Planta (Berl.) 103, 361-364 (1972) 9 by Springer-Verlag 1972
Short Communication
Glucosyhransferase Activity in a Water Extract of Maize Pollen* R. L. Larson and Cynthia M. Lonergan ** Department of Agronomy, University of Missouri, Columbia Received December 13, 1971
Summary. Glucosyltransferase activity was extracted from maize pollen in distilled water. The enzymatic reaction required UDP-glueoso, mereaptoethanol and Ca++, and had a pI-I optimum at 8.2. Either kampferol or quercetin served as a substrate. Michaelis-Menten constants obtained were 0.6 x 10-aM for quereetin and 0.74 x 10-aM for UDP-glucose. Ammonium-sulfato precipitation of the enzyme gave a 4fold purification. The presence of quercetin in the pollen and its 3-glucoside, isoquercitrin, in the husks of maize (Zea mays L.) has long been known but little information is available about the conversion of the aglycone to the corresponding glucoside (Redemann et al., 1950; Sando and Bartlett, 1922). I n a previous publication (Larson, 1971), we reported the presence of quereetin and isoquereitrin and the glucosyltransferase (UDPglucose:quercetin glucosyltransferase) that catalyzes the conversion of quercetin to isoquercitrin in the pollen of maize. Glueosyltransferase activity was demonstrated using intact pollen as a source of the enzyme in the reaction mixture because of difficulties encountered in obtaining an enzymatically active soluble preparation. The purpose of this study was to obtain the enzyme in a soluble, particle-free preparation to facilitate its further purification and an investigation of gene-enzyme relationships and control mechanisms involved in anthocyanin biosynthesis. All attempts to obtain the enzyme free of the pollen in sodium carbonate (Nakamura and Becket, 1951) or dilute buffers were un* Cooperative investigations, Plant Science Research Division, Agricultural Research Service, U.S. Department of Agriculture, and Missouri Agricultural Experiment Station, Columbia. Journal Series No. 6224. ** Formerly Biological Laboratory Technician, University of Missouri. 25a Planta (Berl.),Bd. 103
362
R. L. Larson and C. M. Lonergan:
successful. U l t i m a t e l y , i n c o n t r a s t t o c o n v e n t i o n a l e n z y m e i s o l a t i o n techniques it was possible to extract the glucosyltransferase in distilled water. Pollen was collected from plants in the field at anthesis, sieved with a No. 70-mesh screen, freeze-dried and stored in vacuo at --10 ~ C until used. Isoquercitrin was prepared by previously described methods (Fox et al., 1953), the other chemicals used were those obtained commercially. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. All purification steps were carried out at 4 ~ C. Previously dried pollen was suspended (6 mg/ml) in distilled water and stirred at 4~ for 4 h. The resulting suspension was centrifuged at 26000 • g for 15 rain after which the supernatant solution was freeze dried and stored in vacuo at - - 1 0 ~ C until used. The freeze dried preparation was suspended (10 mg/ml) in 0.005 M phosphate buffer, pH 6.6. In successive steps ammonium sulfate was added to the suspension first to 0.3 M and then to 0.55 M concentrations. The precipitates were collected by centrifugation at 26000 • g, discarding the initial precipitate and saving the fraction precipitating between 0.3 and 0.55 M ammonium sulfate. This fraction was then suspended in 0.005 M phosphate buffer, p t t 7.2 -t- 0.001 M E D T A and dialyzed against distilled water for 6 h. The dialyzed suspension was then freezedried and stored in vacuo at - - 1 0 ~ C until used. To measure glucosyltransferase activity, samples were incubated with shaking at 37 ~ C for 90 min; after this, the isoquercitrin was extracted into ethyl acetate. The ethyl acetate was evaporated and the residue suspended in ethanol and chromatographed on Magnesol 1 (magnesium acid silicate), [Waverly Chemical Co., Guilford, CN, U.S.A.], (Larson, 1969). The yield of isoquercitrin in the reaction mixtures could then be determined, following extraction from Magnesol, by the spectrophotometric methods described in an earlier report (Larson, 1969). The standard reaction mixture contained UDPglucose (1.i • 10-aM), quercetin (0.413 • 10-8M), tris(hydroxymethyl)aminomethane buffer (3.1 • 10-~M), mercaptoethanol (5 • 10-2M), CaC1z (6.25 • 10-aM) and distilled water to 1.6 ml total volume. T h e r e s u l t s in T a b l e 1 d e a r l y i n d i c a t e t h a t g l u c o s y l t r a n s f e r a s e c a n be e x t r a c t e d f r o m t h e p o l l e n i n d i s t i l l e d w a t e r . A c o m p a r i s o n of r e c o v e r y
Table 1. Glucosyltransferase extraction and purification
Crude pollen b Water extract Ammoninm-sulfate
Specific act. a
Total act. ( • 104)
Recovery (%)
Purification
22.3 49.4 82
2.9 4.11 1.44
-146 34
-2.2 3.6
a Micrograms isoquercitrin produced per hour per mg protein. b The activity data for the crude pollen are based on the extractable protein in a given amount of pollen. 1 Mention of a trademark name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U. S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable.
