Use of Protein in Extraction and Stabilization of Nitrate Reductase'

Plant Physiol. (1974) 53, 688-690

Received for publication October 29, 1973 and in revised form December 28, 1973

L. E. SCHRADER, D. A. CATALDO, AND D. M. PETERSON Department of Agronomy and United States Department of Agriculture, Agricultural Research Service, Uni- versity of Wisconsin-Madison, Madison, Wisconsin 53706

ABSTRACT vestigations of the instability phenomenon in corn, oats, and tobacco, and development of a procedure for stabilizing nitrate The in vitro instability of nitrate reductase (EC 1.6.6.1) reductase activity after extraction. activity from leaves of several species of higher plants was investigated. Decay of activity was exponential with time, suggest- ing that an enzyme-catalyzed reaction was involved. The rate of MATERIALS AND METHODS decay of nitrate reductase activity increased as leaf age increased Corn seedlings and oat plants were grown in environmental in all species studied. Activity was relatively stable in certain chambers as previously described (1). Also corn was grown in genotypes of Zea mays L., but extremely unstable in others. the field. Tobacco was planted in soil in the greenhouse, but In all genotypes of Avena sativa L. and Nicotiana tabacum L. was transferred to environmental chambers at 6 weeks of age, studied, nitrate reductase was unstable. Addition of 3% (w/v) and watered daily with nutrient solution described earlier (1). bovine serum albumin or casein to extraction media pre- Nitrate reductase was extracted from tissue and assayed by the vented or retarded the decay of nitrate reductase activity for procedures of Hageman and Hucklesby (6), except that 25 mM several hours. In addition, the presence of bovine serum al- L-cysteine was used in the extraction medium for leaf blades bumin or casein in the enzyme homogenate markedly increased of oats. When casein or bovine serum albumin was added to nitrate reductase activity (up to 15-fold), especially in older the extraction medium, the pH was readjusted to 8.8 before leaf tissue. use. Casein (technical or pure) and bovine serum albumin (fraction V) were obtained from Sargent-Welch2 and Sigma Chemical Co., respectively. In all experiments, duplicate ali- quots of each extract were assayed for 15 min at 30 C. Ex- tracts were stored in an ice bath between assays. RESULTS AND DISCUSSION Nitrate reductase (EC 1.6.6. 1) extracted from leaf blades of Oh43, an inbred line of corn (Zea mays L.), was previously Time course studies on decay of nitrate reductase activity reported (9) to be unstable when stored at 0 C. Decay rate of in extracts from corn and oat leaf blades showed that decay activity increased from 2%/hr in 8-day-old seedlings to was exponential with time when no bovine serum albumin or 31%/hr in 13-day-old seedlings. Attempts to stabilize the ac- casein was added to media for extraction of nitrate reductase tivity were unsuccessful (9). (Fig. 1). The half-life (50% decay) of the enzyme extracted We have recently identified several other genotypes of corn from 79-day-old corn plants and 42-day-old oat plants was 73 which have unstable activity; with oats (Avena sativa L.), wheat and 52 min, respectively. The initial points on the curves (Fig. (Triticum aestivum L.), barley (Hordeum vulgare L.). rye (Se- 1) were obtained by assaying extracts 5 to 10 min after ex- cale cereale L.), and tobacco (Nicotiana tabacum L.), no geno- traction. The 30-min values were obtained immediately after types with stable nitrate reductase activity were found (un- a 15-min centrifugation at 30,000g. Because initial values were published data). Thus, the potential capacity for enzymic re- highest, the necessity for centrifugation before assay may be duction of nitrate may have been grossly underestimated by questioned. Procedures employed by most investigators (6) in vitro assays in many earlier studies. In olders leaves of oats involve extraction followed by centrifugation for at least 15 and corn, normal extraction and in vitro assay (6) may have min. Thus, an hour may elapse between extraction and assay, underestimated nitrate-reducing capacity by as much as 15- even when a small number of samples is analyzed. Particularly fold. Because measurements of nitrate reductase activity have in older tissue, potential activity may be underestimated be- been used as an index in selecting desirable stocks in plant cause as much as half of the activity may be lost during this breeding programs, it is essential that enzyme activity be sta- time. bilized in all extracts for genotypic comparisons (7, 8, 10), or Because loss of nitrate reductase activity was a first order for comparisons of enzyme activity with total accumulation of reaction (Fig. 1), it seemed likely that another enzyme was in- reduced nitrogen in plant parts (2, 4, 5). Hence, we report involved in the inactivation of nitrate reductase. To afford pro-

