Clinical Chemistry 47: 569-574, 2001;
(Clinical Chemistry. 2001;47:569-574.)
© 2001 American Association for Clinical Chemistry, Inc.
Homogeneous Enzyme Immunoassay for Triiodothyronine in Serum
Christina D. Karapitta1,2,
Theodore G. Sotiroudis1,a,
Athanassios Papadimitriou3 and
Aristotelis Xenakis1
1
Industrial Enzymology Unit, Institute of Biological Research & Biotechnology, The National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635 Athens, Greece.
2
MEDICON S.A., 15344 Gerakas, Greece.
3
Department of Nuclear Medicine, Navy’s Hospital, 11521
Athens, Greece.
a Author for correspondence. Fax 30-1-7273758; e-mail
tsotir{at}eie.gr.
 |
Abstract
|
|---|
Background: The concentration of triiodothyronine (T3)
in human serum is extremely low and can be determined only by very
sensitive methods. We developed a homogeneous enzyme immunoassay for
T3 analysis in unextracted serum.
Methods: A T3 derivative was conjugated to the -SH
groups of glycogen phosphorylase b (GPb) from rabbit muscle.
Conjugation caused inhibition of enzyme activity, and the enzyme
conjugate was reactivated upon binding of anti-T3 antibody.
Activation was blocked by the presence of non-antibody-bound
T3; this was the basis for the development of the
homogeneous enzyme immunoassay for T3 by determining GPb
activity fluorometrically.
Results: We used furosemide to block the interaction of
T3 with serum proteins with T3-binding sites,
avoiding any serum treatment step. T3 was measured in the
range 0.3–8 µg/L. T3 values obtained by this assay
correlated well with those obtained by a RIA
(y = 0.97x - 0.07 µg/L;
r = 0.96; n = 92). Within- and between-run
imprecision (CV) was 5–9% for normal and high concentrations and
16–20% for low concentrations.
Conclusions: Chemical modification of GPb with a T3
derivative allows the development of a simple homogeneous enzyme
immunoassay for T3 in unextracted serum.
|
Introduction
|
|---|
The serum concentration of triiodothyronine
(T3)1
is very low
[reference interval, 0.7–2.1 µg/L (1)], and it is
usually determined by RIA (2)(3), heterogeneous
enzyme immunoassays (4), chemiluminescent immunoassays
(5)(6), or electrochemiluminescent immunoassays
(7). Homogeneous enzyme immunoassays have been developed
(8)(9)(10)(11) for various haptens, including thyroxine
(10)(12), but no homogeneous enzyme immunoassay
for T3 analysis has been reported.
We describe here the development of the first homogeneous
enzyme immunoassay for T3, with
T3 conjugated to glycogen phosphorylase b (GPb)
from rabbit muscle. No other enzyme immunoassay has appeared in the
literature that uses hapten conjugated to GP. The binding was carried
out using 4-(maleimidomethyl)-cyclohexane-1-carboxylic acid
N-hydroxysuccinimide ester (SMCC), a reagent containing a
maleimide group that reacts specifically with -SH groups
(13). GP is an allosteric enzyme that catalyzes the
degradative phosphorolysis of glycogen to glucose 1-phosphate. The
rabbit muscle enzyme exists in two interconvertible forms, the
phosphorylated form, GPa, and the dephosphorylated form, GPb. GPb is
inactive and can be activated either by covalent phosphorylation to
form GPa or by no covalent cooperative binding of AMP
(14)(15). GPb consists of two identical
polypeptide chains. GPb from rabbit muscle contains 842 amino acids per
subunit (16) and has nine sulfhydryl groups important for
enzyme activity (17). GP exists in human serum of healthy
subjects predominantly in the b form at very low concentrations (<5
µg/L) (18)(19).
|
Materials and Methods
|
|---|
reagents
All chemicals, as well as the polyclonal
anti-T3 antibody (T-2777) prepared in rabbits
were from Sigma Chemical Co. Sephadex G-25 (fine) was from
Amersham Pharmacia Biotech AB. T3 serum
calibrators were from Bayer Diagnostics. Silica Gel 60 thin-layer
chromatography plates (0.25 mm; cat. no. 5721) were obtained
from Merck.