Glucosyltransferase in Maize Pollen
363
data indicates that the glucosyltransferase that can be assayed in a given amount of pollen can be increased 1.5 fold by extraction. Requirements for glucosyltransferase activity with the extracted enzyme were much the same as those with crude pollen as a source of the enzyme (Larson, 1971). UDP-glucose gave optimum activity as the glucose donor and the optimum incubation temperature and time were 37 ~ C and 90 min. The extracted enzyme has a single p i t optimum of 8.2 in contrast to the p i t optima of 6.2 and 8.2 observed with crude pollen as the enzyme source (Larson, 1971). The divalent metal ion of choice for glucosyltransferase activity was Ca++. Mercaptoethanol satisfied the requirement for a reducing agent when added to the reaction mixture but when added to the extraction or dialysis medium it resulted in loss of enzyme activity. I n contrast to the results of the earlier studies (Larson, 1971), it was possible to obtain Michaelis-Menten constants of 0.6 • 10-4M for quercetin and 0.74• 10-3M for UDP-glueose. The glucosyltransferase catalyzed the formation of the glucoside of kampferol as well as quercetin. Mercuric chloride or p-chloromercuribenzoate markedly inhibited the enzymatic activity. The ability to readily extract enzymes from pollen in water should facilitate the study of the metabolism of pollen. This technique is in contrast to conventional enzyme-isolation methods that require strict control of the p H in the extraction medium. The evidence presented clearly supports further use of the method and purification of the enzyme is in progress. The lack of anthocyanins in the pollen also prompts consideration of the role of this enzyme in the metabolism of pollen. This study was supported in part by a research grant (GB-3992) from the National Science Foundation. The authors would like to thank Mr. James Bussard for technical assistance and Dr. E. H. Coe, Jr., Research Geneticist, U.S. Department of Agriculture for making plant materials available for these studies. References Fox, D. W., Savage, W. L., Wender, S. H.- Hydrolysis of some flavonoid rhamnoglycosides to flavonoid glucosides. J. Amer. chem. Soc. 75, 2504-2505 (1953). Larson, R.L.: Magnesol thin-layer chromatography of flavonoid compounds. J. Chrom. 43, 287-290 (1969). Larson, R. L. 9Glucosylation of quercetin by a maize pollen enzyme. Phytochem. 10, 3073-3076 (1971). Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the Folin phenol reagent. J. biol. Chem. ]93, 265-275 (1951). 25b
P l a n t a (Berl.), Bd. 103
364
R . L . Larson and C. M. Lonergan: Glucosyltransferase in Maize Pollen
Nakamura, G. R., Becker, E. L.: Phosphatases of ragweed pollen. Arch. Biochem. 83, 78-89 (1951). Redemann, C.T., Wittwer, S.H., Ball, C.D., Sell, H.M.: The occurrence of quercetin in the pollen of Zea mays. Arch. Biochem. 25, 277-279 (1950). Sando, C. E., Bartlett, H. H.: Pigments of the Mendelian color types in maize: isoquercitrin from brown-husked maize. J. biol. Chem. 54, 629-645 (1922). Dr. R. L. Larson Research Chemist 304 Curtis Hall Department of Agriculture University of Missouri Columbia, MO, U.S.A. 65201
Cynthia M. Lonergan's present address: Michael Reese Hospital Chicago, Illinois, U.S.A.
