Planta (Berl.) 93, 160--170 (1970) 9 by Springer-Verlag 1970
Studies on the Sub-cellular Location of Particulate Nitrate and Nitrite Reductase, Glutamic Dehydrogenase and other Enzymes in Barley Roots B. J. M~L13r Department of Plant Science, The University, Newcastle upon Tyne Received may 11, June 1, 1970
Summary. The distribution of nitrate and nitrite rcductase, glutamic dehydrogcnasc, cytochrome oxidase, fumarase, peroxidas3 and catalasc in particular fractions of barley roots, separated by differential and density gradient centrifugation, has been determined. Evidence obtained suggests that there are three separate groups of particles, one, the mitochondria, containing cytochrome oxidase, fumarase and glutamic dehydrogenase, one containing catalase, and one containing nitrate and nitrite reductase. The results show that, under certain conditions, the high osmotic pressures obtained in sucrose density gradients may cause artefacts due to the release of enzymes, especially nitrite reductase, from the particles. Introduction
A proportion of the nitrate and nitrite reducing enzymes of barley roots is associated with a particulate fraction (Miflin, 1967). This association does not appear to be the result of adsorption during the isolation of the particles (Miflin, 1968a), or contamination b y bacteria (Miflin, 1970). On the basis of differential centrifugation, this fraction was originally termed the mitochondria. However, there is much evidence to suggest t h a t this fraction is a n y t h i n g but pure. Briedenbach and Beevers (1967) showed t h a t the enzymes of the glyoxylate cycle in the castor bean endosperm, previously believed to be associated with the mitochondria were, in fact, located in particles more dense t h a n mitochondria, termed the glyoxysomes. Preliminary experiments showed t h a t barley root particulate nitrate and nitrate reductase were also not associated with the main b a n d of mitochondria separated b y density gradient centrifugation (Miflin, 1968 b). This paper reports further studies of the distribution of particulate enzymes from root tissue, especially of nitrate and nitrite reductase.
Location of Nitrate and Nitrite Reductase
161
Methods Barley (oar Proctor) was grown by a modification (Miflin, 1970) of the method Epstein a n d Hagen (1952) over a nitrate free n u t r i e n t solution. The enzymes were induced by adding nitrate to 0.01 M 20--44 hours before harvesting the roots. The n u t r i e n t solutions were aerated and changed daily. The roots were 4 - - 6 days old a t harvest. They were ground with a chilled pestle a n d mortar in ice cold isolation medium (0.2 M sucrose, 0.3 5I mannitol, 0.05 3I phosphate (pH 7.8), 0.005 M EDTA, 1% PVP, 0.005 M 31gC1~, 0.001 31 glutathione) without sand. The homogenate was then filtered through 4 layers of nylon gauze (300/400 mesh) and centrifuged at 500 • g for 5 minutes and the pellet discarded. The supernatant was then centrifuged a t 18,000 • g for 10 minutes. In some experiments the preliminary spin was omitted. The 18,000 • g pellet was resuspended in 0.4 M sucrose, 0.001 31 phosphate p H 7.5 0.001 M MgCle and layered on top of the density gradients. The original composition of the gradients is given in the figures relating to the individual experiments. All gradients stood at least 1 hr before use. The gradients were formed in 60 ml Spinco cellulose acetate tubes and centrifuged in a SW 25 head in an L2-50 Spinco (Beckm a n Instruments Ltd., Glenrothes, Scotland) centrifuge at 2 ~ C. At the end of the r u n the b o t t o m of the tubes were pierced and fractions collected by drop counting, or b y volume. All assays were done as soon as possible, nitrate and nitrite reductase were always done on the same day, b u t occasionally it was necessary to determine cytochrome oxidase and glutamic dehydrogenase activity the following day.
