Single Stable Reagent for Creatine Kinase Assay

1971 ◽  
Vol 17 (6) ◽  
pp. 548-550 ◽  
Author(s):  
Charles Bishop ◽  
T M Chu ◽  
Z K Shihabi
Keyword(s):  
Creatine Kinase ◽  
Maximum Velocity ◽  
Creatine Phosphate ◽  
Spectrophotometric Assay ◽  
Kinase Assay ◽  
Reagent Cost ◽  
One Step ◽  
Stable Reagent

Abstract A single stable reagent for spectrophotometric assay of creatine kinase in one step is described. Reagent cost has been decreased by running the reaction at Km conditions for creatine phosphate and relating the activity to maximum velocity.

scholarly journals Molecular docking and density functional theory studies on creatine, guanidinoacetic acid, and their phosphorylated analogues binding to muscle creatine kinase

2020 ◽  
pp. 174751982097858
Author(s):  
M Vraneš ◽  
S Ostojić ◽  
Č Podlipnik ◽  
A Tot
Keyword(s):  
Density Functional Theory ◽  
Creatine Kinase ◽  
Molecular Docking ◽  
Density Functional ◽  
Creatine Phosphate ◽  
Docking Studies ◽  
Muscle Creatine Kinase ◽  
Functional Theory ◽  
Guanidinoacetic Acid ◽  
Muscle Creatine

Comparative molecular docking studies on creatine and guanidinoacetic acid, as well as their phosphorylated analogues, creatine phosphate, and phosphorylated guanidinoacetic acid, are investigated. Docking and density functional theory studies are carried out for muscle creatine kinase. The changes in the geometries of the ligands before and after binding to the enzyme are investigated to explain the better binding of guanidinoacetic acid and phosphorylated guanidinoacetic acid compared to creatine and creatine phosphate.

Evaluation of adenosine 5'-monophosphate and fluoride as adenylate kinase inhibitors in the creatine kinase assay.

1976 ◽  
Vol 22 (7) ◽  
pp. 1078-1083 ◽  
Author(s):  
T G Rosano ◽  
K J Clayson ◽  
P E Strandjord
Keyword(s):  
Creatine Kinase ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Creatine Kinase Activity ◽  
Serum Creatine Kinase ◽  
Assay System ◽  
Kinase Assay ◽  
Rabbit Muscle ◽  
Atp Production

Abstract Adenylate kinase (EC 2.7.4.3) interferes positively in the serum creatine kinase (EC 2.7.3.2) assay when the rate of ATP production is monitored by a coupled enzyme system. A dual assay, measuring creatine kinase and adenylate kinase activity, was used to evaluate AMP and other possible adenylate kinase inhibitors that would permit specific measurement of creatine kinase activity in the presence of adenylate kinase. We found that AMP, routinely included in the creatine kinase assay system to inhibit adenylate kinase, partially inhibits both human serum creatine kinase and purified creatine kinase from rabbit muscle. The amount of creatine kinase inhibition is related directly to the AMP concentration and inversely to the substrate (ADP) concentration. We found that 25 mmol/liter of fluoride inhibits adenylate kinase without measurable effect on creatine kinase activity. We developed a serum creatine kinase assay including fluoride, and compared it with the dual assay system and with two commercial assay kits. Other halides or adenosine 2'-monophosphate did not selectively inhibit adenylate kinase.

Activation of human creatine kinase isoenzymes by pH and various sulfhydryl and chelating agents.

