Correction of right-angle molecular fluorescence measurements for absorption of fluorescence radiation

Christmann, D. R.; Crouch, S. R.; Holland, John F.; Timnick, Andrew.

doi:10.1021/ac50052a019

Cited by 49 publications

(27 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At the end of this time, the contents of each tube were placed into a 1.00-cm quartz fluorescence cell, and the emission spectrum and the steady-state depolarization were quantitated. Additionally, background fluorescence of the cellular membrane fragment suspension was obtained, and correction was made for this effect It was found that correction for the inner filter effect (18) was not necessary, provided that the total absorbance in the cuvette was maintained below 0.4 at the excitation maximum of propranolol. Above concentrations corresponding to this absorbance the inner filter effect became significant, as predicted (18).…”

mentioning

confidence: 99%

Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes.

Cherksey¹,

Zadunaisky²,

Murphy³

1980

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

View full text Add to dashboard Cite

A fluorescence receptor binding assay, based upon the high-affinity fi-adrenergic receptor antagonist propranolol, is utilized to probe the microenvironment of the antagonist-receptor complex in the frog (Rana catesbeiana) erythrocyte membrane. The technique of steady-state fluorescence depolarization is applied to the propranolol-receptor complex, allowing quantitation of the rotational relaxation time of the complex. It is found that the complex is dynamically constrained at 20'C. However, in the temperature range 6-100C a sharp reversible release of constraint is observed. It is further demonstrated that the addition of drugs that are known to specifically disrupt the cytoskeleton (colchicine, vincristine, and vinblastine) causes a similar but irreversible release of constraint at 200C. Cytochalasin B has a much smaller influence on the rotational mobility of the propranolol-receptor complex than do the other drugs that disrupt the cytoskeleton. Amphotericin B is without effect on the rotational constraint of the complex. Binding of the antagonist 13H~dihydroalprenolol is not influenced by colchicine. A model is proposed which postulates that cytoskeletal elements are linked to the antagonist-receptor complex. Antagonist binding does not result in cytoskeleta release, whereas agonist binding is postulated to lead to dissociation of the agonist-receptor complex from the cytoskeleton, thereby activating adenylate cyclase.The importance of the f3-adrenergic receptor system derives from its ubiquitous distribution. The 13-receptor is functionally involved in regulation of processes as diverse as epithelial chloride transport (1) and central nervous system activity (2).It has long been clear that the fl-adrenergic receptor operates through an associated adenylate cyclase, which activity the receptor modulates (3). The molecular mechanism of the receptor-cyclase linkage has been the subject of considerable speculation, and although a number of hypotheses have been advanced, none have been validated or repudiated. Consequently, it is of considerable interest to employ receptor-specific biophysical probes, which allow clarification of the intimate details of the linkage.Propranolol, 1-(isopropylamino)-3-naphthyloxy-2-propanol, is a well-characterized antagonist for the ,3-adrenergic receptor, to which it binds with high affinity (4-6). Its availability as a tritium-labeled compound has made possible its utilization in the characterization of the receptor.The naphthalene nucleus of propranolol makes it a likely fluorescent substrate for the ,B-adrenergic receptor. Many compounds of this general structural type have been applied in the past as fluorescent probes in biological systems (7), and propranolol itself has been shown to fluoresce. This property has in fact been utilized in various pharmacokinetic studies (8,9). We have previously shown (10, 11) that the propranolol fluorescence is sufficiently intense to allow useful determinationThe publication costs of this article were defrayed in part by page char...

show abstract

mentioning

confidence: 99%

Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes.

Cherksey¹,

Zadunaisky²,

Murphy³

1980

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

View full text Add to dashboard Cite

show abstract

“…Cherksey et al: Insertion of Fluorescent/3-Receptors ously that correction for an inner-filter effect is not significant when the total optical absorbance of the sample in the cuvette is below 0.4 units at the excitation maximum of rhodamine (Cherksey et al, 1980), we chose to make all measurements at an optical absorbance of less than 0.1 units. In all experiments, appropriate corrections were made for inner-filter effects (Christmann et al, 1980). Fluorescence spectra were obtained on a modified Aminco Bowman SPF spectrofluorometer.…”

Section: Fluorescence Measurementsmentioning

confidence: 99%

Direct insertion and fluorescence studies of rhodamine-labeled β-adrenergic receptors in cell membranes