Enzyme Assays Nitrate and nitrite reductase were determined as described previously (Miflin, 1967, 1970). Succinate was used as the electron donor for nitrate reductase and the assay carried out under vacuum. Glutamic dehydrogenase (GDH) was assayed by determining the initial rate of decrease in absorption at 340 m~ in the presence of 50 ~ moles (NI-Ia)eSOa 0.6 ~ moles N A D H (or NADPH), 1.5 ~ moles KCN, 250 moles phosphate p i t 7.4 15 ~ moles c~-oxoglutarate and a suitable aliquot of particles made 1% with respect to Triton X-100. The final volume was 3 mls and the rate in the absence of a-oxoglutarate was used as the control. Fumarase was assayed by the method of Racker (1950). Peroxidase was measured b y a modification of the method of Gregory (1966) in which the H202 concentration was decreased to 0.02 3I. This resulted in a 5-fold increase in activity. Catalase was assayed by following the decrease in OD at 240 m ~ after Beers and Sizer (1952). Cytochrome oxidase was determined from the initial rate of decrease in OD at 550 m~ in the presence of 1% Triton X-100. An abitrary unit of activity for the various enzymes is defined as follows in order to allow the results to be presented on the same graphical scale: - cytochrome oxidase 20, nitrite reductase 1, nitrate reductase 0.1, glutamic dehydrogenase 10, Fumarase 10, catalase 2,000 and peroxidase 4,000, all in n moles/ min/ml of gradient.
Results Initial experiments, using a discontinuous multistep sucrose gradient, suggested that nitrite reductase was located in a band around a density e q u i v a l e n t t o 1.8 M s u c r o s e (Fig. 1). T h e m a i n b a n d of m i t o c h o n d r i a l a c t i v i t y as m a r k e d b y c y t o c h r o m e o x i d a s e , g l u t a m i c d e h y d r o g e n a s e a n d f u m a r a s e w a s l o c a t e d a t a d e n s i t y a p p r o x i m a t e l y e q u i v a l e n t t o 1.5 M
162
B.J. Miflin: ~
2"0
w
1"6 1.4 b2
o
~
L__
I'0 20 16
5~
12
ag ~)
>--
E~
8
N55
p I 2 3 4 5 6 7 8 9 I0 II 12 13 fraction number
Fig. 1. Distribution of particulate enzymes on a multistep discontinuous gradient. The gradient was centrifuged for 4 hr at 23,000 r.p.m, fraetionated and nitrite reductase ( e - - o ) , nitrate reductase (A--A), glutamie dehydrogenase ( A - - A ) and cytochrome oxidase (~7--v) assayed. The pellet (p) was resuspended in 3 mls of resuspension buffer and also assayed. The original sucrose concentration of the gradient in relation to the fraction number is given
sucrose, with a denser s u b - b a n d . All of the above activities also occurred i n the pellet. N i t r a t e reductase was f o u n d only i n the pellet. Differential centrifugation, i n which the h o m o g e n a t e was centrifuged a t successively increasing g / m i n values was used to give a series of pellets which were t h e n assayed. The results i n d i c a t e d t h a t the n i t r a t e a n d n i t r i t e reducing enzymes, which co-sediment a t lower g values t h a n g l u t a m i c dehydrogenase a n d fumarase, reside i n a particle separate from the m i t o c h o n d r i a (Table 1). T h e y differ from those shown i n Fig. 1 i n t h a t , Table 1. Distribution of various barley root enzymes in the particulate fractions obtained by differential centri/ugation. Distribution in each/raction given in terms of percent of total particulate activity. Assays as described in the text Maximum centrifugal force (g)
Time (rain)
Nitrite reductase
Nitrate reductase
NADKGDH
Fumarase
Catalase
1,500 4,700 8,400 21,500
5 7 7 15
25 46 21 8
25 51 18 6
9 40 36 15
19 28 27 26
3 14 31 52
Location of Nitrate aad Nitrite Reductase
163
under differential eentrifugation, nitrite and nitrate reductase behave in exactly the same manner, suggesting that the two enzymes reside in the same particle. The latter conclusion appears more likely on teleological grounds. I t seems advantageous to the plant to have the two enzymes located in the same place and to reduce NOs through to NI-Ia without the need to release and transfer nitrite. This viewpoint has recently been supported by experimental evidence showing that nitrate can be reduced through to ammonia b y the particles (Bourne and Miflin, 1970). Results from the sucrose density studies (Fig. 1) also seemed peculiar in t h a t the nitrite reductase activity in the gradient was not associated with any particle band and only with a small protein peak. The location of nitrate and nitrite reductase activities in the pellet does not allow any conclusions to be made because material can arrive at the bottom