1 This research was supported by the College of Agricultural and 2 Mention of a trademark or proprietary product does not consti- Life Sciences, University of Wisconsin-Madison, and by United tute a guarantee or warranty of the product by the United States States Department of Agriculture Cooperative Agreement 12-14- Department of Agriculture, and does not imply its approval to the 100-10,888. exclusion of other products that may also be suitable. 688 Plant Physiol. Vol. 53, 1974 NITRATE REDUCTASE STABILIZATION 689 tection to nitrate reductase from proteolytic enzymes after Table II. Effect ofAdding Casein or Bovinie Serum Albumini to extraction, protein was added to the extraction media. Prelimi- Media for Extraction of Nitrate Reductase from Leaf nary experiments with various proteins (peptone, gelatin, he- Blades of Two Genotypes of Corn at Two Ages moglobin, bovine serum albumin, and casein) showed that Leaf tissue used in experiment A was from corn seedlings 11 bovine serum albumin and casein were most effective in ex- days after planting in an environmental chamber. Tissue for experi- periments with corn and oats. Not only did these proteins help ment B was from field-grown corn at 89 days after planting. The stabilize nitrate reductase after extraction, but the level of ac- third leaf blade from the top of several plants was sampled in tivity was increased, especially in more mature tissue. experiment B. The level of nitrate reductase activity in extracts from oat leaves with no additives decreased rapidly as the age of plants Nitrate Reductase Activity increased (Table I). Nitrate reductase activity was also less stable in extracts from older tissue. Addition of 3% (w/v) Genotype and Treatment Experiment A Experiment B bovine serum albumin partially stabilized the activity. How- ever, casein was superior to bovine serum albumin for stabili- 62' 122 182 64 214 zation of nitrate reductase from oats in all three experiments; j.moles NO2- produtced/g fresh w;I-' hIr-1 3% (w/v) was the optimal concentration (unpublished data). Ohio 43 In addition to stabilization, higher activities were obtained No additive 10.3 8.9 7.7 3.6 1.2 in extracts containing bovine serum albumin or casein (Table 3%, Bovine serum albumin 24.8 24.5 24.1 10.8 7.6 I). This was especially true in older leaves (experiment A) 3c%c Casein 24.2 24.5 24.6 W64A X W182E No additive 19.5 20.0 20.2 9.1 7.6 3% Bovine serum albumin 25.0 24.2 24.0 11.7 10.3 L. 3c% Casein 25.3 25.6 25.9

> _. 1 Minutes between extraction and assay.