purification of rabbit muscle GPb
Rabbit muscle GPb was prepared according to Fischer and Krebs
(20), using 2-mercaptoethanol instead of cysteine, and was
recrystallized at least four times before use. The GPb concentration
was determined spectrophotometrically using the extinction
coefficient E280 nm1% =
13.2 (21).
synthesis and purification of smcc-t3
N-[4-(maleimidomethyl)-cyclohexan-1-yl]triiodothyronyl
carboxamide (MCC-T3) was synthesized by mixing
0.11 mL of 9 mmol/L SMCC and 0.67 mL of 1.5 mmol/L
T3 in dimethylformamide in a final reaction
volume of 1 mL. The reaction was carried out for 90 min at 30 °C.
The product MCC-T3 was purified by thin-layer
chromatography on Silica Gel 60 plates (0.25 mm; Merck) with ethyl
acetate-acetic acid-water (90:10:5 by volume) as mobile phase. The
prominent band at Rf = 0.67 was cut
out, and the product was extracted with methanol. The sample was
filtered, and the filtrate was condensed with a rotary evaporator to a
volume of 1 mL. The product was quantified spectrophotometrically. The
hormone derivative was stored as a methanol solution in the dark at
-20 °C. The above methodology is a modification of similar
published procedures (22)(23).
conjugation of mcc-t3 to GPb
The enzyme conjugate was prepared by the chemical coupling of
MCC-T3 to GPb. In the coupling procedure, 0.4 mL
of GPb (10.3 µmol/L) in 50 mmol/L triethanolamine-HCl buffer
(pH 6.8) was mixed with 0.037 mL of 0.222 mmol/L
MCC-T3 in methanol. The mixture was incubated at
30 °C for 15 min. The enzyme conjugate was purified on a Sephadex
G-25 column equilibrated with 50 mmol/L triethanolamine-HCl buffer (pH
6.8) containing 1 mmol/L EDTA and eluted with the same solution. The
enzyme fractions were collected, and the protein concentration was
determined spectrophotometrically. The enzyme conjugate was stored at
-20 °C after the addition of an equal volume of glycerol containing
2-mercaptoethanol and bovine serum albumin (final concentrations, 0.03
mol/L 2-mercaptoethanol and 1 g/L bovine serum albumin) and
could be used for at least 6 months.
determination of GPb activity
GPb activity in the direction of phosphorolysis of glycogen was
measured using the auxiliary assay system as described by Helmreich and
Cori (24) with some modifications. The final reaction
mixture was 0.6 mL and contained 2.5 kU/L glucose-6-phosphate
dehydrogenase, 0.6 kU/L phosphoglucomutase, 1 mmol/L
NADP+, 1 mmol/L magnesium acetate, 1 µmol/L
glucose 1,6-diphosphate, 16 mmol/L sodium phosphate (pH 7), 1 mmol/L
AMP, 5 g/L glycogen, 0.15 mmol/L EDTA, and GPb, as indicated, in 20
mmol/L Tris-acetate buffer (pH 7.4), containing 0.03 mol/L NaCl, 0.1
mmol/L MgCl2, 0.1 g/L bovine serum albumin, 0.5
g/L gelatin, 0.1 g/L NaN3, and 0.2 mmol/L
N-ethylmaleimide (22). GPb, AMP, and glycogen
were preincubated for 15 min at 30 °C before the reaction was
initiated with this mixture. The reaction was terminated by the
addition of 0.05 mL of 13 g/L sodium dodecyl sulfate. NADPH
formed by the reaction was measured fluorometrically at 25 °C in
10-mm pathlength quartz cuvettes using a Perkin-Elmer 650-40
fluorescence spectrophotometer. The excitation and emission wavelengths
were set at 340 and 460 nm, respectively. The relevant slits were set
at 5 and 10 nm, respectively.
Kinetic data were analyzed by using the nonlinear regression program
GraFit (25).
determination of the number of t3 molecules bound to
GPb
The number of molecules of T3 conjugated per
subunit of GPb was calculated using the method of Saboori et al.