1981 ◽  
Vol 27 (3) ◽  
pp. 402-404 ◽  
Author(s):  
D A Nealon ◽  
S M Pettit ◽  
A R Henderson
Keyword(s):  
Creatine Kinase ◽  
Ethylene Glycol ◽  
Chelating Agents ◽  
Enzyme Stability ◽  
Kinase Assay ◽  
Lag Phase ◽  
Creatine Kinase Isoenzymes

Abstract We report the effect of serum pH and of the presence or absence of mercaptoethanol, N-acetyl-L-cysteine, monothioglycerol, ethylenediaminetetraacetate, and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate on the activation of the human creatine kinase isoenzymes. At the serum pH giving maximal enzyme stability and minimal assay lag phase (Nealon et al., Clin. Chem. 26: 1165-1169, 1980) thiol activation of CK-1 and CK-3 is nearly maximal with monothioglycerol in an optimized creatine kinase assay (Szasz et al., Clin. Chem. 22: 650-656, 1976). However, CK-2 is maximally activated at pH 8.5, a pH at which this isoenzyme is least stable on storage and its assay lag phase is prolonged. These findings suggest irreconcilable problems in the storage, activation, and assay of CK-2.

scholarly journals Creatine kinase expression and creatine phosphate accumulation are developmentally regulated during differentiation of mouse and human monocytes.

1984 ◽  
Vol 159 (3) ◽  
pp. 746-757 ◽  
Author(s):  
J D Loike ◽  
V F Kozler ◽  
S C Silverstein
Keyword(s):  
Creatine Kinase ◽  
Human Lymphocytes ◽  
Creatine Phosphate ◽  
Human Platelets ◽  
In Vitro Cultivation ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Developmentally Regulated ◽  
The Brain

We have studied the expression of creatine kinase (CK) and the accumulation of creatine phosphate during the differentiation of human and mouse peripheral blood monocytes. Mouse monocytes cultured for 24 h do not contain detectable levels of CK and creatine phosphate. However, resident tissue macrophages and inflammatory elicited macrophages obtained from the peritoneal cavities of mice have 70 and 300 mU per mg protein of CK activity and contain 3 and 6 mol of creatine phosphate per mol of ATP, respectively. The major isozyme of CK in these cells has been identified as the brain form. These findings suggest that the differentiation of monocytes into macrophages is associated with the expression of CK and the accumulation of creatine phosphate. We have found a similar pattern in human monocytes. Human blood monocytes, maintained in culture for 24 or 48 h, do not contain detectable levels of CK or creatine phosphate. Monocyte-derived macrophages (monocytes maintained in tissue cultures for 1 to 2 wk) have up to 100 mU per mg protein of CK activity and contain 0.5 mol of creatine phosphate per mol of ATP. Human macrophages express multiple isozymes of CK including the brain (BB) and possibly the mitochondrial forms of this enzyme. Thus, the expression of CK and the accumulation of creatine phosphate in human monocytes is induced by their in vitro cultivation. The induction of CK during in vitro cultivation occurs independently of the concentration of creatine in the medium. However, the size of the creatine phosphate pool varies with respect to extracellular creatine concentration. Creatine phosphate and CK are not detectable in freshly isolated human lymphocytes, polymorphonuclear leukocytes or erythrocytes, but are found in freshly isolated human platelets.

Creatine Kinase: ATP Contaminating Creatine Phosphate Substrate of a Commercial Kit

1973 ◽  
Vol 19 (2) ◽  
pp. 280-281
Author(s):  
Joseph Tesfaye ◽  
Jacques Longpré ◽  
Frank Bodley
Keyword(s):  
Creatine Kinase ◽  
Creatine Phosphate ◽  
Commercial Kit

Characteristics of phosphate-induced Ca2+ efflux from the SR in mechanically skinned rat skeletal muscle fibers

2000 ◽  
Vol 278 (1) ◽  
pp. C126-C135 ◽  
Author(s):  
Adrian M. Duke ◽  
Derek S. Steele
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Muscle Fibers ◽  
Cyclopiazonic Acid ◽  
Creatine Phosphate ◽  
Ruthenium Red ◽  
Rat Skeletal Muscle ◽  
Atpase Inhibitor ◽  
Skeletal Muscle Fibers ◽  
Efflux Pathway