of the gradient either by passing through the gradient or around it (Anderson, 1955). Those activities in the pellet that are also represented in the gradient above could have reached the pellet by the particles impacting on the side of the tube and passing down outside the gradient. However, since nitrate reduetase was not found in the gradient, it is most likely that it is present in very dense particles that have sedimented through the gradient. To a t t e m p t to overcome these difficulties a variety of techniques have been tried. Continuous sucrose gradients up to 2.5 M sucrose were used, but the results did not differ materially from Fig. 1. Self-generating silica sol gradients (Pertoft, 1966) were also tried but, although giving very good density ranges, the particulate fraction coagulated into clumps within the gradient and did not separate into discrete bands. Ficoll, when used in a continuous gradient (18 to 36% (w/w) in 0.4 M sucrose), layered over 2.0 M sucrose, allowed a certain amount of separation, but further resolution using higher concentrations of Ficoll was not practical due to its high viscosity. Ficoll gradients also did not give consistent results (cf. Beaufay etal., 1964). However, with the Fieoll/sucrose gradients a proportion of the nitrate reductase activity stayed on top of the 2.0 M sucrose whereas, with all previous sucrose gradients (both continuous and discontinuous) it went into the pellet. This suggests that the particles are shrunk by passing through the sucrose, which has a higher osmotic pressure, and their density increased. The effect of the high osmotic pressure in sucrose gradients might also cause the release of enzymes as well as the contraction of the particles, explaining the presence of a nitrite reductase band in Fig. 1 in the absence of visible particles. To test this a particulate fraction was prepared b y differential centrifugation and then resuspended in sucrose concentrations of different molarity. After standing for 30 min all the suspensions were made to
165
B. J. YIiflin: 50 40 3O
b I0
~.
0
I
'-'~'/I
2 3 4 5 6 7 8 q I0 II 12 13 14 15 Ib
~ 2s
I
fraction number
E
~5
I
2
3
5
6
8
7
fraction number
N
!
visible particle bandinq
I
2,7
I-5
1
sucrose c o n c ,
I
I0
1.0(N )
I
20
N I
homoqenate
I
30
qradient volume (ml)
Fig. 2. Distribution of particulate enzymes on a three step sucrose gradient. The gradient was centrifuged for 30 min at 23,000 r.p.m, fractionated and nitrite reductase ( e - - e ) , nitrate reductase (a--A), fumarase ( o - - - o ) , glutamic dehydrogenase ( A - - A) and peroxidase ( I - - I ) assayed
0.4 M a n d t h e n a d j u s t e d to equal v o l u m e with 0.4 M sucrose. The particles were centrifuged off a n d resnspended, a n d t h e y a n d t h e supern a t a n t , assayed. I t was f o u n d (Table 2) t h a t a t sucrose c o n c e n t r a t i o n s a b o v e 1.4 M t h e r e is considerable increase in t h e release of n i t r i t e r e d u c t ase a n d a t 2.0 M 60% of t h e a c t i v i t y is lost from t h e particles. The results strongly support the contention that the nitrite reductase band around 1.8 M sucrose in Fig. 1 is due to t h e release of t h e e n z y m e from t h e particles due to osmotic shock as t h e y pass down t h e gradient. Since t h e m i t o c h o n d r i a l fraction a p p e a r s to b a n d a t densities less t h a n 1.7 M a n d n i t r i t e r e d u c t a s e release is r e l a t i v e l y low a t c o n c e n t r a t i o n s less t h a n 1.5 M, two large step, discrete gradients, b a s e d on these observations, were c o n s t r u c t e d a n d t h e particles b a n d e d according to r a t e
Location of Nitrate and Nitrite Reductase
165
50
2 40
._>
20 >-. r
0
-~ I ~ I 2
1 v--4"-"v~ I 4 5 6
3
N
I
I 7
fraction number
9
m
visible particle banding
I
I
2-7
I
Iq
1.5
I'0
sucrose cone. (M)
I 3
I 8
I
I
I
6
9
12
I [5
I
18
gradient volume [ml)
I 21
I 24
27
Fig. 3. Distribution of particulate enzymes on a four step sucrose gradient. The gradient was centrifuged for 30 rain at 23,000 r.p.m, fractionated and nitrite reductase ( e - - e ) , catalase (9169 and eytoehrome oxidase ( v - - v ) assayed Table 2. Osmotic release o] nitrite reductase /tom particles. Particles isolated by differential centri]ugation and washed once in resuspending bu/]er and treated as described in the text. The percent o/ the total recovered activity [ound in the supernatant alter treatment is termed as the percent released. Virtually 100% recovery o/ the initial activity was obtained with all treatments Sucrose cone.
