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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Isoenzyme Analysis
 
Enzymes with the same catalytic activity but with different molecular forms are known as iso(en)zymes. They are generally made up of a number of subunits, and it is the varying combination of the subunits which gives rise to isozymes. An isozyme may be constituted of by the association of similar subunits or dissimilar subunits. They are an essential feature of the biochemical organization of living things. A number of major biological problems such as evolution of population, the transformation, the regulation of gene expression and metabolic regulation in differentiated tissues are understood in the light of isozymes. They may arise due to microheterogeneity in proteins, changes in conformation, genetic mechanisms etc. Isozyme studies in agriculture extend from heterosis to tree breeding.
 
Since the first report in 1959, isomeric forms of a few hundred enzymes have so far been elucidated. This is possible due to a variety of experimental techniques available to separate the different molecular forms of an isozyme. Electrophoresis in starch and polyacrylamide gels is a powerful tool for resolving the isozymes to give a zymogram.
 
 



Principle
Technically, the sample extract is electrophoresed in starch or polyacrylamide buffered (non-denaturing) slab gels at a low temperature (4-8°C). Each lane should be loaded with equal amount of proteins after normalizing the protein content in extract in as small volume as possible (25—50mL). After electrophoresis the gel is incubated in a solution containing all the necessary components for enzyme reaction. The colored reaction products stain the gel where the enzymes are located.
 
Staining Procedure
1.
Conduct electrophoresis in starch or polyacrylamide disc/slab buffer gels   (with   no   SDS)   at   low temperature   as   described   under Polyacrylamide Disc gel electrophoresis.
2.
Immediately after electrophoresis, incubate the gel in the substrate solution(s). The zones where the enzymes are located in the gel are visualized due to the appearance of colored reaction products. After sufficient incubation period, stop the reaction by adding appropriate stop solution and photograph the zymogram.  Otherwise, the relative
position of each visualized band in the gel may be drawn schematically for easy reference.

3.
Enzyme extraction and stain for various isoenzymes are given below:
 
i)   Esterase
ii)  Polyphenol Oxidase (PPO)
iii) Peroxidase
iv) Acid Phosphatase
v) Glutamine Synthetase
vi) Phosphoenol Pyruvate Carboxylase (PEP Case)
vii) Glutamate Dehydrogenase
viii) Indolylacetic Acid Oxidase
ix) Malate Dehydrogenase
x) Sucrose synthase
xi) Aspartate aminotransferase
xii) Glutamate synthase
 
i) Esterase
Enzyme Extract
Homogenize the sample material in 5-fold volume of 10mM sodium phosphate buffer (pH 9.5), 1mM EDTA Na2\ 1mM 2-mercaptoethanol. Centrifuge the homogenate at 10,000 X g for 10 min and use the supernatant as enzyme source. All operations are at 0-4°C. Incubate the gel in a solution given below at 37°C for 20-30min, preferably in dark.
Sodium dihydrogen phosphate        2.8g
Disodium hydrogen phosphate        1.1g
Fast blue RR salt                               0.2g
Alpha-naphthyl acetate                    0.03g
Water to                                            200mL
Stop the enzyme reaction by adding a mixture of methanol: water: acetic acid: ethyl alcohol in the ratio 10:10:2:1.
 


References
1.  Smith, HH et al (1970) J Heredit 61 203.
 
 
ii) Polyphenol Oxidase (PPO)
Enzyme Extract
The PPO is extracted by homogenizing the material in 0.01 M potassium phosphate buffer (pH 7.0) containing 1% non-ionic detergent (Tween 80) at 0°C for 15min. Centrifuge the homogenate at 20,000 X g for 15min at 0°C in a refrigerated centrifuge. Use the supernatant as enzyme source.
Equilibrate the gel for 30min in 0.1% p-phenylenediamine in 0.1M potassium phosphate buffer (pH 7.0) followed by 10mM catechol in the same buffer.
 


References
1.  Jayaraman, K S, Ramanuja, M N, Vijayaraghavan, P K and Vaidyanathan, C S (1987) Food Chem 24 203.
 
 
iii) Peroxidase
Enzyme Extract. See under Teroxidase'
Incubate the gel in the following solutions
Benzidine                          2.08g
Acetic acid                        18mL
Hydrogen peroxide (3%)  100mL
Water                               80mL
Bright blue colored bands appear in gel. When the bands are stained sufficiently, arrest the reaction by immersing the gel into a large volume of 0.67% sodium hydroxide or 7% acetic acid solution for 10 min.
 


References
1.  Reddy, M M and Gasber, E D (1971) Bot Gaz 132158.
 
 
iv) Acid Phosphatase
Enzyme Extract. See under 'Phosphatases'
Wash the gel 3-4 times in 0.1M acetate buffer (pH 5) by changing the buffer every 15min (to lower the pH of the gel to 5). Incubate the gel at 37°C for 2 - 3h in the following solution.
1-Naphthyl phosphate                                                      0.05g
Fast blue RR                                                                        0.05g
Sodium chloride                                                                 1.0g
10% magnesium chloride                                                0.5mL
0.1M Acetate buffer (pH 5.0) to                                       50mL
Reddish-brown bands visible in the gel. Fix the gel using 50% ethanol.
 