The effects of Pi on sarcoplasmic reticulum (SR) Ca2+ regulation were studied in mechanically skinned rat skeletal muscle fibers. Brief application of caffeine was used to assess the SR Ca2+ content, and changes in concentration of Ca2+([Ca2+]) within the cytosol were detected with fura 2 fluorescence. Introduction of Pi (1–40 mM) induced a concentration-dependent Ca2+ efflux from the SR. In solutions lacking creatine phosphate (CP), the amplitude of the Pi-induced Ca2+ transient approximately doubled. A similar potentiation of Pi-induced Ca2+ release occurred after inhibition of creatine kinase (CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced Ca2+ release was almost abolished, whereas Pi-induced Ca2+ release was unaffected. However, introduction of the SR Ca2+ ATPase inhibitor cyclopiazonic acid effectively abolished Pi-induced Ca2+ release. These data suggest that Pi induces Ca2+ release from the SR by reversal of the SR Ca2+ pump but not via the SR Ca2+ channel under these conditions. If this occurs in intact skeletal muscle during fatigue, activation of a Ca2+efflux pathway by Pi may contribute to the reported decrease in net Ca2+ uptake and increase in resting [Ca2+].

Effect of serum pH on storage stability and reaction lag phase of human creatine kinase isoenzymes.

1980 ◽  
Vol 26 (8) ◽  
pp. 1165-1169 ◽  
Author(s):  
D A Nealon ◽  
S M Pettit ◽  
A R Henderson
Keyword(s):  
Creatine Kinase ◽  
Ethylene Glycol ◽  
Storage Stability ◽  
Kinase Assay ◽  
Lag Phase ◽  
Creatine Kinase Isoenzyme ◽  
Sh Groups ◽  
C Storage ◽  
Creatine Kinase Isoenzymes

Abstract We report the effect of serum pH on the storage stability of the human creatine kinase isoenzymes and on the creatine kinase assay lag phase (Szasz et al., Clin. Chem. 22: 650, 1976). We also investigated the effect of including mercaptoethanol, N-acetyl-L-cysteine, monothioglycerol, ethylenediaminetetraacetate, or ethylene glycol bis(betaaminoethyl ether)-N,N,N',N'-tetraacetate at 20, 4, and --20 degrees C. Storage stability of the isoenzymes is profoundly affected by pH. For patients' samples and semi-purified human creatine kinase isoenzymes added to heat-inactivated sera, increasing diluent pH above 7.0 decreases creatine kinase stability. The thiol agents or chelators generally give little or no protection above pH 7.5; at pH 8.5 they contribute significantly to isoenzyme instability. Storage at 4 degrees C provides greater stability than storage at 20 degrees C, particularly in the case of creatine kinase isoenzyme BB. The lag phase was minimum at a serum pH of 6.5, in the presence of 10 mmol of monothioglycerol per liter. Increasing serum pH to 8.5 prolongs the reaction lag phase by about 1 min over the minimum. We recommend that, before they are stored at 4 degrees C, the pH of patients' samples be adjusted to 6.5 and oxidation of SH-groups be minimized by adding monothioglycerol to the sample.

Automated Fluorometric Creatine Kinase Assay. Measurement of 100-Fold Normal Activity without Serum Dilution

1974 ◽  
Vol 20 (5) ◽  
pp. 560-565 ◽  
Author(s):  
J B Armstrong ◽  
J A Lowden ◽  
A L Sherwin
Keyword(s):  
Creatine Kinase ◽  
Normal Activity ◽  
Normal Serum ◽  
Kinase Assay ◽  
Alkaline Ph ◽  
Protein Fluorescence ◽  
Severe Renal Failure ◽  
Additional Advantage ◽  
Serum Dilution ◽  
Sample Dilution

Abstract Creatine kinase (EC 2.7.3.2) activity can be measured fluorometrically by coupling the reaction product, creatine, to ninhydrin at alkaline pH. We have automated this procedure to develop an accurate and precise method, which requires 0.1 ml of sample and can be performed at 60 tests per hour. By using an abbreviated incubation time and a dialyzer in the assay, we have extended the analytical range to 100 times normal serum activity without sample dilution. The dialyzer also eliminates the nonspecific protein fluorescence of the sample. An additional advantage of the method is the reduced cost of reagents in comparison to similar procedures. Serum blanks should be analyzed whenever serum creatine is abnormally high, especially in cases of severe renal failure.