Short Communication
Glucosyhransferase Activity in a Water Extract of Maize Pollen* R. L. Larson and Cynthia M. Lonergan ** Department of Agronomy, University of Missouri, Columbia Received December 13, 1971
Summary. Glucosyltransferase activity was extracted from maize pollen in distilled water. The enzymatic reaction required UDP-glueoso, mereaptoethanol and Ca++, and had a pI-I optimum at 8.2. Either kampferol or quercetin served as a substrate. Michaelis-Menten constants obtained were 0.6 x 10-aM for quereetin and 0.74 x 10-aM for UDP-glucose. Ammonium-sulfato precipitation of the enzyme gave a 4fold purification. The presence of quercetin in the pollen and its 3-glucoside, isoquercitrin, in the husks of maize (Zea mays L.) has long been known but little information is available about the conversion of the aglycone to the corresponding glucoside (Redemann et al., 1950; Sando and Bartlett, 1922). I n a previous publication (Larson, 1971), we reported the presence of quereetin and isoquereitrin and the glucosyltransferase (UDPglucose:quercetin glucosyltransferase) that catalyzes the conversion of quercetin to isoquercitrin in the pollen of maize. Glueosyltransferase activity was demonstrated using intact pollen as a source of the enzyme in the reaction mixture because of difficulties encountered in obtaining an enzymatically active soluble preparation. The purpose of this study was to obtain the enzyme in a soluble, particle-free preparation to facilitate its further purification and an investigation of gene-enzyme relationships and control mechanisms involved in anthocyanin biosynthesis. All attempts to obtain the enzyme free of the pollen in sodium carbonate (Nakamura and Becket, 1951) or dilute buffers were un* Cooperative investigations, Plant Science Research Division, Agricultural Research Service, U.S. Department of Agriculture, and Missouri Agricultural Experiment Station, Columbia. Journal Series No. 6224. ** Formerly Biological Laboratory Technician, University of Missouri. 25a Planta (Berl.),Bd. 103
362
R. L. Larson and C. M. Lonergan:
successful. U l t i m a t e l y , i n c o n t r a s t t o c o n v e n t i o n a l e n z y m e i s o l a t i o n techniques it was possible to extract the glucosyltransferase in distilled water. Pollen was collected from plants in the field at anthesis, sieved with a No. 70-mesh screen, freeze-dried and stored in vacuo at --10 ~ C until used. Isoquercitrin was prepared by previously described methods (Fox et al., 1953), the other chemicals used were those obtained commercially. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. All purification steps were carried out at 4 ~ C. Previously dried pollen was suspended (6 mg/ml) in distilled water and stirred at 4~ for 4 h. The resulting suspension was centrifuged at 26000 • g for 15 rain after which the supernatant solution was freeze dried and stored in vacuo at - - 1 0 ~ C until used. The freeze dried preparation was suspended (10 mg/ml) in 0.005 M phosphate buffer, pH 6.6. In successive steps ammonium sulfate was added to the suspension first to 0.3 M and then to 0.55 M concentrations. The precipitates were collected by centrifugation at 26000 • g, discarding the initial precipitate and saving the fraction precipitating between 0.3 and 0.55 M ammonium sulfate. This fraction was then suspended in 0.005 M phosphate buffer, p t t 7.2 -t- 0.001 M E D T A and dialyzed against distilled water for 6 h. The dialyzed suspension was then freezedried and stored in vacuo at - - 1 0 ~ C until used. To measure glucosyltransferase activity, samples were incubated with shaking at 37 ~ C for 90 min; after this, the isoquercitrin was extracted into ethyl acetate. The ethyl acetate was evaporated and the residue suspended in ethanol and chromatographed on Magnesol 1 (magnesium acid silicate), [Waverly Chemical Co., Guilford, CN, U.S.A.], (Larson, 1969). The yield of isoquercitrin in the reaction mixtures could then be determined, following extraction from Magnesol, by the spectrophotometric methods described in an earlier report (Larson, 1969). The standard reaction mixture contained UDPglucose (1.i • 10-aM), quercetin (0.413 • 10-8M), tris(hydroxymethyl)aminomethane buffer (3.1 • 10-~M), mercaptoethanol (5 • 10-2M), CaC1z (6.25 • 10-aM) and distilled water to 1.6 ml total volume. T h e r e s u l t s in T a b l e 1 d e a r l y i n d i c a t e t h a t g l u c o s y l t r a n s f e r a s e c a n be e x t r a c t e d f r o m t h e p o l l e n i n d i s t i l l e d w a t e r . A c o m p a r i s o n of r e c o v e r y
Table 1. Glucosyltransferase extraction and purification
Crude pollen b Water extract Ammoninm-sulfate
Specific act. a
Total act. ( • 104)
Recovery (%)
Purification
22.3 49.4 82
2.9 4.11 1.44
-146 34
-2.2 3.6
a Micrograms isoquercitrin produced per hour per mg protein. b The activity data for the crude pollen are based on the extractable protein in a given amount of pollen. 1 Mention of a trademark name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U. S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable.