% release of nitrite reductase
0.7 M 1.4 M 1.7 M 2.0 M
27 34 47 60
zonal s e p a r a t i o n (30 rain) r a t h e r t h a n equilibrium d e n s i t y g r a d i e n t eentrifugation. The g r a d i e n t s a n d the results o b t a i n e d from t h e m are shown in Figs. 2 a n d 3. A f u r t h e r modification used in these e x p e r i m e n t s was the omission of the p r e l i m i n a r y s e p a r a t i o n of t h e particles b y differential eentrifugation. A n y damage, clumping or o t h e r a r t e f a c t s caused b y such a e e n t r i f u g a t i o n should t h u s be eliminated. A f t e r grinding, t h e h o m o g e n a t e was filtered t h r o u g h n y l o n mesh a n d l a y e r e d over t h e gradients.
166
B.J. Miflin:
Using this technique 2 major bands and a very small pellet were obtained. A further band at the bottom of the original homogenate was also observed and presumably consisted of small membrane fragments. I n Fig. 2 all of the nitrate reductase activity and most of the nitrite reductase were located in a band resting on the 2.7 M sucrose. Some nitrite reductase remained in the 1.5 M sucrose slightly below the peak of fumarase and N A D H glutamic dehydrogenase activity. Peroxidase activity was almost entirely located in the supernatant fraction of the homogenate. Fumarase and g]utamic dehydrogenase have a small peak of activity corresponding to the nitrite and nitrate reduetase. However, these activities are completely separated from the main nitrite rednctase band by the inclusion of a 1.7 M sucrose band in the gradient (Fig. 3). This band increases the release of nitrite reduetase from the particles. Table 3. Distribution o] pea root nitrite reductase and /umarase on a discontinuous density gradient. Pea roots were homogenized as described in the method section /or barley. The homogenate was ]iltered and layered over a discontinuous gradient of 8 ml 2 . 7 M , 8 ml 1 . 7 M , 6 ml 1 . 5 M and 8 ml 1 . 0 M sucrose. The gradient was centrifuged at 23,000 r.p.m. /or 30 minutes Fraction number
Sucrose cone. (M)
Enzyme activity (units) Nitrite Reductase
Fumarase
2 3 4 5 6 7 8
].7 1.7 1.5 1.5 1.0 1.0 1.0
89 59 40 26 29 27 75
6.0 4.4 25.5 27.4 13.3 10.9 6.1
In this experiment it can be seen that catalase co-sediments with cytochrome oxidase, but there is no evidence of any activity associated with nitrite and nitrate reductase activity. I t is not suggested from these results that cata]ase is a mitochondrial enzyme since several observations exist to show that it is not (Plesniear e t a l . , 1967; Breidenbach e t a l . , ]968). Particularly the results in Table 1 indicate that catalase does not co-sediment with the mitochondrial enzymes under differential centrifugation. I t is probable that catalase containing particles and mitochondria from barley are both too close in density to be resolved on this gradient. Besides the enzymes shown in figures 4 and 5 N A D P H glutamie dehydrogenase has been assayed and found to be correlated with cytochrome oxidase activity.
Location of Nitrate and Nitrite Reductase
167
J o y (personal communication) has also found that particulate nitrite reduetase from pea roots can be separated by differential centrifugation from N A D H glutamie dehydrogenase. The behaviour of pea root nitrite reductase on sucrose density gradients has also been investigated and the results are shown in Table 3. Again there is a clean separation between nitrite reductase and fumarase. The pea particles do not seem to be so sensitive to osmotic pressure as there is no release of nitrite reduetase at the 1.5--1.7 M sucrose interface.
