use of nicotinamide adenine dinucleotide (nad)- dependent glucose-6

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dehydrogenase for the nicotinamide adenine dinucleotide phosphate ... significantly reduces the cost of staining for adenylate kinase, creatine kinase, ...
0038-9 153/80/5503-0173$02200/0 STAIN TECHNOLOGY Copyright O 1980 by The Williams & Wilkins Co

Vol. 55, No. 3 Printed in U. S. A.

USE OF NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD)DEPENDENT GLUCOSE-6-PHOSPHATE DEHYDROGENASE IN ENZYME STAINING PROCEDURES DONALD G. BUTHAND ROBERT W. MURPHY, Department of Biolog, University of Caltfornta, Los Angeles, Caltfornia 90024 ABSTRACT.Substitution of nicotinamide adenine dinucleotide dependent glucose-6-phosphate dehydrogenase for the nicotinamide adenine dinucleotide phosphate dependent enzyme has produced identical results in a number of enzyme-linkedelectrophoretic staining procedures. This substitution significantly reduces the cost of staining for adenylate kinase, creatine kinase, glucosephosphate isomerase, mannosephosphate isomerase: phosphoglucomutase, and pyruvate kinase activity by utilizing NAD rather than the more expenslve NADP.

Enzyme-linked staining methods have greatly expanded the number of gene-loci that can be studied via electrophoretic techniques. These staining methods use an exogenous enzyme to trap and change a product of the enzyme in question into some secondary product that ultimately can be visualized (Harris and Hopkinson 1976). Glucose-6-phosphate dehydrogenase (G-6-PDH) is the enzyme most commonly used in linkage staining; it is coupled to the phenazine-tetrazolium system (Brewer 1970, Harris and Hopkinson 1976). Initial enzymatic steps yield the substrate glucose-6-phosphate, which then may be acted upon by G-6-PDH. T h e reduced pyridine nucleotide involved in this step reduces an added electron carrier (phenazine methosulfate) which further reduces the tetrazolium dye. T h e resulting formazan precipitate indicates the location of enzyme activity. An alternative procedure involves the formation of adenosine-5'-triphosphate (ATP) in the initial reaction. ATP can then act as a cofactor for hexokinase (also added) activity which yields glucose-6-phosphate. Additional staining reactions proceed through the phenazine-tetrazolium system as outlined above (Brewer 1970). Most enzyme staining procedures using G-6-PDH as a linking enzyme require nicotinamide adenine dinucleotide phosphate (NADP) as the pyridine nucleotide (Brewer 1970, Shaw and Prasad 1970, Selander et al. 1971, Ayala et al. 1972, Harris and Hopkinson 1976), i.e. the G-6-PDH is NADP-dependent. However, a number of investigators have found that a G-6-PDH that utilizes nicotinamide adenine dinucleotide (NAD) as the pyridine nucleotide can achieve the same goal, although this information has largely remained unpublished. The cost of NADP is often ten times that of NAD and substitution of the latter can result in substantial financial savings where a large number of individuals are being screened for relevant gene products. We have modified a number of commonly used enzyme assays to use NADdependent G-6-PDH, and thus NAD, and present them for use in vertebrate studies. PROCEDURES Six enzyme assays previously described using NADP-dependent G-6-PDH are re-described below using an NAD-dependent form of the enzyme (Product #3992, 173

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Worthington Biochemical Corporation, Freehold, NJ 07728 USA). G. R. Joswiak (personal communication) has indicated that NADP-NAD-dependent G-6-PDH may also be used (Product G-2256, Sigma Chemical Company, St. Louis, MO 63178 USA). The following components of the phenazine-tetrazolium system were also obtained from Sigma: nicotinamide adenine dinucleotide (NAD, Sigma N7004), nitro blue tetrazolium (NBT, Sigma N-6876), 3-(4,5-dimethylthiazolyl-2)2,5-phenyl tetrazolium bromide (MTT, Sigma M-2128), phenazine methosulfate (PMS, Sigma P-9625). Substrates and other components obtained from Sigma are noted. The following formulations are recommended for demonstration of the enzymes named. Adenylate kinase (EC 2.7.4.3); modified from Fildes and Harris (1966): 0.2 M Tris-HC1, pH 8.0 0.1 M MgClz. 6Hz0 Adenosine-5'-diphosphate (Sigma A-8 146) D-Glucose (Sigma G-5000) Hexokinase (Sigma H-5000) 30 U/ml G-6-PDH 10 mg/ml NAD 10 mg/ml NBT 10 mg/ml PMS Creatine kinase (EC 2.7.3.2); modified from Shaw and Prasad (1970): 0.2 M Tris-HC1, pH 7.0 0.1 M MgC12.6 H 2 0 Adenosine-5'-diphosphate (Sigma A-8 146) D-Glucose (Sigma G-5000) Hexokinase (Sigma H-5000) Phosphocreatine (Sigma P-6502) 30 U/ml G-6-PDH 10 mg/ml NAD 10 mg/ml NBT 10 mg/ml PMS Glucosephosphate isomerase (EC 5.3.1.9); modified from DeLorenzo and Ruddle (1969) : 0.2 M Tris-HC1, pH 7.0 50.0 ml 5.0 ml 0.1 M MgC12.6Hz0 Fructose-6-phosphate (Sigma F-3627) 0.04 g 1.0 ml 30 U/ml G-6-PDH 10 mg/ml NAD 1.5 ml 0.5 ml 10 mg/ml NBT 0.5 ml 10 mg/ml PMS