(26) for determination of iodine in iodoproteins.
assay of t3
For T3 analysis the followed reagents were
used:
- Tris-acetate buffer, pH 7.4 (stock solution), containing 200 mmol/L
Tris-acetate buffer (pH 7.4), 0.3 mol/L NaCl, 1 mmol/L
MgCl2, 1 g/L bovine serum albumin, 5 g/L gelatin,
1 g/L NaN3, and 2 mmol/L
N-ethylmaleimide
- Antibody reagent, containing polyclonal anti-T3
antibody and 1.2 mmol/L furosemide in 20 mmol/L Tris-acetate buffer
- Blank antibody reagent, containing 1.2 mmol/L furosemide in 20 mmol/L
Tris-acetate buffer
- Enzyme reagent, containing GPb-T3 conjugate, 15
mmol/L AMP, and 75 g/L glycogen
- Substrate reagent, containing 3.33 kU/L glucose 6-phosphate
dehydrogenase, 0.8 kU/L phosphoglucomutase, 1.33 mmol/L
NADP+, 1.33 mmol/L magnesium acetate, 1.33
µmol/L glucose 1,6-diphosphate, 21 mmol/L sodium phosphate (pH 7) in
27 mmol/L Tris-acetate buffer
- T3 serum calibrators, which were provided as
lyophilized human serum-based preparations containing 0.99 g/L sodium
azide as preservative and 0, 0.5, 1, 2, 4, and 8 µg/L of
L-triiodothyronine. T3 human serum
calibrators were those provided for the Technicon Immuno
1® System (Bayer Diagnostics)
The assay was performed as follows: 0.1 mL of calibrator or serum
sample and 0.01 mL of antibody or blank antibody reagent were incubated
at 30 °C for 30 min. Enzyme reagent (0.04 mL) was then added, and
the mixture was incubated for 15 min at 30 °C. Finally, 0.45 mL of
the substrate reagent was added. After an additional incubation at
30 °C for 1 h, the reaction was stopped by the addition of 0.05
mL of 13 g/L sodium dodecyl sulfate, and the NADPH formed was
determined fluorometrically as described above.
For each sample, the fluorescence intensity in the presence
(F; addition of 0.01 mL of antibody reagent) and absence of
antibody (Fo; addition of 0.01 mL of blank antibody reagent)
was measured. The calibration curve was constructed by plotting
F - Fo vs the concentrations of the
T3 calibrator.
ria
For comparison, we assayed T3 using the
T3-solid-RIA reagent set (3),
from the Institute of Radioisotopes and Radiodiagnostic Products.
subjects
Sera from blood samples without anticoagulant were obtained from
patients who had attended Navy’s Hospital, Athens, Greece. Ninety-two
consecutive routine patients (41 men and 51 women) who required
laboratory thyroid testing were used for the precision and method
comparison study, following the procedures approved by the ethics
committee of Navy’s Hospital. The mean ages of the total patient
group, and the men and the women as separate groups were 42.5 years
(range, 19–71 years) for the total patient group, 39.8 years (range,
23–61 years) for the men, and 44.8 years for the women (range, 19–71
years).
|
Results
|
|---|
GPb reacted with MCC-T3 at a GPb-to-amide
mol/mol ratio of 1:2. This reaction ratio was found to be the optimum
under the present experimental conditions (data not shown).
Determination of iodine indicated that 1.2 molecules of
T3 were bound per enzyme subunit. The activity of
the enzyme conjugate was 
10% of the original activity prior to
conjugation. When excess polyclonal anti-T3
antibody bound to the GPb-T3 conjugate, the
enzyme regained activity, reaching 65% of its original value. This
activation of GPb-T3 conjugate was decreased in
the presence of non-antibody-bound T3. The
observed decrease in activity depended on the T3
concentration. This was the basis for the proposed homogeneous enzyme
immunoassay for the determination of T3.
In an effort to understand the mechanism of inhibition of GPb by
MCC-T3 conjugation, a comparative study of the
kinetic parameters of the native and modified enzyme in presence and
absence of polyclonal anti-T3 antibody was
undertaken. As shown in Table 1
, the conjugation led to increases in
Km values for both substrates,
Pi and glycogen, and for AMP. In the
presence of anti-T3 antibody, only the
Km value for AMP was increased
1.5-fold. In parallel, the conjugated enzyme lost its allosteric
character (Hill coefficient for AMP, n 
1). The binding of
anti-T3 antibody to GPb-T3
conjugate induced an enhancement of the
Vmax values for
Pi, glycogen, and AMP.
