scholarly journals Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro

2021 ◽  
Author(s):  
Dirk Steinritz ◽  
Robin Lüling ◽  
Markus Siegert ◽  
Harald Mückter ◽  
Tanja Popp ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Enzyme Activity ◽  
Sulfur Mustard ◽  
Critical Role ◽  
Chemical Warfare Agent ◽  
Cysteine Residue ◽  
Special Focus ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase

AbstractCreatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.

scholarly journals Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study

2021 ◽  
Author(s):  
Dirk Steinritz ◽  
Robin Lüling ◽  
Markus Siegert ◽  
Julia Herbert ◽  
Harald Mückter ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Dna Adducts ◽  
Sulfur Mustard ◽  
Ex Vivo ◽  
Immunomagnetic Separation ◽  
Chemical Warfare Agent ◽  
Cysteine Residue ◽  
Islamic State ◽  
Rat Skin

AbstractSulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys282 in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.

scholarly journals Alkylated albumin-derived dipeptide C(-HETE)P derivatized by propionic anhydride as a biomarker for the verification of poisoning with sulfur mustard

2021 ◽  
Author(s):  
Annika Richter ◽  
Markus Siegert ◽  
Horst Thiermann ◽  
Harald John
Keyword(s):  
Sulfur Mustard ◽  
Limit Of Detection ◽  
Thiol Group ◽  
Chemical Warfare Agent ◽  
Cysteine Residue ◽  
Selected Reaction Monitoring ◽  
Delayed Wound Healing ◽  
Propionic Anhydride ◽  
Product Ions

AbstractSulfur mustard (SM) is a banned chemical warfare agent recently used in the Syrian Arab Republic conflict causing erythema and blisters characterized by complicated and delayed wound healing. For medical and legal reasons, the proof of exposure to SM is of high toxicological and forensic relevance. SM reacts with endogenous human serum albumin (HSA adducts) alkylating the thiol group of the cysteine residue C34, thus causing the addition of the hydroxyethylthioethyl (HETE) moiety. Following proteolysis with pronase, the biomarker dipeptide C(-HETE)P is produced. To expand the possibilities for verification of exposure, we herein introduce a novel biomarker produced from that alkylated dipeptide by derivatization with propionic anhydride inducing the selective propionylation of the N-terminus yielding PA-C(-HETE)P. Quantitative derivatization is carried out at room temperature in aqueous buffer within 10 s. The biomarker was found to be stable in the autosampler at 15 °C for at least 24 h, thus documenting its suitability even for larger sets of samples. Selective and sensitive detection is done by micro liquid chromatography-electrospray ionization tandem-mass spectrometry (μLC-ESI MS/MS) operating in the selected reaction monitoring (SRM) mode detecting product ions of the single protonated PA-C(-HETE)P (m/z 379.1) at m/z 116.1, m/z 137.0, and m/z 105.0. The lower limit of detection corresponds to 32 nM SM in plasma in vitro and the limit of identification to 160 nM. The applicability to real exposure scenarios was proven by analyzing samples from the Middle East confirming poisoning with SM. Graphical abstract

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.

scholarly journals Immunoassay of muscle-specific creatine kinase with a monoclonal antibody and application to myogenesis and muscular dystrophy

1983 ◽  
Vol 213 (2) ◽  
pp. 417-425 ◽  
Author(s):  
G E Morris ◽  
L P Head
Keyword(s):  
Monoclonal Antibody ◽  
Creatine Kinase ◽  
Enzyme Activity ◽  
Total Protein ◽  
Plasma Concentrations ◽  
Thigh Muscle ◽  
Cell Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Activity Measurements

A competition e.l.i.s.a. (enzyme-linked immunosorbent assay) is described that enables direct measurement of the muscle-specific polypeptide of chick creatine kinase (M-CK) in extracts of differentiating muscle-cell cultures and in blood plasma samples, even in the presence of embryonic, or brain-type, creatine kinase. The characteristics of the assay can be considerably improved by the use of a monoclonal antibody, CK-ART, instead of rabbit antisera, and we offer an explanation for this in terms of heterogeneity of antibody affinities in polyclonal antisera. In addition to native enzyme, the assay will measure creatine kinase unfolded and inactivated by 8 M-urea treatment. During chick muscle differentiation in vitro, M-CK increased from 7.5% of the total creatine kinase at 24h to 76.0% at 143h, in good agreement with isoenzyme separation data. As a percentage of the total cell protein, M-CK increased by 156-340-fold over the same period and constituted 0.38-0.56% of the total protein in late cultures. E.l.i.s.a. measurements on 17-20-day embryonic thigh-muscle extracts, which contain almost exclusively M-CK, agree well with enzyme activity and radioimmunoassay. M-CK constituted 0.7-1.6% of the total protein in 17-19-day embryonic thigh muscle. Plasma M-CK concentrations in normal 2-8-week-old chickens were found to be in the range 0.5-0.9 micrograms/ml. Plasma concentrations of 32-56 micrograms/ml were found in 8-week-old dystrophic chickens by both e.l.i.s.a. and enzyme-activity measurements. The results suggest that inactive or unfolded forms of M-CK do not normally exist, in any significant amounts, in cell and tissue extracts or in freshly prepared samples of plasma.