NAD-DEPENDENT G-6-PDH IN ENZYME STAINS

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Mannosephosphate isomerase (EC 5.3.1.8); modified from Nichols et a/. (1973): 0.2 M Tris-HC1, pH 8.0 50.0 ml Mannose-6-phosphate (Sigma M-8754) 0.05 gm 50 U/ml Glucosephosphate isomerase (Sigma P-5381) 1.0 rnl 30 U/ml G-6-PDH 1.0 ml 1.5 ml 10 mg/ml NAD 0.5 ml 10 mg/ml M T T 10 mg/ml PMS 0.5 ml Phosphoglucornutase (EC 2.7.5.1); modified from Spencer et al. (1964): 0.2 M Tris-HC1, p H 8.0 0.1 M MgC12.6Hz0 Glucose- 1-phosphate (Sigma G-7000) 30 U/ml G-6-PDH 10 mg/ml NAD 10 mg/ml NBT 10 mg/ml PMS

50.0 ml 5.0 ml 0.1 g 0.5 ml 1.5 ml 0.5 ml 0.5 ml

Pyruvate kinase (EC 2.7.1.40); modified from Brewer (1970): 0.2 M Tris-HC1, pH 8.0 0.1 M MgC12.6HzO Adenosine 5'-diphosphate (Sigma A-8146) D-Glucose (Sigma G-5000) Hexokinase (Sigma H-5000) Phospho(eno1)pyruvate (Sigma P-7 127) 30 U/ml G-6-PDH 10 mg/ml NAD 10 mg/ml NBT 10 mg/ml PMS

(Note: this PK stain also resolves adenylate kinase gene products.) While enzyme activity can be visualized by simply mixing the staining components and applying the mixture to a starch gel slice, considerable improvement in resolution can be achieved by using an agar overlay method (Brewer 1970) to localize staining components.

We have applied both NAD-dependent and NADP-dependent forms of G-6PDH in enzyme stains in electrophoretic studies of many groups of fishes (e.g. Buth 1979), amphibians and reptiles and in a minimal number of birds and mammals. In all cases, identical results were achieved in terms of staining clarity. The only difference, therefore, is the substantial cost benefit of substituting NAD for NADP with NAD-dependent G-6-PDH in these stains. A number of additional existing enzyme stains, especially the kinases, could also be modified to accept an NAD-dependent G-6-PDH component. The use of this

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less expensive step should be taken into consideration when developing new enzyme-linked staining methods.

ACKNOWLEDGMENTS We thank G. S. Whitt and D. P. Philipp for the initial suggestion of modifying the stains in question in the manner described. Helpful contributions by S. D. Ferris, G. R. Joswiak, J. Lubina, and R. Matson are appreciated. This study was supported in part by the National Science Foundation (NSF DEB 75-16773 to D. G. Buth and NSF DEB 77-03259 to G. C. Gorman). REFERENCES Ayala, F. J., Powell, J. R., Tracey, M. L., Mourao, C. A. and Perez-Salas, S. 1972. Enzyme variability in the Drosophila willistoni group. IV. Genic variation in natural populations of Drosophzla willistoni. Genetics 70: 113- 139. Brewer, G. J. 1970. An Introduction to Isozyme Techniques. Academic Press, New York. Buth, D. G. 1979. Duplicate gene expression in tetraploid fishes of the tribe Moxostomatini (Cypriniformes, Catostomidae). Comp. Biochem. Physiol. 63B: 7-12. DeLorenzo, R. J. and Ruddle, F. H. 1969. Genetic control of two electrophoretic variants of glucosephosphate isomerase in the mouse. Biochem. Genet. 3: 151-162. Fildes, R. A. and Harris, H. 1966. Genetically determined variation of adenylate kinase in man. Nature 209: 261-263. Harris, H. and Hopkinson, D. A. 1976. Handbook of Eruyme Electrophoresis in Human Genetics. NorthHolland Publ. Co., Amsterdam. Nichols, E. A,, Chapman, V. M. and Ruddle, F. H. 1973. Polymorphism and linkage for mannosephosphate isomerase in Mus mutculus. Biochem. Genet. 8: 47-53. Selander, R. K., Smith, M. I]., Yang, S. Y., Johnson, W. E. and Gentry, J. B. 1971. IV. Biochemical polymorphism and systematics in the genus Peromyscus. I. Variation in the old-field mouse (Peromyscuspolionotus). Studies in Genetics VI. Univ. Texas Publ. 7103; 49-90. Shaw, C. R. and Prasad, R. 1970. Starch gel electrophoresis of enzymes- a compilation of recipes. Biochem. Genet. 4: 297-320. Spencer, N., Hopkinson, D. A. and Harris, H. 1964. Phosphoglucomutase polymorphism in man. Nature 204: 742-745.