Characterization of the pyridoxal phosphate site in glycogen phosphorylase b from rabbit muscle

Biochemistry ◽  
1978 ◽  
Vol 17 (25) ◽  
pp. 5359-5367 ◽  
Author(s):  
Shoji Shimomura ◽  
Toshio Fukui
Keyword(s):  
Glycogen Phosphorylase ◽  
Pyridoxal Phosphate ◽  
Rabbit Muscle ◽  
Glycogen Phosphorylase B

scholarly journals The nature of the binding site for aromatic compounds in glycogen phosphorylase b

1975 ◽  
Vol 147 (2) ◽  
pp. 369-371 ◽  
Author(s):  
G Soman ◽  
G Philip
Keyword(s):  
Chemical Modification ◽  
Binding Site ◽  
Aromatic Compounds ◽  
Glycogen Phosphorylase ◽  
Relative Effectiveness ◽  
Rabbit Muscle ◽  
Substituent Constant ◽  
Chemically Modified ◽  
Glycogen Phosphorylase B ◽  
Hydrophobic Nature

The inhibition of rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan--orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) by aromatic compounds was examined with 15 compounds. The relative effectiveness of the inhibitors correlated well with increasing substituent constant, pi, indicating the hydrophobic nature of the binding site. The inhibition was not affected by the ionic-strength variation of the assay mixtures. The results predict that the course of chemical modification of this enzyme and the properties of the derivatives depend on the nature of the reagent and on the incorporated groups. Many of the dissimilar and sometimes contradictory results reported for chemical-modification studies and for chemically modified phosphorylase b are explained by the findings presented in the paper.

scholarly journals Homogeneous Enzyme Immunoassay for Triiodothyronine in Serum

2001 ◽  
Vol 47 (3) ◽  
pp. 569-574 ◽  
Author(s):  
Christina D Karapitta ◽  
Theodore G Sotiroudis ◽  
Athanassios Papadimitriou ◽  
Aristotelis Xenakis
Keyword(s):  
Enzyme Immunoassay ◽  
Glycogen Phosphorylase ◽  
Serum Proteins ◽  
Rabbit Muscle ◽  
Glycogen Phosphorylase B ◽  
Low Concentrations ◽  
Enzyme Conjugate ◽  
High Concentrations ◽  
Treatment Step ◽  
Homogeneous Enzyme Immunoassay

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.

Time-resolved diffraction studies on glycogen phosphorylase b

1992 ◽  
Vol 340 (1657) ◽  
pp. 245-261 ◽  
Keyword(s):  
Glycogen Phosphorylase ◽  
Pyridoxal Phosphate ◽  
Data Sets ◽  
Laue Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Glycogen Phosphorylase B ◽  
Partial Data ◽  
Spread Function ◽  
Diode Array Spectrophotometer

Glycogen phosphorylase catalyses the reversible phosphorylation of glycogen to give glucose-1-phosphate in a reaction mechanism promoted by the 5'-phosphate of the cofactor pyridoxal phosphate. The reaction with the small substrate heptenitol has been probed using Laue diffraction at the Synchrotron Radiation Source, Daresbury. The reaction was initiated following photolysis from a caged phosphate compound 3,5-dinitrophenylphosphate (DNPP). In measurements on photolysis in the crystal using a diode array spectrophotometer approximately 7 mM cage (and hence phosphate) was released from a 21 mM solution with five flashes from a xenon flash lamp. In an experiment with the home source it was shown that DNPP is stable in the crystal under conditions of X-ray measurements and that on flashing sufficient phosphate is released to promote catalysis within 24 h. In a similar experiment with the synchrotron and Laue diffraction, data were recorded before and then 3 min, 15 min and 1 h after initiation of the reaction. Theoretical analysis of the point spread function arising from partial data-sets, numerical calculations with ideal data and the experimental results have shown the importance of low-resolution terms for the interpretation of Laue difference maps. Inclusion of terms obtained from unscrambling the wavelength harmonic overlaps led to significant improvement. The maps showed heptenitol bound at the catalytic site but no evidence for catalysis under these conditions. A rational for the lack of reaction and suggestions for future experiments with improved data are outlined.

scholarly journals Studies on the Pyridoxal Phosphate Site in Glycogen Phosphorylase b

1976 ◽  
Vol 65 (2) ◽  
pp. 521-527 ◽  
Author(s):  
Manuel CORTIJO ◽  
Juan LLOR ◽  
Juan S. JIMENEZ ◽  
Francisco GARCIA-BLANCO
Keyword(s):  
Glycogen Phosphorylase ◽  
Pyridoxal Phosphate ◽  
Glycogen Phosphorylase B
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