptors on thyroid plasma membranes.
Unlabeled cholera toxin inhibits [125I]thyrotropin binding to thyrotropin receptors on thyroid plasma membranes. Maximal inhibition by cholera toxin does not exceed 40%, whereas unalbeled thyrotropin completely inhibits [125I]thyrotropin binding to these same membranes. Kinetic analyses of the binding data are compatible with the view that the cholera toxin decreases the number of receptor sites available to thyrotropin and that the mechanism by which the cholera toxin inhibits [125I]thyrotropin binding to these receptor sites involves both competitive and noncompetitive elements.
Adenylyl Cyclases↗
Structure of an exophthalmos-producing factor derived from thyrotropin by partial pepsin digestion.
Previously reported experiments (Winand, R.J., and Kohn, L.D. (1970) J. Biol. Chem. 245, 967-975; Kohn, L.D., and Winand, R.J. (1971) J. Biol. Chem 246, 6570-6575) have demonstrated that partial pepsin digestion of bovine thyrotropin preparation yields a fragment of the thyrotropin molecule which is exophthalmogenic but has negligible or no thyroid-stimulating activity. In the present report this exophthalmogenic derivative of the thyrotropin molecule is shown to contain two major polypeptide components with approximate molecular weights of 14,000 and 6,000. Amino acid analyses, carbohydrate analyses, and tryptic digestion experiments indicate that this exophthalmogenic factor is composed of an intact or nearly intact beta subunit of thyrotropin and an NH2-terminal fragment of the alpha subunit of thyrotropin. Neither polypeptide component of the exophthalmogenic factor has the in vivo exophthalmogenic activity of the intact structure. In vitro the intact exophthalmogenic derivative of the thyrotropin molecule can bind to the thyrotropin receptor on thyroid membranes less efficiently than thyrotropin but significantly better than either its own polypeptide components or the alpha or beta subunits of thyrotropin. The exophthalmogenic factor and its parent thyrotropin molecule can stimulate adenylate cyclase activity in retro-orbital tissue membranes from guinea pigs, a mammalian model of exophthalmos; its polypeptide components have little or no such activity.
Adenylyl Cyclases↗
Thyrotropin receptors in thyroid plasma membranes. Characteristics of thyrotropin binding and solubilization of thyrotropin receptor activity by tryptic digestion.
Biologically active bovine 125I-thyrotropin preparations have been prepared, characterized, and used to evaluate the optimal conditions for thyrotropin binding to bovine thyroid plasma membranes in vitro. Binding of 125I-TSH has a pH optimum around 6.0 and is sensitive to the choice and concentration of buffer. Binding is inhibited by salts, especially those containing magnesium and calcium ions; magnesium concentrations optimal for adenylate cyclase assays (2 to 5 mM) result in 85 to 98% inhibition of binding. Binding is temperature sensitive. At 37 degrees binding has its highest initial level; however, instability of the membrane at this temperature causes a rapid loss of binding activity. Binding at 0 degrees is optimal in 30 min and at the same level as initial binding at 37 degrees; since there is no decrease in binding activity, it has been chosen as the optimal temperature. Thyrotropin, luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin have relative binding affinities for the thyrotropin receptors of 100, 10, 2, and less than 0.5, respectively. In all of these characteristics, 125I-thyrotropin at 1.5 x 10(-5) M concentrations has the same properties of binding to bovine plasma membranes as do [3H]thyrotropin preparations which have been previously characterized (Amir, S.M., Carraway, T.F., Jr., Kohn, L.D., and Winand, R.V. (1973) J. Biol. Chem. 248, 4092-4100) and used to study binding at 5 x 10(-6) M concentrations. 125I-TSH binding as a function of hormone concentration results in curved Scatchard plots; however, Hill plots of these same binding data are linear and have a slope of 0.65. Taken together, these data suggest that the heterogeneity in thyrotropin binding constants which is evident in the Scatchard plot reflects a negatively cooperative relationship among the thyrotropin receptor sites, i.e. decreased hormonal affinity as hormone concentrations increase. Adenylate cyclase studies yield kinetic plots which also exhibit negative cooperativity; corrections for thyrotropin bound under the adverse binding conditions of the adenylate cyclase assays suggest that Km values for thyrotropin in this enzymatic assay are compatible with binding constants measured by the 125I-thyrotropin preparations. Tryptic digestion destroys binding activity on the thyroid plasma membrane but releases specific thyrotropin receptor activity into the supernatant phase. Chromatography on Sephadex G-100 indicates that this solubilized receptor fragment has a molecular weight between 15,000 and 30,000.