View this table:
[in this window]
[in a new window]
|
Table 1. Kinetic parameters of native GPb and
GPb-T3 conjugate in the absence and presence of polyclonal
anti-T3
antibody.1
|
|
The present assay for T3 analysis used 0.1 mL of
serum in a 0.6-mL final assay volume. The effect of this amount of
serum on the activity of the native GPb, the conjugate
GPb-T3, and the reactivation of the latter by
anti-T3 antibody was examined. In all cases, the
presence of serum induced an enzyme activity loss of 30% compared
with the values determined in the absence of serum. It is worth noting
that the presence of serum in the assay did not alter the percentage of
reactivation of GPb-T3 conjugate upon binding of
anti-T3 antibody. Moreover, the small amount of
GPb (<5 µg/L) present in human serum (18)(19)
did not influence the determination of T3 by the
present immunoassay because the T3 concentration
was determined by the relative value of increase of the enzyme
conjugate activity in the presence of anti-T3
antibody (F - Fo). Both values,
Fo and F, included the activity of endogenous
serum GP.
Furosemide was used for displacing T3 from serum
binding proteins (27). The optimum concentration of
furosemide was determined by mixing various concentrations of
displacing agent with human serum containing 10 µg/L
T3 and measuring the T3
concentration as described previously (27). Furosemide, at a
final concentration of 0.1 mmol/L in
serum-anti-T3 antibody-furosemide solution,
provided maximum T3 displacement without
interfering with the phosphorylase assay. T3
displacement was estimated by calculation of the analytical recovery.
The mean analytical recovery of three separate samples was 96% (range,
95–98%).
To select the optimum concentration ratio of enzyme conjugate to
anti-T3 antibody that could be used to determine
the T3 concentration with the desired sensitivity
and accuracy, an optimization procedure similar to the one applied for
the analysis of theophylline in serum (11) was performed. We
first followed the time course of the activation of
GPb-T3 conjugate by polyclonal
anti-T3 antibody. Activation of
GPb-T3 was complete after incubation of the
conjugate and the anti-T3 antibody for 30 min at
30 °C (data not shown). Preliminary experiments using various
concentrations of GPb-T3 conjugate (0.03–0.2
mg/L) indicated that 0.09 mg/L was the optimal concentration under the
chosen conditions. The GPb-T3 conjugate (0.09
mg/L) was then mixed with gradually increasing amounts of polyclonal
anti-T3 antibody, and the enzyme reactivation was
determined. As shown in Fig. 1
, the enzyme activity increased with increasing antibody
concentration. Because the immunoassay must be able to discriminate
among the T3 concentrations likely to be
encountered when patient specimens are assayed, the optimum ratio of
anti-T3 antibody to enzyme conjugate in the
presence of different T3 concentrations within
the desired assay range was determined. Fig. 2
illustrates an assessment of the system’s ability to
discriminate among hypothyroidic, normothyroidic, and
hyperthyroidic T3 concentrations (0.6, 1.8, and
5.5 µg/L T3, respectively). Although the peak
antibody signal occurred between 0.1 and 0.2 µL for the given amount
of the GPb-T3 conjugate, 0.1 µL of antibody was
used for the T3 immunoassay.
Fig. 3
shows a representative calibration curve for
the homogeneous enzyme immunoassay for T3 using
the six T3 serum calibrators.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 1. Activation of GPb-T3 activity by various
concentrations of polyclonal anti-T3 antibody.
Activity of the enzyme conjugate was measured using a final
GPb-T3 conjugate concentration of 0.09 mg/L and
anti-T3 antibody as indicated. F and
Fo, fluorescence intensity in the presence or absence of
antibody, respectively.
|
|
View larger version (10K):
[in this window]
[in a new window]
|
Figure 3. Calibration curve for T3.
Activity of enzyme conjugate was measured using a final concentration
of 0.09 mg/L GPb-T3 conjugate and 0.1 µL of polyclonal
anti-T3 antibody. The T3 assay was performed as
described in Materials and Methods.
|
|
The detection limit of the present immunoassay was 0.15 µg/L
T3. This value was determined by calculating the
concentration of T3 that would give a response
equal to 2 SD above that of T3 zero calibrator
(0.0 µg/L).
