Heterogeneity of rabbit muscle creatine kinase and limited proteolysis by proteinase K

By using sodium dodecyl sulphage/polyacrylamide-gel electrophoresis it was shown that rabbit muscle creatine kinase, both in a homogenate and purified, appears to be composed of a mixture of two peptides (mol.wts. 42100 and 40300) differing in length by about 15 amino acids. It is found that low concentrations of proteinase K from the fungus Tritirachium album can cleave about 38 amino acids from each chain of creatine kinase, leaving two large fragments (mol.wts 37700 and 35500). Scission of the whole enzyme was found to be concomitant with complete loss of enzyme activity. MgADP in the presence of absence of creatine slowed the rate of proteolysis by about 50%, but the transition-state analogue complex creatine-NO3—MgADP appeared to protect completely. The time course for the proteolytic inactivation in the presence of this complex, but not in its absence, was biphasic.

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The effect of limited proteolysis on rabbit muscle creatine kinase

Biochemical Journal ◽

10.1042/bj1990239 ◽

1981 ◽

Vol 199 (1) ◽

pp. 239-244 ◽

Cited By ~ 12

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.

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Folding pathway for partially folded rabbit muscle creatine kinase

Biochemistry and Cell Biology ◽

10.1139/o01-129 ◽

2001 ◽

Vol 79 (4) ◽

pp. 479-487 ◽

Cited By ~ 14

Author(s):

Yong-Doo Park ◽

Wen-Bin Ou ◽

Tian-Wei Yu ◽

Hai-Meng Zhou

Keyword(s):

Creatine Kinase ◽

Guanidine Hydrochloride ◽

Polyacrylamide Gel Electrophoresis ◽

Hydrophobic Surface ◽

Size Exclusion ◽

Folding Pathway ◽

Rabbit Muscle ◽

Muscle Creatine Kinase ◽

Muscle Creatine ◽

Partially Folded State

Rabbit muscle creatine kinase (CK) was modified by 5,5'-dithio-bis(2-nitrobenzoic acid) accompanied by 3 M guanidine hydrochloride denaturation to produce a partially folded state with modified thiol groups. The partially folded CK was in a monomeric state detected by size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and intrinsic fluorescence studies. After dithiothreitol (DTT) treatment, about 70% CK activity was regained with a two-phase kinetic course. Rate constants calculated for regaining of activity and refolding were compared with those for CK modified with various treatments to show that refolding and recovery of activity were synchronized. To further characterize the partially folded CK state and its folding pathway, the molecular chaperone GroEL was used to evaluate whether it can bind with partly folded CK during refolding, and 1-anilinonaphthalene-8-sulfonate was used to detect the hydrophobic surface of the monomeric state of CK. The monomeric state of CK did not bind with GroEL, although it had a larger area of hydrophobic surface relative to the native state. These results may provide different evidence for the structural requirement of GroEL recognition to the substrate protein compared with previously reported results that GroEL bound with substrate proteins mainly through hydrophobic surface. The present study provides data for a monomeric intermediate trapped by the modification of the SH groups during the refolding of CK. Schemes are given for explaining both the partial folding CK pathway and the refolding pathway.Key words: creatine kinase; partially folded state; reactivation; refolding; GroEL; intermediate.

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Monoclonal antibody studies of creatine kinase. The ART epitope: evidence for an intermediate in protein folding

Biochemical Journal ◽

10.1042/bj2570461 ◽

1989 ◽

Vol 257 (2) ◽

pp. 461-469 ◽

Cited By ~ 25

Author(s):

G E Morris

Keyword(s):

Amino Acids ◽

Monoclonal Antibody ◽

Creatine Kinase ◽

Specific Binding ◽

Trypsin Digestion ◽

Proteinase K ◽

Enzyme Linked Immunosorbent Assay ◽

Muscle Creatine Kinase ◽

Western Blots ◽

Methionine Residues

Chemical cleavage at cysteine residues with nitrothiocyanobenzoic acid shows that the last 98 amino acids of the 380-amino-acid sequence of chick muscle creatine kinase are sufficient for binding of the monoclonal antibody CK-ART. Removal of the last 30 amino acids by cleavage at methionine residues with CNBr results in loss of CK-ART binding. CK-ART binding is also lost when these C-terminal methionine residues are oxidized to sulphoxide, but binding is regained on reduction. Proteinase K ‘nicks’ native CK at a single site near the C-terminus and two fragments of 327 amino acides and 53 amino acids can be separated by subsequent SDS or urea treatment. CK-ART still binds normally to ‘nicked’ CK, which is enzymically inactive. After treatment with either urea (in a competition enzyme-linked immunosorbent assay) or SDS (on Western blots), however, CK-ART binds to neither of the two fragments, although these treatments do not affect binding to intact CK. This suggests that parts of both CK fragments contribute to the CK-ART epitope. CK-ART is both species- and isoenzyme-specific, binding only to chick M-CK. The only C-terminal regions containing chick-specific sequences are residues 300-312 and residues 368-371, the latter group being close to the essential methionine residues. We suggest that one, or possibly both, of these regions is involved in forming the conformational epitope on the surface of the CK molecule which CK-ART recognizes. Native CK is resistant to trypsin digestion. The C-terminal half of urea-treated and partly-refolded CK is also resistant to trypsin digestion, whereas the N-terminal half is readily digested. The results suggest a C-terminal region which can refold more rapidly than the rest of the CK molecule and provide evidence for an intermediate in CK refolding.

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

Biochemistry and Cell Biology ◽

10.1139/o05-166 ◽

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.

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Exosite-dependent regulation of factor VIIIa by activated protein C

Blood ◽

10.1182/blood-2003-01-0126 ◽

2003 ◽

Vol 101 (12) ◽

pp. 4802-4807 ◽

Cited By ~ 21

Author(s):

Chandrashekhara Manithody ◽

Philip J. Fay ◽

Alireza R. Rezaie

Keyword(s):

Protein C ◽

Time Course ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Limited Proteolysis ◽

Activated Protein C ◽

Coagulation Cascade ◽

Factor Va ◽

Factor Viiia

AbstractActivated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80–loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.

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

Biochemistry and Cell Biology ◽

10.1139/o02-132 ◽

2002 ◽

Vol 80 (4) ◽

pp. 427-434 ◽

Cited By ~ 7

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 waterorganic 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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