Glucosyltransferase in Maize Pollen
363
data indicates that the glucosyltransferase that can be assayed in a given amount of pollen can be increased 1.5 fold by extraction. Requirements for glucosyltransferase activity with the extracted enzyme were much the same as those with crude pollen as a source of the enzyme (Larson, 1971). UDP-glucose gave optimum activity as the glucose donor and the optimum incubation temperature and time were 37 ~ C and 90 min. The extracted enzyme has a single p i t optimum of 8.2 in contrast to the p i t optima of 6.2 and 8.2 observed with crude pollen as the enzyme source (Larson, 1971). The divalent metal ion of choice for glucosyltransferase activity was Ca++. Mercaptoethanol satisfied the requirement for a reducing agent when added to the reaction mixture but when added to the extraction or dialysis medium it resulted in loss of enzyme activity. I n contrast to the results of the earlier studies (Larson, 1971), it was possible to obtain Michaelis-Menten constants of 0.6 • 10-4M for quercetin and 0.74• 10-3M for UDP-glueose. The glucosyltransferase catalyzed the formation of the glucoside of kampferol as well as quercetin. Mercuric chloride or p-chloromercuribenzoate markedly inhibited the enzymatic activity. The ability to readily extract enzymes from pollen in water should facilitate the study of the metabolism of pollen. This technique is in contrast to conventional enzyme-isolation methods that require strict control of the p H in the extraction medium. The evidence presented clearly supports further use of the method and purification of the enzyme is in progress. The lack of anthocyanins in the pollen also prompts consideration of the role of this enzyme in the metabolism of pollen. This study was supported in part by a research grant (GB-3992) from the National Science Foundation. The authors would like to thank Mr. James Bussard for technical assistance and Dr. E. H. Coe, Jr., Research Geneticist, U.S. Department of Agriculture for making plant materials available for these studies. References Fox, D. W., Savage, W. L., Wender, S. H.- Hydrolysis of some flavonoid rhamnoglycosides to flavonoid glucosides. J. Amer. chem. Soc. 75, 2504-2505 (1953). Larson, R.L.: Magnesol thin-layer chromatography of flavonoid compounds. J. Chrom. 43, 287-290 (1969). Larson, R. L. 9Glucosylation of quercetin by a maize pollen enzyme. Phytochem. 10, 3073-3076 (1971). Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the Folin phenol reagent. J. biol. Chem. ]93, 265-275 (1951). 25b
P l a n t a (Berl.), Bd. 103
364
R . L . Larson and C. M. Lonergan: Glucosyltransferase in Maize Pollen
Nakamura, G. R., Becker, E. L.: Phosphatases of ragweed pollen. Arch. Biochem. 83, 78-89 (1951). Redemann, C.T., Wittwer, S.H., Ball, C.D., Sell, H.M.: The occurrence of quercetin in the pollen of Zea mays. Arch. Biochem. 25, 277-279 (1950). Sando, C. E., Bartlett, H. H.: Pigments of the Mendelian color types in maize: isoquercitrin from brown-husked maize. J. biol. Chem. 54, 629-645 (1922). Dr. R. L. Larson Research Chemist 304 Curtis Hall Department of Agriculture University of Missouri Columbia, MO, U.S.A. 65201
Cynthia M. Lonergan's present address: Michael Reese Hospital Chicago, Illinois, U.S.A.