References
1.  De, K K and Roy, S C (1984) Theor appl Genetics 68 285.
 
 
v) Glutamine Synthetase
Enzyme Extract. See under 'Glutamine synthetase'
Mix the following solutions:
Tris Sigma 7-9 (Sigma T.1328)                        0.1M       36mL
Imidazole-HCl                                                      1M          10mL
Hydroxylamine HCl                                            0.8M       10mL
Magnesium Monosodium glutamate                              3M          0.74mL
Adjust to pH 7.7 with NaOH.                              0.8M       21mL
Incubate the gel at 32°C for 10 min.
Add 14.5mL of 0.2M ATP solution to the above mixture and continue the incubation for 50min. Terminate the reaction by adding 80mL of a mixture consisting of ferric chloride hexahydrate (55g/L), trichloroacetic acid (20g/L) and Cone. HCl (21mL/L).
The isozymes appear as purple bands against yellow background and are not permanent unless stored at 4°C.
 


References
1.  Atkins, A, Pate, J S and Shelp, B J (1984) Plant Physiol 76705.
 
 
vi) Phosphoenol Pyruvate Carboxylase (PEP Case)
 Enzyme Extract
Homogenize the plant tissue in ice-cold 100mM Tris-HCl (pH 7.8), 10mM magnesium chloride, 10mM sodium bicarbonate, 2% polyvinyl pyrrolidone, 1mM EDTA and 15mM 2-mercaptoethanol. Centrifuge at 30,000 X g for 45 min and use the supernatant.
Incubate the gel in a solution of 100mM Tris-HCl (pH 8.0), 10mM magnesium chloride, 200mM calcium chloride, 10mM sodium bicarbonate and 5mM phosphoenol pyruvate for 30 min at 40°C. During the incubation white bands of precipitate develop indicating the position of enzymes on the gel. The phosphate released by the enzyme action is precipitated as white calcium phosphate in the gel and is stable for many hours.
 


References
1. Francis, K and Gnanam, A (1979) Experientia 35 23.
 
 
vii) Glutamate Dehydrogenase
Enzyme Extract. See under 'Glutamate dehydrogenase
Incubate the gels in a solution containing 8mM monosodium glutamate, 0.2mM methylthiazol tetrazolium (MTT) or nitrobluetetrazolium (NBT), 0.1mM phenazine methosulphate, 0.2mM NAD, 1.2mM sodium cyanide in 200mM Tris-HCl buffer pH 7.5. The staining solution for glutamate dehydrogenase (NADP-dependant) contains 0.2mM NADP instead of NAD.
 


References
1.  Nash, D T and Davies, M E (1975) Phytochem 14 2113.
 
 
viii) Indolylacetic Acid Oxidase (See under IAA oxidase)
Enzyme Extract
Incubate the gel in staining mixture of 1mg potassium indoleacetate, 0.08mg 2,3,6-trichlorophenol and 2mg Fast blue BB per mL in 60mM phosphate buffer (pH 6.0) at 30°C for sufficient time (preferably overnight).
 


References
1.  Endo, T (1968) Plant Cell Physiol 9333.
 
 
ix) Malate Dehydrogenase
Enzyme Extract
Proceed as described previously.
First preincubate the gels for 15-20 min in 200mM Tris-HCl (pH 7.5) buffer. Then transfer the gel to a solution containing 16mM L-malate, 0.2mM NAD, 0.25mM methylthiazol tetrazolium, 0.8mM phenazine methosulfate, 4mM MgCl2 and 1.2mM sodium cyanide in 200mM Tris-HCl (pH 7.5) buffer. To stain MDH (NADP-dependant), use NADP instead of NAD.
 


References
1.  Honold, G R, Farkas, G L, Stahmann, M A (1966) Cereal Chem 43 517.
 
 
x) Sucrose synthase
Enzyme Extract
Proceed as described previously for assay
Incubate the gels in a reaction mixture containing 20mM HEPES-KOH buffer (pH 7.5), 0.1 M sucrose and 2mM UDP for 30 min at 37°C. Rinse thoroughly in distilled water and incubate in 1N NaOH containing 1% (w/v) triphenyl tetrazolium chloride (TTC) for 10 minutes to develop color. Sucrose synthase bands appear red colored due to formation of formazon.
 


References
1.  Morell, M and Copeland, S (1985) Plant Physiol 78 149.
 
 
xi) Aspartate aminotransferase
Enzyme Extract
As described previously for assay
Incubate the gels in 100mM Tris-HCl buffer (pH 7.5) containing 40mM aspartate, 5mM 2-oxoglutarate and 1 mg/ml Fast violet B salt, in darkness for 30 min at 40°C.
 


References
1.  Grifith, SM and Vance, CP (1989) Plant Physiol 90 1622.
 
 
xii) Glutamate synthase
Enzyme Extract
As described previously for assay
Preequilibrate the gels in 200mM potassium phosphate buffer (pH 7.5) for 30 minutes. Incubate in 100mM potassium phosphate buffer (pH 7.5) containing 30mM NAD(P)H, 15mM 2-oxoglutarate and 15mM glutamine for 60 minutes at room temperature. Rinse the gels 3 times in 100mM Tris-HCl (pH 8.5) and incubate in a staining solution containing 50mM Tris -HC1 (pH 8.5), 5mg/ml nitroblue tetrazolium (NBT) and 0.6mg/ml phenazine methosulphate (PMS). GOGAT activity is indicated by negative staining (clear bands on a purple gel) due to the presence of NAD+.
 


References
1.  Matoh, T, Ida, S and Takahashi, E (1980) Plant Cell Physiol 21 1461.
 
     
 
 
     




     
 
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