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.

scholarly journals Human ‘creatine kinase conversion factor’ identified as a carboxypeptidase

1984 ◽  
Vol 221 (2) ◽  
pp. 465-470 ◽  
Author(s):  
R J Edwards ◽  
D C Watts
Keyword(s):  
Creatine Kinase ◽  
Conversion Factor ◽  
Electrophoretic Pattern ◽  
Beta Subunits ◽  
Rabbit Muscle ◽  
Activity Profile ◽  
Muscle Creatine Kinase ◽  
Electrophoretic Patterns ◽  
Carboxypeptidase N ◽  
Low Activity

The effect of partially purified ‘creatine kinase conversion factor’ on rabbit muscle creatine kinase is shown to be that of a carboxypeptidase, removing the C-terminal lysine residue from both subunits. These changes fully explain the three-banded electrophoretic patterns of the partially and the fully modified rabbit and human enzymes. The factor also produces a similar electrophoretic pattern with haemoglobin A; comparison with the effects of carboxypeptidases A and B permits the inference that the C-terminal residues of both alpha- and beta-subunits are removed. Small synthetic peptides are poor or non-substrates. A low activity with hippuryl-L-lysine may be due to contamination of the preparation with carboxypeptidase N. The possibility has been excluded that the action of conversion factor on creatine kinase involves modification of the protein thiol groups. Mr, substrate-specificity, pH-activity profile and the effects of metal ions distinguish creatine kinase conversion factor from carboxypeptidases A, B and N. On the basis of this evidence it is proposed to give the conversion factor the provisional name of carboxypeptidase K.

scholarly journals Properties and mechanism of action of creatine kinase from ox smooth muscle. Anion effects compared with pyruvate kinase

1973 ◽  
Vol 135 (2) ◽  
pp. 265-276 ◽  
Author(s):  
B. Focant ◽  
D. C. Watts
Keyword(s):  
Creatine Kinase ◽  
Smooth Muscle ◽  
Pyruvate Kinase ◽  
Conformational Changes ◽  
Intermediate Stage ◽  
Hill Coefficient ◽  
Rabbit Muscle ◽  
Difference Spectra ◽  
Muscle Creatine Kinase ◽  
Quaternary Complex

1. An improved purification procedure for the brain-type creatine kinase from ox smooth muscle is described. 2. Michaelis constants show the characteristic dependence on the concentration of the second substrate: the derived constants are compared with those for the enzyme from ox brain. 3. Inhibition by iodoacetamide gives a biphasic curve and the total extent of the reaction depends on the enzyme concentration. The rate of inhibition at pH8.6 is not affected by creatine plus MgADP or by a range of simple anions. Addition of creatine plus MgADP plus either NO3- or Cl- ions affords 71.5 and 44% protection respectively. ADP could be replaced by 2-deoxy-ADP but not by αβ-methylene ADP, XDP, IDP, GDP or CDP. Nucleotides that did not protect would not act as substrates. 4. Difference-spectra measurements support the interpretation that addition of NO3- ions to the enzyme–creatine–MgADP complex causes further conformational changes in the enzyme accompanying the formation of a stable quaternary enzyme–creatine–NO3-–MgADP complex that simulates an intermediate stage in the transphosphorylation reaction. However, the enzyme structure is partially destabilized by quaternary-complex formation. IDP apparently fails to act as a substrate because it cannot induce the necessary conformational change. This behaviour is compared with that of rabbit skeletal muscle creatine kinase. 5. With pyruvate kinase from rabbit muscle, anions activate in the absence of an activating cation and either inhibit or have no effect in its presence. 6. Both activation and inhibition were competitive with respect to the substrate, phosphoenolpyruvate, and curved double-reciprocal plots were obtained. The results may be interpreted in terms of co-operatively induced conformational changes, and this is supported by difference-spectra measurements. However, the Hill coefficient of 1 was not significantly altered. 7. Inhibition by lactate plus pyruvate is less than additive, indicating that both bind to the same site on the enzyme, whereas that by lactate plus NO3- is additive, indicating binding at separate sites. It is inferred that a quaternary enzyme–pyruvate–NO3-–MgADP complex could form, but no evidence was obtained to suggest that it possessed special properties comparable with those found with creatine kinase. The implications of these findings for the unidirectional nature of the mechanism of pyruvate kinase is discussed. 8. Lactate or α-hydroxybutyrate could not act instead of pyruvate to form a stable quaternary complex, although both activate the K+-free enzyme. Only the former inhibits the K+-activated enzyme. The activating cation both lowers the Michaelis constant for phosphoenolpyruvate and tightens up the specificity of its binding site.

scholarly journals Identification by protein microsequencing of a proteinase-V8-cleavage site in a folding intermediate of chick muscle creatine kinase

1991 ◽  
Vol 280 (3) ◽  
pp. 809-811 ◽  
Author(s):  
G E Morris ◽  
P J Jackson
Keyword(s):  
Creatine Kinase ◽  
Enzyme Activity ◽  
Glutamic Acid ◽  
Cleavage Site ◽  
Protein Refolding ◽  
Folding Intermediate ◽  
Muscle Creatine Kinase ◽  
Glutamic Acid Residue ◽  
Muscle Creatine

We have identified by protein microsequencing a glutamic acid residue (Glu-166) in a folding intermediate of chick muscle creatine kinase that is very sensitive to cleavage by staphylococcal proteinase V8. Most other glutamic acid residues, including Glu-168, are already partly protected from proteolytic attack at this stage. After the final stages of protein refolding, when enzyme activity is recovered, Glu-166 is also resistant to proteolysis.

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