Discussion The separation techniques used in this study suggest the presence of at least two, and probably three, types of particle in pea and barley roots. The first are the mitochondria which have been identified on the basis that they contain cytochrome oxidase and fumarase. This identification has been confirmed by observation of their structure under the electron microscope. The second group are those particles containing nitrite and nitrate reductase activity. The nature and the origin of these particles is not positively known. Preliminary electron microscopic evidence shows that the fraction is rich in membranous vesicles. These vesicles may exist in vivo, or could result from cell membranes, such as the endoplasmie reticulum rolling up into spheres upon rupture of the cell. Should the isolated particles represent in vivo particles they could be nitrosomes. The term nitrosome might cause some confusion since it has already been used in a slightly different context by Sims et al. (1968) to refer to the physical association of the two enzymes in yeast which are coordinately induced and repressed. In yeast there is no evidence that the enzymes are in a membrane bound particle sedimenting in a centrifuge. In barley, although both of the enzymes are inducible, the term nitrosome is not meant to imply that they are coordinately controlled. I n fact, the evidence suggests that the two enzymes are not so controlled in higher plants (Ingle etal., 1966; Schrader, 1969) and in barley particulate nitrite and nitrate reductase are differentially affected by oxygen tension (Miflin, 1970). The one point of similarity between the yeast and barley nitrosome is that in both eases it is envisaged that the two enzymes are in a close physical association forming a functionally interactive system catalysing the reduction of nitrate to ammonia. Another possibility is that since nitrite reductase is associated with chloroplasts in the leaves (l~itenour et al., 1967) the activities could be associated with proplastids in the root. This has not been eliminated, partly because of the difficulty of finding a known marker enzyme for the proplastids, however, Breiden-
168
B.J. Miflin:
baeh et al. (1968) report the density of castor bean endosperm proplastids as less than glyoxysomes (eatalase containing bodies) and, if barley root proplastids have similar density, they would not be found at the 1.7, 2.7 M sucrose interface. Further researeh is needed to identify the particles definitely. The next enzyme in the nitrogen ehain-glutamie dehydrogenase is not in the "nitrosome", but in the mitochondria. Although this has long been postulated on the evidence of differential eentrifugation, this is not in itself sufficient and the present results with density gradient eentrifugation provide the necessary confirmation t h a t this is so. I t has been speculated t h a t N A D H and NADPI-I dependent glutamie dehydrogenases m a y act in different directions in vivo, however, our results suggest t h a t they have the same distribution in the cell. The third group of particles postulated are those containing eata.lase. As suggested previously, it is considered on the basis of Table 1 and Fig. 3 t h a t barley root tissue contain eatalase containing particles (peroxisomes) separate from, but of a similar density, to mitoehondria. Breidenbaeh et al. (1968) mention t h a t maize roots have eatalase containing particles intermediate in density between the catalase containing glyoxysomes of the castor bean endosperm and mitoehondria. Peroxidase has been shown to be a particulate enzyme on the basis of histochemieal evidence, but only a small proportion of it can be sedimented in higher plant preparations (Plesnicar et al., 1967). In barley only a small minority of the activity is particle bound and it is distributed in a similar manner to eatalase in the large step density gradients. There is no evidence of any association of peroxidase with the nitrosomes. The extent of the particulate bound enzymes in the overall nitrogen economy of the cell is not known. I t nmst be re-emphasized that the majority of both the nitrate and nitrite reduetase is found in the supernatant fraction after homogenisation and eentrifugation. The relationship of the nitrosomal enzymes to the rest of the nitrate and nitrite reductase in the cell is not absolutely elear. One possibility is that the particle is an artefact caused by accidental enclosure of cytoplasmic enzymes in membranous vesicles formed upon rupturing the eelI. This is unlikely since the particulate nitrate reduetase can use sneeinate as an electron donor and the supernatant one cannot. Further, nltrasonieation and repeated freezing and thawing are not able to completely release nitrate reduetase from the particle (Miflin, 1970). A converse hypothesis is that the supernatant enzymes originate from the particles due to damage during extraction. A parallel for this is the ease of peroxidase which is considered in vivo to be located in the peroxisomes and yet, upon fraetionation, is found largely in the particle-free supernatant. Although
