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An improved nonfluorescent stain for enolase activity on cellulose acetate strips
ANALYTICAL
BIOCHEMISTRY
An Improved
82, 240-242
Nonfluorescent on Cellulose
(1977)
Stain for Enolase Acetate Strips
Activity
A method is described for staining enolase activity on cellulose acetate strips using nitroblue tetrazolium. Simultaneous staining of adenylate kinase is prevented by using only catalytic amounts of ADP and by including AMP in the staining solution.
Methods of staining for enolase activity following electrophoresis usually measure the loss of NADH fluorescence obtained when phosphoenolpyruvate, the product of the enolase reaction, is converted to pyruvate and then reduced to lactate, fluorescence changes being observed with ultraviolet light (1). This procedure is inconvenient as the fluorescence changes are relatively short-lived, and a positive staining procedure for enolase would therefore be useful. A common method of staining for dehydrogenases relies on the formation of the intensely colored diformazan produced when reduced nicotinamide compounds react with nitroblue tetrazolium (2), consequently, the site of enolase activity can be made visible by coupling to a dehydrogenase enzyme. This can be achieved by coupling phosphoenolpyruvate production to glucosed-phosphate production via pyruvate kinase and hexokinase and then using glucosed-phosphate dehydrogenase to produce NADPH. A staining procedure for enolase activity in immunoprecipitates and starch gels based on this method has recently been described (3). However, this staining procedure also stains areas of adenylate kinase activity (3). We have developed a positive staining procedure based on the above method which is specific for enolase activity. MATERIALS
AND METHODS
The staining solution contained: 50 mM Tris-HCl, pH 7.5, 2 mM MgClz, 1 mM glucose, 0.1 mM ADP, 20 mM AMP, 0.5 mM NADP, pyruvate kinase (4 unit/ml), hexokinase (3 unit/ml), glucose-6-phosphate dehydrogenase (1.4 unit/ml), nitroblue tetrazolium (0.4 mg/ml), and 1.4 mM 2phosphoglycerate. Methyl phenazonium methosulfate solution (2.4 mg/ml) was added to a final concentration of 1% (v/v) immediately before use. Rabbits were killed by decapitation, and organs were removed and homogenized as a 2:l (v/w) homogenate with a Willems Polytron in 50 mM Tris-HCl, pH 7.5, containing 2 mM MgC&; the 100,OOOg supernatants were used for electrophoresis. Approximately 5 ,ul of supernatant 240 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN ooO3-2697
241
SHORT COMMUNICATIONS
containing from 0.02 to 0.04 unit of enolase activity were applied near the cathode to a strip of cellulose acetate 7 cm long and 4 cm wide. Electrophoresis was conducted at 8 mA for 50 min in barbital acetate buffer, ionic strength 0.05, pH 8.4. After electrophoresis the strips were immersed in the above staining solution; color appears within 15 min at 40°C. RESULTS AND DISCUSSION
The above staining procedure shows bands of enolase activity in each rabbit organ examined (heart, kidney, brain, muscle, and liver). No color is observed if 2-phosphoglycerate is omitted, showing that the staining procedure is specific for enolase activity. The appearance of adenylate kinase activity is prevented by the inclusion of excess AMP and by use of catalytic levels of ADP, the latter being regenerated by glucose and hexokinase. Omission of AMP and use of higher levels of ADP leads to the appearance of extra bands of activity which are not dependent on the presence of 2-phosphoglycerate and which are presumably due to adenylate kinase; these bands are seen in all organs tested except brain. The above improved staining procedure shows one band of enolase activity in all organs except brain, the latter shows three bands of activity. Under the present conditions of electrophoresis there was no difference in the mobility of enolase activity from heart, muscle, liver, and kidney; in each case activity remained relatively close to the cathode. Brain tissue showed one band in common with the cathodic band from other tissues, one rapidly moving anodic band, and one intermediate band. The rapidly moving band presumably represents the brain-specific form of enolase described by Rider and Taylor (4) and Pearce et al. (5), which has recently been equated with the brain-specific protein 14-3-2 (3,6), while the intermediate band represents a heterodimer form of the enzyme (4). ACKNOWLEDGMENTS This work was supported by the Multiple Sclerosis Society of Great Britain and Northern Ireland and by the Welsh Scheme for the Development of Health and Social Research. D. M. Mackay is thanked for expert technical assistance. Professor C. N. Hales is thanked for his interest and encouragement.
REFERENCES 1. 2. 3. 4.
Rider, C. C., and Taylor, C. B. (1974) B&him. Biophys. Acta Wilkinson, .I. H. (1970) Isoenzymes, pp. 47-50. Chapman and Bock, E., and Dissing, J. (1975) Stand. J. Immunol. 4: Suppl. Rider, C. C., and Taylor, C. B. (1975) Biochem. Biophys. 814-820. 5. Pearce, J. M., Edwards, Y. H.. and Harris, H. (1976) Ann. 39,263-275.
365,285-300.
Hall, London. 2, 31-36. Res. Hum.
Commun. Genet.
66,
London
242 6. Marangos, Commun.
SHORT
COMMUNICATIONS
P. J., Zomely-Neurath, 68: 1309-1316.
C., and York,
C. (1976)
Biochem.
Bk@ys.
i&s.