Within-run imprecision was estimated by analyzing three samples with
low, normal, and above-normal T3 concentrations,
eight times each; the CVs were 5.0–21%. Between-run
imprecision was measured in duplicate for three samples with low,
normal, and above-normal T3 concentrations on 6
different days; the CVs were 6.1–16% (Table 2
).
Analytical recovery studies were carried out by adding
T3 to T3-free human sera
(28) to give samples with T3
concentrations of 0.5, 1.3, 3.5, 4.5, and 6.5 µg/L. Analytical
recoveries were 93%, 107%, 108%, 102%, and 106%, respectively.
To evaluate the applicability of the present immunoassay, we compared
the values for 92 serum samples as measured with our homogeneous enzyme
immunoassay with the values obtained by a RIA (Fig. 4
). The regression equation of the obtained plot was:
y = 0.97x - 0.07 µg/L (range
examined, 0.3–8 µg/L T3); the correlation
coefficient (r) was 0.96.
|
Discussion
|
|---|
The development of the first homogeneous enzyme immunoassay for
the determination of T3 concentrations in human
serum was based on the conjugation of T3 to GPb.
MCC-T3 was covalently bound to the -SH groups of
GPb. This modification induced inhibition of the enzyme activity,
whereas binding of excess polyclonal anti-T3
antibody to the modified enzyme led to reactivation. One -SH group was
modified per enzyme subunit. The enzyme lost its allosteric character,
and the Km values for AMP and
Pi were increased 13- and 11-fold, respectively.
It is known that cysteine-318 is near the allosteric binding site, and
its accessibility was calculated to be 77.8% in the GPb dimer
by the program NACCESS (29). The -SH group of cysteine-318
is 9.03 Å from atom N1 of adenosine and 14.7 Å from the phosphorus of
the phosphate group in AMP [the distances were calculated using the
program CONTACT (30)]. All of the above, in addition to the
fact that the conjugation reaction was instantaneous, led to the
conclusions that cysteine-318 was the target of modification by
MCC-T3 and that this modification was the cause
of the observed enzyme inhibition. The binding of
anti-T3 antibody to the conjugate caused
conformational changes that led to an 1.3- to 1.4-fold increase in
the Vmax values for AMP and
Pi.
The advantage of the present homogeneous enzyme immunoassay is its
simplicity: homogeneous enzyme immunoassays do not require physical
separation of free and antibody-bound components to determine the
fraction of bound conjugate (31). Moreover, it is
sufficiently sensitive and accurate for determining serum
T3 concentrations between 0.3 and 8 µg/L, as do
other currently available immunoassays for T3
analysis. In addition, it could be adapted to an automated analyzer. In
this case, the storage characteristics and stability of the reagents
should be determined over a wide range of laboratory conditions and a
long period of time. Our results compare favorably with those obtained
by a RIA. Furosemide was successfully used for blocking the binding of
T3 to serum proteins
(27)(32). The compound used most frequently to
block the binding of T3 to these proteins,
8-anilino-1-naphthalenesulfonic acid, was excluded because it could
interfere in the absorbance measurements (33) and inhibit
GPb (34). Serum contains a small amount of GPb, <5
µg/L, which does not influence the determination of
T3 with our enzyme immunoassay. The measurement
of T3 is not affected by serum volume and serum
components, which may cause inhibition of GPb, because the
T3 concentration is determined by the relative
increase in the enzyme conjugate activity in the presence of
anti-T3 antibody.
In conclusion, modification of the -SH groups of GPb from rabbit
muscle by a T3 derivative led to the development of the
first homogeneous enzyme immunoassay for T3 analysis. The
binding of MCC-T3 to GPb inhibited the enzyme activity,
whereas binding of anti-T3 antibody to the
GPb-T3 led to reactivation. The activation of
GPb-T3 was gradually decreased in the presence of
increasing concentrations of non-antibody-bound T3. More
robust clinical investigation of this potential novel immunoassay is
necessary to demonstrate its clinical utility because several
medications or abnormal serum constituents might distort the assay
signal.
|
Acknowledgments
|
|---|
This work was supported by the Greek General Secretariat of
Research and Technology (Grant Y
EP 55) and by MEDICON S.A. We thank
Dr. N.G. Oikonomakos for valuable suggestions.
|
Footnotes
|
|---|
1 Nonstandard abbreviations: T3, triiodothyronine; GP, glycogen phosphorylase; SMCC, 4-(maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester; and MCC-T3, N-[4-(maleimidomethyl)-cyclohexan-1-yl]triiodothyronyl carboxamide. 
|
References
|
|---|
-
Tietz NW eds. Clinical guide to laboratory tests, 3rd ed 1995:612 WB Saunders Philadelphia. .