et al. 1985

View full text Add to dashboard Cite

Previous studies utilizing the fluorescence of propanolol as a probe for the beta-adrenergic receptor showed that this receptor is motionally constrained. To further study the beta-adrenergic receptor in situ we have reinserted rhodamine-labeled beta-receptors into cell membranes. This report presents documentation of their insertion and physiologic viability. Beta-receptors were purified by affinity chromatography (10,000-fold), then fluorescently labeled with tetramethyl rhodamine isothiocyanate, repurified (55,000-fold) and incubated with rat pancreatic islet cells. The binding of 3H-dihydroalprenolol by these cells was increased from a Bmax of 168 +/- 2 to 309 +/- 20 fmol/mg protein with no change in Kd. Various treatments which remove peripheral membrane proteins, e.g. NaOH, lithium diiodosalicylate, and trypsinization, did not alter binding by the cells with inserted receptors indicating that the receptors inserted into cell membranes. In islet cells treated with Koshland's reagent I, beta-adrenergic binding was completely abolished, but following incubation with isolated beta-receptors, the cells bound beta-adrenergic radioligand with a Bmax of 100 fmol/mg protein, indicating functionality on the part of the inserted receptors. Furthermore, insertion of isolated receptors into frog erythrocytes resulted in increased production of cAMP in response to added isoproterenol. In pancreatic islet cells, incubation with labeled receptors caused the fluorescence to shift in wavelength with increased intensity indicating a shift from an aqueous to a lipid environment, probably into the membrane. Using fluorescence (P), it was found that the inserted receptors became motionally constrained to a P of 0.38 (limiting Po = 0.42) during the first 15 min, remaining so for at least 2 hr. Colchicine (5 micrograms/ml) caused a decrease in P to 0.18 indicating release of constraint. Isoproterenol (10(-5)M) caused a rapid decrease to P = 0.15. This effect was blocked by propranolol. Propranolol itself (10(-5) M) had no effect. These results indicate that the labeled receptors rapidly insert into cell membranes and also support the view that agonist activation of the receptor causes an increase in receptor mobility, presumably due to release of constraint from cytoskeleton elements.

show abstract

“…All fluorescence measurements were made with a computer-centered spectrofluorimeter (Holland, Teets & Timnick, 1973;Christman, Crouch, Holland & Timnick, 1980) as described by Schroeder and Gob (1980). Trans-parinarate, cis-parinarate, and 1,6-diphenyl-l,3,5-hexatriene were incorporated into liver plasma membranes at probe to phospholipid molar ratios of 1:100 or less as described elsewhere (Schroeder, Holland & Vagelos, 1976;Schroeder & Gob, 1979, 1980.…”

Section: Fluorescence Measurementsmentioning

confidence: 99%

Hormonal effects on fatty acid binding and physical properties of rat liver plasma membranes

Schroeder

1982

J. Membrain Biol.

View full text Add to dashboard Cite

The fluorescent fatty acids, trans-parinaric and cL*-parinaric acid, were used as analogs of saturated and unsaturated fatty acids in order to evaluate binding of fatty acids to liver plasma membranes isolated from normal fed rats. Insulin (10 8 to 10 -6 M) decreased trans-parinaric acid binding 7 to 26% while cis-parinaric acid binding was unaffected. Glucagon (10-4 M) increased transparinaric acid binding 44%. The fluorescence polarization of transparinarate, cis-parinarate and 1,6-diphenyl 1,3,5-hexatriene was used to investigate effects of triiodothyronine, insulin and glucagon on the structure of liver plasma membranes from normal fed rats or from rats treated with triiodothyronirze or propylthiouracil. The fluorescence polarization of trans-parinarate, cis-parinarate~ and 1,6-diphenyl-l,3,5-hexatriene was 0.300_+ 0.004, 0.251 + 0.003, and 0.302 + 0.003, respectively, in liver plasma membranes from control rats and 0.316 + 0.003, 0.276 + 0.003 and 0.316 +_ 0.003, respectively, in liver plasma membranes from hyperthyroid rats (p < 0.025, n = 5). Propylthiouracil treatment did not significantly alter the fluorescence polarization of these probe molecules in the liver plasma membranes. Thus, liver plasma membranes from hyperthyroid animals appear to be more rigid than those of control animals. The effects of triiodothyronine, insulin and glucagon added in vitro to isolated liver plasma membrane preparations were also evaluated as follows: insulin (10 ~o M) and triiodothyronine (10 ~o M) increased fluorescence polarization of trans-parinaric acid, cis-parinaric acid and 1,6-diphenyl-l,3,5-hexatriene in liver plasma membranes while glucagon (10 -l~ M) had no effects. These hormonal effects on probe fluorescence polarization in liver plasma membranes were abolished by pretreatment of the rats for 7 days with triiodothyronine. Administration of triiodothyronine (10 -lo M) in vitro increased the fluorescence polarization of trans-parinaric acid in liver plasma membranes from propylthiouracil-treated rats. Thus, hyperthyroidism appeared to abolish the in vitro increase in polarization of probe molecules in the liver plasma membranes. Temperature dependencies in Arrhenius plots of absorption-corrected fluorescence and fluorescence polarization of trans-parinaric acid, cis-parinaric acid and 1,6-diphenyl-l,3,5-hexatriene were noted near 25 ~ in liver plasma membranes from triiodothyroninetreated rats and near 18 ~ in liver plasma membranes from propylthiouracil-treated rats. In summary, hormones such as triiodothyronine, insulin and glncagon may at least in part exert their biological effects on metabolism by altering the structure of the liver plasma membranes.

show abstract

Correction of right-angle molecular fluorescence measurements for absorption of fluorescence radiation

Cited by 49 publications

References 10 publications

Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes.

Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes.

Direct insertion and fluorescence studies of rhodamine-labeled β-adrenergic receptors in cell membranes

Hormonal effects on fatty acid binding and physical properties of rat liver plasma membranes

Contact Info

Product

Resources

About