Adenylyl Cyclases↗
Characteristics of a solubilized thyrotropin receptor from bovine thyroid plasma membranes.
The thyrotropin receptor from bovine thyroid plasma membranes has been solubilized using lithium diiodosalicylate, and an assay to measure thyrotropin binding to the solubilized receptor has been developed. Both the solubilized thyrotropin receptor and the thyrotropin receptor on thyroid plasma membranes have effectively identical nonlinear Scatchard plots and negatively sloped Hill plots, i.e. both preparations have receptors which appear to exhibit a similar negatively cooperative relationship. Although the pH optimum of thyrotropin binding to the solubilized receptor is the same as that of the thyroid plasma membrane receptor, pH 6.0, the pH dependency curve of the solubilized receptor is slightly different in its outline. Thyrotropin binding to the solubilized receptor is less sensitive to salt inhibition than is binding to the thyroid plasma membrane receptor; however, optimal binding remains at 0 degrees. The relative affinities of thyrotropin and two glycoprotein hormones which can be considered structural analogs, luteinizing hormone and human chorionic gonadotropin, are 100:10:5, respectively, toward plasma membrane receptors, but 100:25:40 toward the solubilized receptors. The solubilized receptor preparation is heterogeneous in size in that it has binding components with molecular weights of 286,000, 160,000, 75,000, and 15,000 to 30,000. Tryptic digestion converts all three higher molecular weight components to the 15,000 to 30,000 molecular weight species, and the 15,000 to 30,000 molecular weight receptor component has all of the binding properties of the solubilized receptor preparation before tryptic digestion including an identical nonlinear Scatchard plot. It has the same size as and coelutes from Sephadex G-100 with a 15,000 to 30,000 molecular weight receptor released by tryptic digestion of bovine thyroid plasma membranes or tryptic digestion of bovine or dog thyroid cells in culture. The tryptic fragment of the solubilized receptor or preparations has been purified almost 250-fold by affinity chromatography on thyrotropin-Sepharose columns. The binding activity is lost when the solubilized thyrotropin receptor preparation is exposed to beads of neuraminidase-Sepharose or conconavalin A-Sepharose.
Animals↗
Thyrotropin effects on thyroid cells in culture. Effects of trypsin on the thyrotropin receptor and on thyrotropin-mediated cyclic 3':5'-AMP changes.
Dog, human, and bovine thyroid cells in culture have been shown to develop follicle-like structures when cells are cultured in conditions of confluency and when cells are incubated in the presence of bovine thyrotropin or N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate during the first 24 to 48 hours after trypsinization. If thyrotropin is added 48 hours after trypsinization, these cells do not form follicle-like structures but remain as a monolayer culture. Although thyroid cells which grow as a monolayer have a thyrotropin receptor on their plasma membranes with the same in vitro binding properties as the thyrotropin receptor on the plasma membranes of the follicle-forming thyroid cells, there is a 1- to 2-fold greater number of receptors per mg of membrane protein when follicle-forming and monolayer cultures are compared...
Animals↗
Experimental exophthalmos. Binding of thyrotropin and an exophthalmogenic factor derived from thyrotropin to retro-orbital tissue plasma membranes.
Biologically active preparations of 125I-thyrotropin, [3H]thyrotropin, and the [3H]exophthalmogenic factor derived from thyrotropin by partial pepsin digestion have been used to study the binding properties of the thyrotropin receptor on guinea pig retro-orbital tissue plasma membranes. In regard to the optimal conditions of binding, pH, buffer, salt concentrations, and temperature, these properties are the same as those described in any accompanying report concerning thyrotropin binding to bovine thyroid plasma membranes (Tate, R.L., Schwartz, H.I., Holmes, J.M., Kohn, L.D., and Winand, R.J. (1975) J. Biol. Chem. 250, 6509-6515). In addition, thyrotropin receptors on the retro-orbital tissue plasma membranes are similar to thyrotropin receptors on bovine thyroid plasma membranes in their apparent negative cooperativity and in their relative affinities for luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin. In contrast, gamma-globulin from patients with malignant exophthalmos enhances binding when added to incubation mixtures containing the retro-orbital tissue plasma membranes but not when added to those containing thyroid plasma membranes. Normal gamma-globulin and gamma-globulin from Graves' disease patients without exophthalmos do not have this property. The gamma-globulin itself does not bind to the membrane except in the presence of thyrotropin or its exophthalmogenic factor derivative. Tryptic digestion of the retro-orbital tissue membranes releases specific thyrotropin and exophthalmogenic factor binding activity into the supernatant phase. Chromatography on Sephadex G-100 indicates that this trypsin-released receptor activity has a molecular weight of 75,000 or greater, rather than 15,000 to 30,000 for the trypsin-released receptor activity from bovine thyroid membranes (Tate, R.L., Schwartz, H.I., Holmes, J.M., Kohn, L.D., and Winand, R.J. (1975) J. Biol. Chem. 250, 6509-6515).