D. A. HULLIN R. J. THOMPSON Department of Medical Biochemistry Welsh National School of Medicine Heath Park, Cardiff CF4 4XN England Received November 23, 1976; accepted
May
16, 1977
BIOCHEMISTRY
An Improved
82, 240-242
Nonfluorescent on Cellulose
(1977)
Stain for Enolase Acetate Strips
Activity
A method is described for staining enolase activity on cellulose acetate strips using nitroblue tetrazolium. Simultaneous staining of adenylate kinase is prevented by using only catalytic amounts of ADP and by including AMP in the staining solution.
Methods of staining for enolase activity following electrophoresis usually measure the loss of NADH fluorescence obtained when phosphoenolpyruvate, the product of the enolase reaction, is converted to pyruvate and then reduced to lactate, fluorescence changes being observed with ultraviolet light (1). This procedure is inconvenient as the fluorescence changes are relatively short-lived, and a positive staining procedure for enolase would therefore be useful. A common method of staining for dehydrogenases relies on the formation of the intensely colored diformazan produced when reduced nicotinamide compounds react with nitroblue tetrazolium (2), consequently, the site of enolase activity can be made visible by coupling to a dehydrogenase enzyme. This can be achieved by coupling phosphoenolpyruvate production to glucosed-phosphate production via pyruvate kinase and hexokinase and then using glucosed-phosphate dehydrogenase to produce NADPH. A staining procedure for enolase activity in immunoprecipitates and starch gels based on this method has recently been described (3). However, this staining procedure also stains areas of adenylate kinase activity (3). We have developed a positive staining procedure based on the above method which is specific for enolase activity. MATERIALS
AND METHODS
The staining solution contained: 50 mM Tris-HCl, pH 7.5, 2 mM MgClz, 1 mM glucose, 0.1 mM ADP, 20 mM AMP, 0.5 mM NADP, pyruvate kinase (4 unit/ml), hexokinase (3 unit/ml), glucose-6-phosphate dehydrogenase (1.4 unit/ml), nitroblue tetrazolium (0.4 mg/ml), and 1.4 mM 2phosphoglycerate. Methyl phenazonium methosulfate solution (2.4 mg/ml) was added to a final concentration of 1% (v/v) immediately before use. Rabbits were killed by decapitation, and organs were removed and homogenized as a 2:l (v/w) homogenate with a Willems Polytron in 50 mM Tris-HCl, pH 7.5, containing 2 mM MgC&; the 100,OOOg supernatants were used for electrophoresis. Approximately 5 ,ul of supernatant 240 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN ooO3-2697
241
SHORT COMMUNICATIONS
containing from 0.02 to 0.04 unit of enolase activity were applied near the cathode to a strip of cellulose acetate 7 cm long and 4 cm wide. Electrophoresis was conducted at 8 mA for 50 min in barbital acetate buffer, ionic strength 0.05, pH 8.4. After electrophoresis the strips were immersed in the above staining solution; color appears within 15 min at 40°C. RESULTS AND DISCUSSION
The above staining procedure shows bands of enolase activity in each rabbit organ examined (heart, kidney, brain, muscle, and liver). No color is observed if 2-phosphoglycerate is omitted, showing that the staining procedure is specific for enolase activity. The appearance of adenylate kinase activity is prevented by the inclusion of excess AMP and by use of catalytic levels of ADP, the latter being regenerated by glucose and hexokinase. Omission of AMP and use of higher levels of ADP leads to the appearance of extra bands of activity which are not dependent on the presence of 2-phosphoglycerate and which are presumably due to adenylate kinase; these bands are seen in all organs tested except brain. The above improved staining procedure shows one band of enolase activity in all organs except brain, the latter shows three bands of activity. Under the present conditions of electrophoresis there was no difference in the mobility of enolase activity from heart, muscle, liver, and kidney; in each case activity remained relatively close to the cathode. Brain tissue showed one band in common with the cathodic band from other tissues, one rapidly moving anodic band, and one intermediate band. The rapidly moving band presumably represents the brain-specific form of enolase described by Rider and Taylor (4) and Pearce et al. (5), which has recently been equated with the brain-specific protein 14-3-2 (3,6), while the intermediate band represents a heterodimer form of the enzyme (4). ACKNOWLEDGMENTS This work was supported by the Multiple Sclerosis Society of Great Britain and Northern Ireland and by the Welsh Scheme for the Development of Health and Social Research. D. M. Mackay is thanked for expert technical assistance. Professor C. N. Hales is thanked for his interest and encouragement.
REFERENCES 1. 2. 3. 4.
Rider, C. C., and Taylor, C. B. (1974) B&him. Biophys. Acta Wilkinson, .I. H. (1970) Isoenzymes, pp. 47-50. Chapman and Bock, E., and Dissing, J. (1975) Stand. J. Immunol. 4: Suppl. Rider, C. C., and Taylor, C. B. (1975) Biochem. Biophys. 814-820. 5. Pearce, J. M., Edwards, Y. H.. and Harris, H. (1976) Ann. 39,263-275.
365,285-300.
Hall, London. 2, 31-36. Res. Hum.
Commun. Genet.
66,
London
242 6. Marangos, Commun.
SHORT
COMMUNICATIONS
P. J., Zomely-Neurath, 68: 1309-1316.
C., and York,
C. (1976)
Biochem.
Bk@ys.
i&s.
D. A. HULLIN R. J. THOMPSON Department of Medical Biochemistry Welsh National School of Medicine Heath Park, Cardiff CF4 4XN England Received November 23, 1976; accepted
May
16, 1977