-
Chopra IJ, Ho RS, Lam R. An improved radioimmunoassay of triiodothyronine in serum: its application to clinical and physiological studies. J Lab Clin Med 1972;80:729-739.[Web of Science][Medline]
[Order article via Infotrieve]
-
Petrou PS, Kakabakos SE, Koupparis MA, Christofidis I. Antibody coating approach involving gamma globulins from non-immunized animal and second antibody antiserum. J Immunoassay 1998;19:271-293.[Web of Science][Medline]
[Order article via Infotrieve]
-
Groskopf W, Hsu S, Sohn L. A fully automated assay for total T3 utilizing the Abbot IMx analyzer [Abstract]. Clin Chem 1988;34:1210.
-
Le Q, Ka M, Chiu L, Toivola B.. Laboratory evaluation of thyroid assays on the Immulite® automated immunoassay system [Abstract]. Clin Chem 1996;42(Suppl 6):156.[Web of Science]
-
Dayal S, Arnold K, Aziz A, Monfiston B, Kaiser A, Hemans W, et al. Automated FT4, FT3, T3, Tuptake 3rd Gen TSH and T4 immunoassays on the Bayer ADVIA® Integrated Modular SystemTM [Abstract]. Clin Chem 2000;46(Suppl 6):A122.
-
Sanchez-Carbayo M, Mauri M, Alfayete R, Miralles C, Soria F. Analytical and clinical evaluation of TSH and thyroid hormones by electrochemiluminescent immunoassays. Clin Biochem 1999;32:395-403.[Web of Science][Medline]
[Order article via Infotrieve]
-
Rubenstein KE, Schneider RS, Ullman EF. Homogeneous enzyme immunoassay. A new immunochemical technique. Biochem Biophys Res Commun 1972;47:846-851.[Web of Science][Medline]
[Order article via Infotrieve]
-
Rowley GL, Rubenstein KE, Huisjen J, Ullman EF. Mechanism by which antibodies inhibit hapten-malate dehydrogenase conjugates. An enzyme immunoassay for morphine. J Biol Chem 1975;250:3759-3766.[Abstract/Free Full Text]
-
Ullman EF, Yoshida RA, Blakemore JI, Maggio E, Leute R. Mechanism of inhibition of malate dehydrogenase by thyroxine derivatives and reactivation by antibodies. Homogeneous enzyme immunoassay for thyroxine. Biochim Biophys Acta 1979;567:66-74.[Medline]
[Order article via Infotrieve]
-
Chang J, Gotcher S, Gushaw JB. Homogeneous enzyme immunoassay for theophylline in serum and plasma. Clin Chem 1982;28:361-367.[Free Full Text]
-
Ullman EF, Blakemore J, Leute RK, Eimstad W, Jaklitsch A. Homogeneous enzyme immunoassay for thyroxine [Abstract]. Clin Chem 1975;21:1011.
-
Yoshitake S, Yamada Y, Ishikawa E, Masseyeff R. Conjugation of glucose oxidase from Aspergillus niger and rabbit antibodies using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide. Eur J Biochem 1979;101:395-399.[Web of Science][Medline]
[Order article via Infotrieve]
-
Graves DJ, Wang JH.
-Glucan phosphorylases—chemical and physical basis of catalysis and regulation. Boyer PD eds. The enzymes, 3rd ed 1972;Vol. 7:432-482 Academic Press New York. .
