scholarly journals A study of the role of the reactive thiol group of rabbit muscle creatine kinase with a chromophoric reporter group

1978 ◽  
Vol 171 (1) ◽  
pp. 269-272 ◽  
Author(s):  
M A Keighren ◽  
N C Price
Keyword(s):  
Creatine Kinase ◽  
Conformational Changes ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase ◽  
Transition State Analogue ◽  
Muscle Creatine ◽  
Reporter Group

Substrate- and ligand-induced conformational changes were studied in a series of thiol-modified derivatives of rabbit muscle creatine kinase that retained different amounts of enzymic activity. The results indicate that the ‘reactive’ thiol group of the enzyme is required for the conformational changes associated with formation of a ‘transition-state analogue’ complex.

scholarly journals The effect of limited proteolysis on rabbit muscle creatine kinase

1981 ◽  
Vol 199 (1) ◽  
pp. 239-244 ◽  
Author(s):  
N C Price ◽  
S Murray ◽  
E J Milner-White
Keyword(s):  
Creatine Kinase ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Thiol Group ◽  
Proteinase K ◽  
Cross Linking ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase ◽  
Transition State Analogue ◽  
Muscle Creatine

Creatine kinase from rabbit muscle is inactivated by limited proteolysis with proteinase K from Tritirachium album. Gel-filtration and cross-linking studies showed that the limited proteolysis did not affect the molecular weight of the enzyme under non-denaturing conditions, but did cause changes in the reactivity of the reactive thiol group on each subunit and in the ability of the enzyme to form a ‘transition-state analogue’ complex in the presence of magnesium acetate plus ADP plus creatinine plus NaNO3.

Dynamic Asymmetry and the Role of the Conserved Active-Site Thiol in Rabbit Muscle Creatine Kinase

Biochemistry ◽  
2014 ◽  
Vol 54 (1) ◽  
pp. 83-95 ◽  
Author(s):  
Casey H. Londergan ◽  
Rachel Baskin ◽  
Connor G. Bischak ◽  
Kevin W. Hoffman ◽  
David M. Snead ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Active Site ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase ◽  
Muscle Creatine

scholarly journals The refolding of denatured rabbit muscle creatine kinase. Search for intermediates in the refolding process and effect of modification at the reactive thiol group on refolding

1982 ◽  
Vol 201 (1) ◽  
pp. 171-177 ◽  
Author(s):  
N C Price ◽  
E Stevens
Keyword(s):  
Creatine Kinase ◽  
Thiol Group ◽  
Rabbit Muscle ◽  
Protein Fluorescence ◽  
Muscle Creatine Kinase ◽  
Folding Process ◽  
Process Modification ◽  
Muscle Creatine

A number of aspects of the refolding of denatured rabbit muscle creatine kinase have been studied. Addition of substrates has no effect on the rate or extent of regain of activity. The changes in protein fluorescence during refolding broadly parallel the regain of activity. A study of the susceptibility of the enzyme to proteolysis during refolding indicates that there is no significant accumulation of folded, but inactive, intermediates in the folding process. Modification of the reactive thiol group on each subunit of the enzyme by small reagents such as iodoacetate or iodoacetamide prior to denaturation has only a small effect on the rate of subsequent refolding. However, modification by the bulky reagent 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate has a very large effect on the ability of the enzyme to refold after denaturation.

scholarly journals The reaction of rabbit muscle creatine kinase with some derivatives of iodoacetamide

1979 ◽  
Vol 177 (2) ◽  
pp. 603-612 ◽  
Author(s):  
N C Price
Keyword(s):  
Creatine Kinase ◽  
Thiol Group ◽  
Rabbit Muscle ◽  
Binding Studies ◽  
Muscle Creatine Kinase ◽  
Hybrid Enzyme ◽  
Complete Inactivation ◽  
Muscle Creatine ◽  
Derivatives Of ◽  
Lines Of Evidence

The dimeric enzyme creatine kinase from rabbit muscle was treated with three derivatives of iodoacetamide that are capable of introducing fluorescent groups into the enzyme. All the three reagents (4-iodoacetamidosalicylate (IAS), 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulphonate (IAEDANS) and 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate (IAANS)) were shown to react at the same single thiol group on each enzyme subunit, leading to complete inactivation of the enzyme. The reaction with IAS was extremely rapid by comparison with the reaction with iodoacetamide or iodoacetate, but various lines of evidence suggest that IAS is not a true affinity label. However, kinetic and binding studies indicate that salicylate itself probably binds at the nucleotide-binding site on the enzyme. As the size of the modifying reagent increased, the first thiol group reacted more rapidly than the second; this trend was more pronounced at 0 degree C than at 25 degree C. With the largest modifying reagent used (IAANS), the pronounced biphasic nature of the modification reaction permitted the preparation of a hybrid enzyme in which only one subunit was modified, but a study of the thiol-group reactivity showed that this hybrid enzyme preparation underwent subunit rearrangement.

Sodium barbital is a slow reversible inactivator of rabbit-muscle creatine kinase

2006 ◽  
Vol 84 (2) ◽  
pp. 142-147
Author(s):  
Feng Shi ◽  
Tong-Jin Zhao ◽  
Hua-Wei He ◽  
Jie Li ◽  
Xian-Gang Zeng ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Energy Homeostasis ◽  
Inactivation Kinetics ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase ◽  
Reversible Inactivation ◽  
Inhibition Effect ◽  
Sodium Barbital ◽  
Muscle Creatine ◽  
Detailed Kinetics

As a depressant of the central nervous system, the clinical effect of sodium barbital has been extensively studied. Here we report on sodium barbital as an inhibitor of rabbit-muscle creatine kinase (CK), which plays a significant role in energy homeostasis in the muscles. Although sodium barbital gradually inhibits the activity of CK with increased concentration, the inhibition effect can be completely reversed by dilution, indicating that the inactivation process is reversible. Detailed kinetics analysis, according to a previously presented theory, indicates that sodium barbital functions as a non complexing inhibitor, and its inhibition effect on CK is a slow reversible inactivation. In this study, a kinetic model of the substrate reaction is presented, and the microscopic rate constants for the reaction of sodium barbital with the free enzyme and the enzyme–substrate complexes are determined. Kinetic analysis reveals that sodium barbital might compete with both creatine and ATP, but mainly with creatine, to inhibit the activity of CK. The results suggest that CK might be a target for sodium barbital in vivo.Key words: creatine kinase; inactivation; kinetics; sodium barbital.

Conformational changes and inactivation of rabbit muscle creatine kinase in dimethyl sulfoxide solutions

2002 ◽  
Vol 80 (4) ◽  
pp. 427-434 ◽  
Author(s):  
Wen-bin Ou ◽  
Ri-Sheng Wang ◽  
Hai-Meng Zhou
Keyword(s):  
Creatine Kinase ◽  
Dimethyl Sulfoxide ◽  
Conformational Changes ◽  
Sodium Dodecyl ◽  
Rabbit Muscle ◽  
Hydrophobic Surfaces ◽  
Muscle Creatine Kinase ◽  
Organic Mixture ◽  
Dmso Concentration ◽  
Partial Unfolding

The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (<65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water–organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation.Key words: creatine kinase, dimethyl sulfoxide, denaturation, activity, conformation.

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