Animals↗
Stimulation of adenylate cyclase activity in retro-orbital tissue membranes by thyrotropin and an exophthalmogenic factor derived from thyrotropin.
Retro-orbital tissue membranes have been shown to have adenylate cyclase activity which can be stimulated by thyrotropin and by an exophthalmogenic factor derived from the thyrotropin molecule by partial pepsin digestion. This stimulable activity is maximal after 15 min and is optimal in the presence of 3 mM magnesium and 1.5 mM ATP. Calcium salts are exquisitely inhibitory to the hormonal stimulation; sodium, lithium, and ammonium salts are significantly less inhibitory. Thyrotropin and the exophthalmogenic factor induce similar maximal levels of stimulation but a 4- to 5-fold higher concentration of exophthalmogenic factor is required to achieve this level. Fluoride stimulates adenylate cyclase activity 2- to 3-fold higher than either thyrotropin or the exophthalmogenic factor; thyrotropin, luteinizing hormone, the beta subunit of thyrotropin, and the alpha subunit of thyrotropin have relative activities for stimulation of cyclase activity of 100:2:2 less than 0.5. Several other polypeptide and glycoprotein hormones have no effect. The gamma-globulin from patients with malignant exophthalmos has no significant effect on cyclase activity either alone or in the presence of maximal levels of thyrotropin or the exophthalmogenic factor; this gamma-globulin does, however, stimulate cyclase activity at submaximal hormone levels. Trypsin not only destroys the hormone-stimulable adenylate cyclase activity on retro-orbital tissue plasma membranes, but also destroys it on the 15,000 to 30,000 molecular weight receptor fragment released from the membranes by the tryptic action.
Adenylyl Cyclases↗
Structural, kinetic, and renaturation properties of an induced hydroxypyruvate reductase from Pseudomonas acidovorans.
A hydroxypyruvate reductase has been induced in Pseudomonas acidovorans by growth on glyoxylate. The enzyme has been purified to homogeneity as assessed by the criteria of analytical ultracentrifugation and analytical disc gel electrophoresis. It has a molecular weight of approximately 85,000 and is composed of two identical subunits. The subunits are not interconnected by disulfide bonds although the enzyme has 4 mol of half-cystine per mol of enzyme. The enzyme catalyzes the reversible conversion of hydroxypyruvate to D(minus)-glycerate in the presence of NADH. Glyoxylate cannot replace hydroxypyruvate as a substrate and is a competitive inhibitor of hydroxypyruvate reduction. The activity of the enzyme toward hydroxypyruvate is anion-modulated; the activity of the enzyme toward D(minus)-glycerate is unaffected by anions but is increased by tris-(hydroxymethyl)aminomethane. The subunits of the induced hydroxypyruvate reductase can be renatured. After the enzyme is dissociated in solutions of 6.0 M guanidine hydrochloride containing 0.1 M 2-mercaptoethanol, optimum renaturation occurs when subunits are diluted into a renaturation solvent consisting of 0.04 M Trischloride, pH 7.4, containing 25% glycerol, 25 mM 2-mercaptoethanol, and 0.14 MM NADH. NAD is an inhibitor of renaturation and therefore cannot substitute for NADH. The optimal temperature of dilution and subsequent incubation is 15 degrees, and increases in protein concentration up to 1.2 mg/ml, the highest concentration tested, improve both the rate of renaturation and the yield of active material. The half-time of renaturation at a protein concentration of 1.2 mg/ml was 1 min. The kinetics of renaturation is second order, i.e., is compatible with a bimolecular reaction preducted by the association of two similar subunits. The physical and kinetic parameters of the renatured protein are the same as those of the native enzyme.
Alcohol Oxidoreductases↗
Integrated steady state rate equations and the determination of individual rate constants.
