Peptide sequences and relative reactivity of the reactive sulfhydryl groups of rabbit muscle phosphorylase

Biochemistry ◽  
1970 ◽  
Vol 9 (23) ◽  
pp. 4480-4486 ◽  
Author(s):  
Allen M. Gold ◽  
David Blackman
Keyword(s):  
Sulfhydryl Groups ◽  
Relative Reactivity ◽  
Rabbit Muscle ◽  
Muscle Phosphorylase

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.

Kinetic mechanism of phosphorylase a, II. Isotope exchange studies at equilibrium

1970 ◽  
Vol 48 (7) ◽  
pp. 755-758 ◽  
Author(s):  
H. D. Engers ◽  
W. A. Bridger ◽  
N. B. Madsen
Keyword(s):  
Exchange Rates ◽  
Isotope Exchange ◽  
Initial Rate ◽  
Kinetic Mechanism ◽  
Rabbit Muscle ◽  
Glucose Inhibition ◽  
Rate Limiting Step ◽  
Equilibrium Exchange ◽  
Muscle Phosphorylase ◽  
Rate Limiting

In order to confirm the kinetic mechanism which was proposed for rabbit muscle phosphorylase a on the basis of initial rate studies and UDP-glucose inhibition experiments, isotope exchange studies at equilibrium were performed, both in the presence and absence of the modifier AMP.Both the 14C-glucose [Formula: see text] and the [Formula: see text]1-phosphate equilibrium exchange rates increased to a maximum as the concentrations of the varied substrates became saturating, either in the presence or absence of AMP. The plateaus observed in these experiments indicate the lack of inhibition of the exchange of one pair of substrates when the concentration of the other substrate pair was raised, and confirms the proposed random addition of substrates to the enzyme.The fact that similar exchange rates were observed for either reaction direction reinforced the concept that rapid equilibrium conditions apply to the phosphorylase a mechanism; i.e. the interconversion of the ternary complexes tends to be the rate-limiting step in the reaction sequence.Maximal velocities determined from initial rate data reported in the previous paper agreed with those calculated from isotope exchange rates.

scholarly journals Amino-terminal sequence of rabbit muscle phosphorylase

FEBS Letters ◽  
1975 ◽  
Vol 55 (1-2) ◽  
pp. 120-123 ◽  
Author(s):  
Koiti Titani ◽  
Philip Cohen ◽  
Kenneth A. Walsh ◽  
Hans Neurath
Keyword(s):  
Rabbit Muscle ◽  
Terminal Sequence ◽  
Amino Terminal ◽  
Muscle Phosphorylase ◽  
Amino Terminal Sequence
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