-
Sotiroudis TG, Cazianis CT, Oikonomakos NG, Evangelopoulos AE. Effect of sodium cholate on the catalytic and structural properties of phosphorylase b. Eur J Biochem 1983;131:625-631.[Web of Science][Medline]
[Order article via Infotrieve]
-
Titani K, Koide A, Hermann J, Ericsson LH, Kumar S, Wade RD, et al. Complete amino acid sequence of rabbit muscle glycogen phosphorylase. Proc Natl Acad Sci U S A 1977;74:4762-4766.[Abstract/Free Full Text]
-
Zarkadas CG, Smile LB, Madsen NB. Sulphydryl groups of muscle phosphorylase. Thiol sequences and subunit structure. J Mol Biol 1968;38:245-247.[Web of Science][Medline]
[Order article via Infotrieve]
-
Krause EG, Will H, Böhm M, Wollenberger A. The assay of glycogen phosphorylase in human blood serum and its application to the diagnosis of myocardial infarction. Clin Chim Acta 1975;58:145-154.[Web of Science][Medline]
[Order article via Infotrieve]
-
Rabitzsch G, Noll F, Hofmann U, Krause EG, Armbruster FP. Basal concentration of the isoenzyme BB of the glycogen phosphorylase b in human blood. Clin Chim Acta 1993;214:109-111.[Web of Science][Medline]
[Order article via Infotrieve]
-
Fischer EH, Krebs EG. Muscle phosphorylase b. Methods Enzymol 1962;5:369-373.[Web of Science]
-
Kastenschmidt LL, Kastenschmidt J, Helmreich E. Subunit interactions and their relationship to the allosteric properties of rabbit skeletal muscle phosphorylase b. Biochemistry 1968;7:3590-3608.[Medline]
[Order article via Infotrieve]
-
Yamamoto R, Hattori S, Inukai T, Matsuura A, Yamashita K, Kosaka A, Kato K. Enzyme immunoassay for thyroxine and triiodothyronine in human serum, with use of a covalent chromatographic separation method. Clin Chem 1981;27:1721-1723.[Abstract/Free Full Text]
-
Köhrle J, Rasmussen UB, Rokos H, Leonard JL, Hesch RD. Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine. J Biol Chem 1990;265:6146-6154.[Abstract/Free Full Text]
-
Helmreich E, Cori CF. The role of adenylic acid in the activation of phosphorylase. Proc Natl Acad Sci U S A 1964;51:131-138.[Free Full Text]
-
Leatherbarrow RJ. GraFit, Ver. 3.0 [Computer
Program]. Staines, UK: Erithakus Software, 1992..
-
Saboori AM, Rose NR, Butscher WG, Burek CL. Modification of a nonincinerative method for determination of iodine in iodoproteins. Anal Biochem 1993;214:335-338.[Web of Science][Medline]
[Order article via Infotrieve]
-
Groskopf W, Green B, Sohn L, Hsu S. Furosemide as a displacing agent in assay of total triiodothyronine [Editorial]. Clin Chem 1991;37:587-588.[Free Full Text]
-
Brown ML, Metheany J. Use of 8-anilino-1-naphalenesulfonic acid in radioimmunoassay of triiodothyronine. J Pharm Sci 1974;63:1214-1217.[Web of Science][Medline]
[Order article via Infotrieve]
-
Hobbard SJ, Thornton JM. NACCESS [Computer Program] 1993 Department of Biochemistry and Molecular Biology, University College London London. .
-
Collaborative Computational Project, Number 4. The CCP4
Suite: programs of protein crystallography. Acta Crystallogr
1994;D50:760–3..
-
Selby C. Interference in immunoassay. Ann Clin Biochem 1999;36:704-721.
-
Stockigt JR, Lim CF, Barlow JW, Wynne KN, Mohr VS, Topliss DJ, et al. Interaction of furosemide with serum thyroxine-binding sites: in vivo and in vitro studies and comparison with other inhibitors. J Clin Endocrinol Metab 1985;60:1025-1031.[Abstract/Free Full Text]
-
Yu X-C, Margolin W. Inhibition of assembly of bacterial cell division protein FtsZ by the hydrophobic dye 5,5'-bis-(8-anilino-1-naphthalenesulfonate). J Biol Chem 1998;273:10216-10222.[Abstract/Free Full Text]
-
Madsen NB, Shechosky S, Fletterick RJ. Site-site interactions in glycogen phosphorylase b probed by ligands specific for each site. Biochemistry 1983;22:4460-4465.[Medline]
[Order article via Infotrieve]
Top
Abstract
Introduction
), 1.8
µg/L (•), and 5.5 µg/L (
). F and
FT3, fluorescence intensity in the absence
and presence of T3, respectively.