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

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.

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

Clinical Chemistry ◽

10.1093/clinchem/26.8.1165 ◽

1980 ◽

Vol 26 (8) ◽

pp. 1165-1169 ◽

Cited By ~ 8

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.

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Creatine kinase in serum: 3. Further study of adenylate kinase inhibitors.

Clinical Chemistry ◽

10.1093/clinchem/23.10.1888 ◽

1977 ◽

Vol 23 (10) ◽

pp. 1888-1892 ◽

Cited By ~ 31

Author(s):

G Szasz ◽

W Gerhardt ◽

W Gruber

Keyword(s):

Creatine Kinase ◽

Adenylate Kinase ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Specific Inhibitor ◽

Kinase Assay ◽

Lag Phase ◽

Diadenosine Pentaphosphate ◽

Phosphoryl Groups ◽

Inhibitor Combination

Abstract In search of an appropriate inhibitor to suppress the interference of adenylate kinase with the creatine kinase assay, we found that the combination diadenosine pentaphosphate (10 mumol/liter) and AMP (5 mmol/liter) is a better inhibitor than is fluoride (25 mmol/liter). The latter inhibits adenylate kinase uncompetitively and weakly (Ki = 2.5 mmol/liter), and must be incorporated in the starting reagent, and at 30 degrees C it becoms fully effective only after a lag phase of 6 min. In this concentration, fluoride inhibits adenylate kinase from erythrocytes, muscle, liver or platelets by 94, 92, 88, and 87%, respectively, and creatine kinase by 8%. Bromide and chloride also inhibit creatine kinase. Attempts to replace AMP by a specific inhibitor of liver adenylate kinase failed. Homologs of diadenosine pentaphosphate with either fewer or more phosphoryl groups in the polyphosphate bridge inhibited even more weakly than did the pentaphosphate. Platelets can significantly contribute to adenylate kinase activity in serum. The inhibitor combination inhibited adenylate kinase from platelets by 90%.

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Creatine Kinase: Stability, Inactivation, Reactivation

Clinical Chemistry ◽

10.1093/clinchem/23.4.646 ◽

1977 ◽

Vol 23 (4) ◽

pp. 646-652 ◽

Cited By ~ 37

Author(s):

Leo G Morin

Keyword(s):

Creatine Kinase ◽

Prolonged Exposure ◽

Lag Phase ◽

Reversible Inactivation ◽

Decay Constants ◽

Creatine Kinase Isoenzymes ◽

Oxidation Reduction ◽

Phase Variations

Abstract I determined the in vitro biological half-lives or decay constants for creatine kinase isoenzymes at various temperatures. Values at 37 °C are consistent with values reported by others in vivo, which suggests that in vivo ir¬reversible inactivation is primarily thermal. Reversible inactivation appears to be an oxidation-reduction phe¬nomenon. Proteins and some inactivators (urate, cate¬cholamines) retard irreversible inactivation and preserve isoenzyme integrity. Dilution and thiols promote reversal of inactivity. Mercaptoethanol is the preferred thiol, par¬ticularly for storage and reactivation of isoenzyme MB. MB is sensitive to light and to freeze-thawing. I recommend that specimens be cooled promptly after drawing, that mercaptoethanol (10 mmol/liter) be added, and that they be stored refrigerated. Avoid prolonged exposure to light and freezing. A model of inactivation is proposed, which is based on the assumed existence of four monomer types: active, denatured, oxidized, and insulated. The model is consistent with dilution and thiol reactivation, lag phase variations, and subtype heterogeneity.

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Human creatine kinase: Isoenzymes and logistics of energy distribution

Scandinavian Journal of Clinical and Laboratory Investigation ◽

10.3109/00365518409083619 ◽

1984 ◽

Vol 44 (7) ◽

pp. 611-615 ◽

Cited By ~ 13

Author(s):

Christer Sylvén ◽

Eva Jansson ◽

Anders Kallner ◽

Kim Böök

Keyword(s):

Creatine Kinase ◽

Energy Distribution ◽

Creatine Kinase Isoenzymes

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Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)39237-3 ◽

1990 ◽

Vol 265 (12) ◽

pp. 6921-6927 ◽

Cited By ~ 2

Author(s):

R C Haas ◽

A W Strauss

Keyword(s):

Creatine Kinase ◽

Nuclear Genes ◽

Mitochondrial Creatine Kinase ◽

Creatine Kinase Isoenzymes

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Creatine kinase isoenzymes in cerebrospinal fluid in a case of brain damage.

Clinical Chemistry ◽

10.1093/clinchem/22.8.1405 ◽

1976 ◽

Vol 22 (8) ◽

pp. 1405-1407 ◽

Cited By ~ 15

Author(s):

P M Bayer ◽

F Gabl ◽

G Granditsch ◽

K Widhalm ◽

H Zyman ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Creatine Kinase ◽

High Activity ◽

Brain Damage ◽

Acute Phase ◽

Clinical Importance ◽

Total Activity ◽

Spinal Fluid ◽

Consumption Coagulopathy ◽

Creatine Kinase Isoenzymes

Abstract We present a case of a 11/2-year-old boy with toxic enteritis, consecutive consumption coagulopathy, and sever brain damage. During the acute phase we found high activity of the BB isoenzyme of creatine kinase in cerebrospinal fluid, but not in the serum. Isoenzyme MM could also be found in the spinal fluid (37.9% of the total activity). We conclude that analysis for creatine kinase isoenzymes in spinal fluid is of clinical importance.

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The effects of osmotic pressure changes on the germination of Dictyostelium discoideum spores

Canadian Journal of Microbiology ◽

10.1139/m77-176 ◽

1977 ◽

Vol 23 (9) ◽

pp. 1170-1177 ◽

Cited By ~ 15

Author(s):

David A. Cotter

Keyword(s):

Ethylene Glycol ◽

Dictyostelium Discoideum ◽

Room Temperature ◽

Thermal Inactivation ◽

Sucrose Concentration ◽

Lag Phase ◽

Activation Process ◽

Sucrose Solutions ◽

Spore Population ◽

Pressure Changes

Polyalcohols such as ethylene glycol and glycerol at 3 M penetrate and activate spores of Dictyostelium discoideum incubated at room temperature. Higher concentrations of ethylene glycol result in lysis upon suspension of spores in dilute phosphate buffer. Erythritol and arabitol at 3 M do not penetrate or activate D. discoideum spores.Air-dried spores or those incubated in 2 M sucrose solutions are not activated with the usual heat treatment of 45 °C for 30 min. The plasmolyzed spores are activated at temperatures above 45 °C when heated in the presence of 2 M sucrose for 30 min. The temperature for maximum activation and the temperature for thermal inactivation of spores are raised 7–10 °C in high sucrose concentrations. Long-term incubation of heat-activated spores in 2 M sucrose solutions does not result in a return to dormancy.Moderate sucrose concentrations near 0.2 M do not block the heat-induced activation process but must be removed from the spore population to prevent a return to dormancy within 6 h. Other polyhydric compounds at 0.25 M concentration also cause spore deactivation within 6 h of room temperature incubation. Oxygen uptake of spores undergoing deactivation in 0.18 M sucrose is inhibited as compared to control levels. Moderate concentrations of sucrose do not block the early events of postactivation lag and the spores accumulate at the end of the lag phase. The longer the spores remain unswollen at the end of the postactivation lag phase, the greater the percentage of spores which return to dormancy. The effects of moderate sucrose concentration (lowered water activity) are not duplicated by the same quantity of Ficoll, indicating that the colligative properties of the sucrose solutions are responsible for deactivation.

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