Integrated steady state rate equations have been used to determine the kinetic constants (Vs, Ks, Vp, and Kp) and rate constants (k1, k2, k3, and k4) of the reversible enzyme mechanism: (see article). The fumarase reaction has been used as a model to illustrate the procedures for determining these constants. In contrast to initial velocity studies, the values of the constants have been obtained by examining the enzyme reaction in only one direction rather than in both forward and reverse directions. To accomplish this, a new procedure is described for fitting data to integrated rate equations which eliminates problems encountered when data are analyzed graphically. The advantages of examining on enzyme reaction in one direction with these new procedures allow this method to be extended to the examination of enzymes with simple mechanisms where initial velocities are difficult to measure because either the substrate or product is not readily available, or because the reaction is not readily reversible.
Animals↗
Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles.
In the preceding paper the preparation and characterization of antiserum to purified D-lactate are described. In this paper the effects of the antibody on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native Escherichia coli ML 308-225 membrane vesicles and ML 308-225dld-3 vesicles reconstituted with D-lactate dehydrogenase are described. The results demonstrate that D-lactate dehydrogenase is inaccessible to antibody in native ML 308-225 vesicles, but readily accessible to antibody in reconstituted dld-3 vesicles. The findings indicate that D-lactate dehydrogenase is located on the inner surface of native ML 308-225 vesicles and on the outer surface of reconstituted dld-3 vesicles. The results with the native vesicle preparations also provide further evidence that virtually none of the vesicles is inverted or sufficiently damaged to allow access of antibody to D-lactate dehydrogenase. In addition, experiments are presented which demonstrate that an impermeable electron carrier, reduced 5-N-methylphenazonium-3-sulfonate, drives active transport in native ML 308-225 vesicles as well as its permeable analogue reduced phenazine methosulfate. Thus, reduction of the respiratory chain from either side of the vesicle membrane is able to drive active transport. Ca2+, Mg2+-stimulated ATPase is also inaccessible to antibody in ML 308-225 vesicles unless the preparation is subjected to ultrasonic sound, incubated in Tris buffer at pH 9.0, or homogenized vigorously. Moreover, as opposed to D-lactate dehydrogenase and cytochrome b1, ATPase is readily lost from the membrane during the preparation of vesicles.
Animals↗
Immunochemical properties of the membrane-bound D-lactate dehydrogenase from Escherichia coli.
The preparation, characterization, and purification of antibody against the membrane-bound D-lactate dehydrogenase solubilized and purified from Escherichia coli ML 308-225 are described. The antibody is highly specific for the flavin-linked D-lactate dehydrogenase, and incubation of the enzyme with antiserum results in marked inhibition of enzymatic activity. By means of a radioimmune assay, it is demonstrated that membrane vesicles prepared from E. coli ML 308-225dld-3 contain catalytically inactive material which cross-reacts with native D-lactate dehydrogenase. In the following paper, the effects of this antiserum on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native and reconstituted membrane vesicles are examined.
Animals↗
Relationship of thyrotropin to exophthalmos-producing substance: formation of an exophthalmos-producing factor by pepsin digestion of mouse pituitary tumor and human thyrotropin preparations.
The present report demonstrates that mouse tumor thyrotropin preprarations have exophthalmogenic activity. In addition it shows that the exophthalmogenic activity of mouse tumor thyrotropin can be increased by partial pepsin digestion, whereas the thyroid stimulating activity of mouse tumor thyrotropin is rapidly destroyed by such treatment. Thus, after 30 min of pepsin digestion, mouse tumor thyrotropin has 130% of its initial exophthalmogenic action but only 10% of its thyroid stimulating activity. Preparations of human thyrotropin are similarly sensitive to partial pepsin digestion, i.e., there is a rapid destruction of thyroid stimulating activity but a very much slower destruction of exophthalmogenic activity. Thus, after 30 min of pepsin digestion, preparations of human thyrotropin retain 80% of their exophthalmogenic activity but only 20-30% of their thyroid stimulating action. Since these results are analogous to those obtained in studies of the partial pepsin digestion of bovine thyrotropin (1), partial pepsin digestion of both purified human and purified mouse tumor thyrotropin preparations should yield an exophthalmogenic fragment of the TSH molecule devoid of thyroid stimulating action.
Animals↗
Exophthalmogenic activity of the beta subunit of thyrotropin.
The exophthalmogenic activity of the beta subunit of bovine thyrotropin is only 10% to 20% that of the thyrotropin molecule or of an exophthalmogenic factor produced by partial pepsin digestion of purified thyrotropin preparations. The alpha subunit of thyrotropin, luteinizing hormone, and both subunits of luteinizing hormone have no exophthalmogenic activity.
Animals↗
Crystalline alpha2 beta2 complexes of tryptophan synthetase of Escherichia coli. A comparison between the native complex and the reconstituted complex.
Explore the source record for details and available documents.
Chromatography, Ion Exchange↗