Myofibrillar end of the creatine phosphate energy shuttle

1984 ◽  
Vol 247 (5) ◽  
pp. C424-C432 ◽  
Author(s):  
F. Savabi ◽  
P. J. Geiger ◽  
S. P. Bessman
Keyword(s):  
Creatine Kinase ◽  
Adenylate Kinase ◽  
Psoas Muscle ◽  
Creatine Phosphate ◽  
Force Of Contraction ◽  
Physiological Concentration ◽  
Complete Relaxation ◽  
Order Of Magnitude ◽  
Contraction And Relaxation

Isometric contraction and relaxation of glycerinated rabbit psoas muscle fibers containing native creatine kinase (CK) and ATPase activities were studied. Energy for contraction and relaxation was provided either by ADP + creatine phosphate (CP) or ATP alone, and the effectiveness of these additions on rate and maximum force of contraction and relaxation were compared. In the presence of 250 microM ADP, physiological concentration of CP (10 mM) produced faster and stronger contraction and faster and more complete relaxation than equimolar or even higher concentrations of ATP. When contraction was initiated by addition of ADP to fibers preincubated with 10 mM CP, the apparent Km for ADP was 1.18 +/- 0.24 mM. If the fibers were preincubated with ADP and contraction initiated by addition of 10 mM CP, the apparent Km for ADP was more than an order of magnitude smaller (76.0 +/- 4 microM). The observed Km for ADP for contraction was about half the Km for CP in solution (151.5 microM). The apparent Km for CP for rate of contraction was 2.67 +/- .046 mM independent of sequence of addition of ADP. Since these experiments were done in the presence of P1,P5-diadenosine 5'-pentaphosphate, a powerful inhibitor of adenylate kinase, the role of this enzyme in the process was not significant. These observations support the idea of compartmentation of myofibrillar CK in close function with myosin ATPase as part of the phosphoryl creatine energy shuttle.

Metabolism of the perfused rabbit heart. V. Verapamil-induced release of creatine kinase

1982 ◽  
Vol 60 (7) ◽  
pp. 952-959 ◽  
Author(s):  
Miguel A. Chiong
Keyword(s):  
Creatine Kinase ◽  
Adenine Nucleotides ◽  
Systolic Pressure ◽  
Creatine Phosphate ◽  
Rabbit Heart ◽  
Left Ventricular ◽  
Calcium Stores ◽  
Ventricular Performance ◽  
Permeability Characteristics ◽  
Energy Stores

The effects of verapamil (VER), at concentrations of 0, 10−9, 10−8, 10−7, and 5 × 10−7 M (or 0, 0.5, 5, 50, and 250 ng/mL) were studied in the isolated rabbit heart during 70 min of aerobic perfusion with a standard Krebs–bicarbonate medium at 37 °C. The studied variables were left ventricular performance (RPP, heart rate times left ventricular (LV) systolic pressure), coronary sinus flow (CSF), oxygen uptake [Formula: see text], rate of creatine kinase (CK) release, and energy stores (glycogen, creatine phosphate (CP), ATP, and total adenine nucleotides (TAN)).The results show that (i) VER depressed RPP in a dose-related manner; (ii) [Formula: see text] declined as VER concentration increased except in the 5 × 10−7 M group which showed a paradoxical increase in O2 uptake; (iii) CSF was only slightly decreased by VER with the exception of the 5 × 10−7 M group, which showed an increase in flow; (iv) VER was associated with increments in the rates of CK release in a dose-related fashion (2, 4, 15, and 29 times the rate observed in the untreated group), and (v) VER was associated with slight decrease in glycogen levels, but no changes in CP or adenine nucleotides.It is concluded that, in our preparation, VER caused marked increases in the rate of CK loss in the absence of depletion of total energy stores. The data suggest that the drug affects the permeability characteristics of the sarcolemma, perhaps via localized depletion of calcium stores.

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