The sulfhydryl groups of muscle phosphorylase. I. Number and reactivity

1968 ◽  
Vol 46 (6) ◽  
pp. 609-615 ◽  
Author(s):  
M. L. Battell ◽  
L. B. Smillie ◽  
N. B. Madsen
Keyword(s):  
Glycogen Phosphorylase ◽  
Monomer Unit ◽  
Sodium Dodecyl ◽  
Spectrophotometric Titration ◽  
Iodoacetic Acid ◽  
Enzymic Activity ◽  
Sulfhydryl Groups ◽  
Rabbit Muscle ◽  
Complete Inactivation ◽  
Protein Sulfhydryl Groups

The sulfhydryl groups of glycogen phosphorylase from rabbit muscle have been investigated with respect to their reactivity with p-chloromercuribenzoate (PCMB), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), cystine, N-ethylmaleimide (NEM), iodoacetic acid, and iodoacetamide. By spectrophotometric titration, PCMB reacts with 6 of the 18 half-cystine residues in native phosphorylase b, and with 12–14 of the 36 half-cystine residues in native phosphorylase a, and causes complete inactivation in both cases. Reaction of approximately 6 more residues in phosphorylase b (12 in phosphorylase a) occurs when the protein is denatured with HCl or sodium dodecyl sulfate (SDS).DTNB, cystine, and iodoacetic acid react with only two sulfhydryl groups in native phosphorylase b, and do not cause any loss of activity. Iodoacetamide can also react with two sulfhydryl groups without causing any loss of enzymic activity, but activity is lost in proportion to the extent of titration of the next four groups. DTNB reacts with 12 groups in SDS-denatured phosphorylase b but with only 9 groups in urea- or guanidine HCl-denatured enzyme. NEM and iodoacetic acid react with 14 groups in urea-denatured phosphorylase b. Two experiments are reported that provide additional confirmation of previous reports of the absence of disulfide bridges in phosphorylase.We conclude that two sulfhydryl groups per mole of phosphorylase b can react with any reagent without loss of enzymic activity. When the correct reagent and conditions are chosen, a further class of four sulfhydryl groups reacts, resulting in complete loss of enzymic activity. Thus, only two specific sulfhydryl groups on each monomer unit need be titrated to inactivate glycogen phosphorylase and cause it to dissociate. Upon denaturation of phosphorylase by several of the usual protein denaturants, another class of sulfhydryl groups reacts readily, the exact number depending on the reagent and the conditions, but the complete titration of all 18 sulfhydryl groups per molecule of phosphorylase b occurs only under special conditions. These results are of general relevance to procedures employing various protein denaturants and sulfhydryl reagents for the titration of protein sulfhydryl groups.

Modifizierung von Glycerinaldehyd-3-phosphat-Dehydrogenase aus Kaninchen-Skelettmuskel mit dem Coenzymanalogen [3-(3-Bromacetylpyridinio)-propyl]-adenosin-pyrophosphat / Modification of Glyceraldehyde-3-phosphate Dehydrogenase from Rabbit Skeletal Muscle by [3-(3-Bromoacetylpyridinio)-propyl]-Adenosine Pyrophosphate

1977 ◽  
Vol 32 (1-2) ◽  
pp. 85-92b ◽  
Author(s):  
Gerhard Dietz ◽  
Christoph Woenckhaus ◽  
Rainer Jaenicke ◽  
Inge Schuster
Keyword(s):  
Skeletal Muscle ◽  
Quaternary Structure ◽  
Enzymic Activity ◽  
Rabbit Skeletal Muscle ◽  
Rabbit Muscle ◽  
Irreversible Loss ◽  
Muscle Enzyme ◽  
Active Site Cysteine ◽  
Complete Inactivation ◽  
Inhibited Enzyme

The NAD analogue [3-(3-acetylpyridinio)-propyl] adenosine pyrophosphate forms enzymically inactive complexes with glyceraldehyde-3-phosphate dehydrogenase from yeast and rabbit skeletal muscle. In the latter enzyme four mol of the analogue are bound with equal affinity inhibiting the enzyme in a competitive way: KI = 0.3 mM as compared to the dissociation constant KD=O.6 mм. The brominated derivative [3- (3-bromoacetylpyridinio) -propyl] adenosine pyrophosphate is co­valently bound to both enzymes causing irreversible loss of enzymic activity. Complete inactivation of the enzyme from muscle requires two moles of the analogue per mol of tetramer. The remaining two sites are still able to bind two mol of NAD+ without regain of enzymic activity. In the case of the yeast enzyme four mol of the analogue are bound. Inactivation of the rabbit muscle enzyme is accompanied by the disappearance of two out of four highly reactive sulfhydryl groups; in the yeast enzyme the four active site cysteine residues are still able to react with DTNB1 the reactivity being diminished significantly. Hybrid formation between the native enzymes from yeast and skeletal muscle is not affected by the modification of the enzyme. Similarly the sedimentation properties of the covalently modi­fied enzyme are indistinguishable from those of the native molecule. This indicates that both the native and the irreversibly inhibited enzyme are identical regarding their quaternary structure.

The Number, Location, and Reactivity of the Cysteine Residues of Sturgeon Muscle Aldolase

1972 ◽  
Vol 50 (2) ◽  
pp. 111-119 ◽  
Author(s):  
P. J. Anderson
Keyword(s):  
Iodoacetic Acid ◽  
Sulfhydryl Groups ◽  
Cysteine Residues ◽  
Rabbit Muscle ◽  
Nitrobenzoic Acid ◽  
Catalytic Role ◽  
Aldolase Activity ◽  
Activity Loss

Sturgeon muscle aldolase contains six cysteine residues per subunit. These residues appear to occur in homologous positions to six of the eight cysteine residues of rabbit muscle aldolase. Three of the six residues can react with either iodoacetic acid or 5,5′-dithiobis-(2-nitrobenzoic acid) in the absence of denaturing agents. Reaction of three residues with iodoacetic acid inactivates the enzyme. The presence of substrate protects one of these residues and in this case no activity loss is observed. However, the results indicate that the effects on activity are due to the addition of the modifying group rather than to a loss of sulfhydryl groups. No residue corresponding to the previously demonstrated substrate-protected cysteine of rabbit aldolase was located in the sturgeon enzyme, which demonstrated that this residue was not essential to aldolase activity. It therefore appears unlikely that cysteine residues have a direct or an auxiliary catalytic role in aldolase activity.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

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.

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