0 Table III. Effect of Adding Bovine Serum Albumini or Caseini to Media for Extraction of Nitrate Reductase from Leaves of Tobacco Planits 0 E The ninth leaf from the bottom of a 10-week-old tobacco plant L.-0 D (cv. Wisconsin 38) with 15 fully expanded leaves was used for this study. _ 0 Z o CY Nitrate Reductase Activity Treatment 1151 242 302 0 60 120 180 pnmoles N02- producedlg fresh utI-I hr-1 MINUTES BETWEEN EXTRACTION No additive 1.2 0.6 0.4 AND ASSAY 3c/% Bovine serum albumin 11.1 10.1 9.6 FIG. 1. In vitro decay of nitrate reductase activity in extracts 3%G Casein 12.8 11.8 10.2 from leaf blades of corn (79-day-old plants cv. Oh43) and oats (42-day-old plants cv. Jaycee). Extraction and assay procedures are Minutes between extraction and assay. described in "Materials and Methods." No bovine serum albumin or casein was added to the extraction media. where a 15-fold increase in activity was obtained with 3% (w/v) casein in the extraction medium. This enhancement was Table 1. Effect of Addinig Proteinis or Sorbitol to Media for less marked in younger tissue (experiments B and C). Either Extraction of Nitrate Reductase from Leaf Blades of technical or pure casein provided similar results (unpublished Oat Planits of Various Ages data). Sorbitol (3% w/v) added as an osmoticum (experiment Oat plants (cv. Orbit) for experiments A, B, and C were grown A) had no effect. Thus, the results obtained with bovine serum in environmental chambers and used at 90, 61, and 46 days after albumin or casein were not attributed to an osmotic effect planting, respectively. during extraction. Preliminary experiments with an inbred line of corn (Oh43) Nitrate Reductase Activity having unstable nitrate reductase activity (9), and an F1 hybrid (W64A x W182E) having relatively stable activity indicated Treatment Experiment A Experiment B Experiment C that 3% (w/v) bovine serum albumin or casein was optimal for 481 108 168 75 135 195 60 120 180 both extraction and stabilization of nitrate reductase (unpublished data). Activity from 11-day-old Oh43 seedlings was dou- .smtoles N02- produced, g fresh wi-1 - hr-I bled by addition of 3% (w/v) bovine serum albumin or casein No additive 0.7 0.2 0.0 3.9 2.91 2.31 6.6 5.1 3.7 to the extraction medium (Table II, experiment A), whereas 3%Bovineserum 6.6 5.2 4.2 7.2 6.77 6.6 17.1 16.7 14.9 activity was increased 3-fold in 89-day-old field-grown plants albumin (experiment B). Activity was stabilized by bovine serum albu- 3%c Casein 10.4 9.6 9.4 9.9 9.9 9.5 20.5 20.1 18.6 min or casein for at least 2 hr after the initial assay in the 3%- Sorbitol 0.6 0.2 0.0 seedlings, but some decay occurred after 2.5 hr in extracts from older leaf blades. Activity in the hybrid (W64A X W182E) 1 Minutes between extraction and assay. was increased about 30% by addition of bovine serum albumin 690 SCHRADER, CATALDO, AND PETERSON Plant Physiol. Vol. 53, 1974 or casein. Stability was not affected in the already stable seed- casein effects such a marked increase in nitrate reductase ac- ling extracts, whereas bovine serum albumin caused a slight tivity in extracts from older tissue, but several possibilities are reduction in decay rate of nitrate reductase activity in extracts being examined. An earlier report (3) suggested that a grind- from older leaf blades. ing medium containing 0.1% (w/v) bovine serum albumin de- It should be noted that Oh43 has been classified previously creased binding of nitrate reductase to broken plastids and or- as low in nitrate reductase activity (7, 8). However, when bo- ganelles from tobacco leaves. Addition of these proteins may vine serum albumin or casein was added to the extraction me- prevent an inhibitor from binding to nitrate reductase (and dium, high levels of nitrate reductase were extracted (Table thereby inactivating the enzyme), or a higher concentration of II). These high values compare favorably with the highest val- protein in the extract may prevent dissociation of the nitrate ues obtained previously for any genotypes (7, 8). In studying reductase molecule. the effect of bovine serum albumin on nitrate reductase extracted from 26 genotypes of corn at several ages, the level of Acknowledgments-The technical assistance of D. E. Elskamp and R. D. activity was increased in all genotypes at every sampling (un- Vogelzang is gratefully acknowledged. published data), but genotypes with unstable activity showed the greatest increase. A 3-fold difference in nitrate reductase LITERATURE CITED activity was observed among the genotypes when 3% (w/v) 1. BEEVERS, L., L. E. SCHRADER, D. FLESHER, AND R. H. HAGETMAN. 1965. The bovine serum albumin was added to the extraction medium as role of light and nitrate in the induction of nitrate reductase in radish compared to a 10-fold difference among genotypes extracted cotyledons and maize seedlings. Plant Physiol. 40: 691-698. in the absence of bovine serum albumin. Thus, genotypic dif- 2. CROY, L. I. AND R. H. HAGEMAN. 1970. Relationship of nitrate reductase activity to grain protein production in wheat. Crop Sci. 10: 280-285. ferences still existed, but nitrate reductase in genotypes with 3. DALLING, M. J., -N. E. TOLBERT, AND R. H. HAGEMAN. 1972. Intracellular unstable activity has been underestimated in the past when con- location of nitrate reductase and nitrite reductase. I. Spinach and tobacco ventional extraction procedures (6) have been employed. leaves. Biochim. Biopliys. Acta 283: 502-512. Nitrate reductase extracted from a tobacco leaf in the ab- 4. DECKARD, E. L., R. J. LAMBERT, AND R. H. HAGEMAN-. 1973. Nitrate reductase activity in corn leaves as related to yields of grain and grain protein. Crop sence of bovine serum albumin or casein also was unstable with Sci. 13: 343-350. a half-life of 115 min (unpublished data). Nitrate reductase 5. EILRICH, G. L. AND R. H. HAGEMNAN. 1973. Nitrate reductase activity and its activity was increased about 10-fold by the addition of 3% relationship to accumulation of vegetative and grain nitrogen in wN-heat (w/v) bovine serum albumin or casein in the medium for ex- (Triticum vulgare L.). Crop Sci. 13: 59-66. 6. HAGENIAN, R. H. AND D. P. HuCKLESBY. 1971. Nitr-ate reductase from higher traction (Table III). Stability of nitrate reductase also was im- plants. Methods Enzymol. 23: 491-503. proved by the addition of either protein. 7. HAGEMAN, R. H., J. F. ZIESERL, JR., AN-D E. R. LENG. 1963. Levels of nitr-ate It seems likely that increased stabilization of nitrate reduc- reductase activity in inbred lines and Fl hybrids in maize. Nature 197: tase in the presence of bovine serum albumin or casein resulted 263-265. 8. SCHRADER, L. E., D. M. PETERSON, E. R. LENG, AND R. H. HAGEMAN. 1966. from protecting nitrate reductase from an inactivating or pro- Nitrate reductase activity of maize hybrids and their parental inbreds. teolytic enzyme, although some other mechanism(s) for pro- Crop Sci. 6: 169-173. tection by the proteins may be functioning. Levels of proteo- 9. WARNER, R. L., R. H. HAGENIAN, J. W. DUDLEY, AND R. J. LAMBERT. 1969. lytic activity in extracts from stable and unstable genotypes of Inheritance of nitrate reductase activity in Zea mays L. Proc. Nat. Acad. Sci. U.S.A. 62: 785-792. corn are being investigated, and we are attempting to isolate 10. ZIESERL, J. F., W. L. RIVENBARK, AND R. H. HAGEMAN. 1963. Nitrate an inactivating enzyme from these extracts. reductase activity, protein content, and yield of four maize hybrids at It is not clear why the presence of bovine serum albumin or varying plant populations. Crop Sci. 3: 27-32.