Location of Nitrate and Nitrite Reductase
169
it is possible t h a t a proportion of the n i t r i t e a n d n i t r a t e reductase i n the s u p e r n a t a n t m a y be derived in this way, there is evidence to suggest t h a t a dissimilatory n i t r a t e reductase is present i n the particles t h a t is n o t in the s u p e r n a t a n t (Miflin, 1970). A further difficulty i n considering the roles of these enzymes in vivo is the evidence t h a t in the presence of a high level of nitrate, cells m a y over-produce n i t r a t e reductase a n d the actual in vivo reduction rate m a y be considerably less t h a n t h a t possible b y the total a m o u n t of enzymes extracted (Ferrari a n d Varner, 1969). This n o n - f u n c t i o n a l n i t r a t e reductase m a y n o t be equally dist r i b u t e d between the particulate a n d soluble phases. The author is pleased to acknowledge helpful discussions with Dr. K. Joy and the financial assistance of th~ A.R.C. in partial support of this work. References Anderson, N. G. : Studies on isolated cell components. Exp. Cell l~es. 9, 446--459 (1955). Beaufay, It., Jacques, P., Badhuin, P., Sellinger, 0. Z., Bertheret, J., Duve, C. de: Resolution of mitochondrial fraction of rat liver into three distinct populations of cytoplasmic particles by means of density gradient equilibration in various gradients. Bioehem. J. 92, 1 8 4 ~ 0 5 (1964). Beers, R. F., Jr., Sizer, I. W.: A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. biol. Chem. 195, 133 (1952). Bourne, W. F., Miflin, B. J. : ~anuscript in preparation (1970). Breidenbach, t~. W., Beevers, H. : Association of the glyoxylate cycle enzymes in a novel subeellular particle from castor bean endosperm. Biochem. biophys. Res. Commun. 27, 462--469 (1967). - - K a h n , A., Beevers, H.: Characterization of glyoxysomes from castor bean endosperm. Plant Physiol. 43, 705--713 (1968). Epstein, E., Hagen, C. E. : A kinetic study of the absorption of alkali cations by barley roots. Plant Physiol. 27, 657---474 (1952). Ferrari, T. E., Varner, J. E. : i n vivo measurement of nitrate reductase activity. Plant Physiol. 41, Abs. No 176 (1969). Gregory, tl. P. F. : A rapid assay for peroxidase activity. Biochem. J. 101, 582--584 (1966). Ingle, J., Joy, K. W., Hageman, R. H. : The regulation of activity of the enzymes involved in the assimilation of nitrate by higher plants. Biochem. J. 190, 577--588 (1966). Miflin, ]3. J. : Distribution of nitrate and nitrite reductase in barley. Nature (Lond.) 214, 1133 (1967). - - Nitrate reducing enzymes in barley. In: Recent aspects of nitrogen metabolism in plants (ed. Hewitt, E. J. and Cutting, C. V.), p. 85. London: Academic Press 1968. - - Separation of barley root mitochondria into different fractions by density gradient centrifugation ]3ioehem. J. 108, 49 (1968b). - - Nitrite and nitrate reductase systems in barley roots. P~ev. roum. Biochim. 7, 53--60 (1970). Pertoft, H. : Gradient eentrifugation in a colloidal silica polysaccharide medium. ]3iochim. biophys. Acta (Amst.) 126, 594--596 (1960). 12 ])lanta(Bcrl.), Bd. 93
170
B. J. Miflin: Location of Nitrate and Nitrite Reductase
Plesnicar, M., Bonner, W.D., Storey, B. T. : Peroxidase associated with higher plant mitochondria. Plant Physiol. 42, 366--370 (1967). Racker, E.: Spectrophotometric measurements of the enzymic formation of fumaric and eis-aconitic acids. Bioehim. biophys. Aeta (Amst.) 4, 211--214 (1950). Ritenour, G. L., Joy, K. W., Burming, J., Hageman, R. H. : Intracellularlocalization of nitrate reductase, nitrite reductase and glutamic dehydrogenase in green leaf tissue. Plant Physiol. 42, 233--237 (1967). Schrader, L. E. : Induction of nitrate and nitrite reductases in corn seedlings. Plant Physiol. 44, Abs. No 175 (1969). Sims, A. P., Folkes, B. •., Bussey, A. H. : Mechanisms involved in the regulation of nitrogen assimilation in micro-organisms and plants. In: Recent aspects of nitrogen metabolism in plants (ed. Hewitt, E. J. and Cutting, C. V.), p. 91. London: Academic Press 1968. B. J. ~iflin Department of Plant Science The University Newcastle upon Tyne NE 1 7 RU, U.K.
Studies on the Sub-cellular Location of Particulate Nitrate and Nitrite Reductase, Glutamic Dehydrogenase and other Enzymes in Barley Roots B. J. M~L13r Department of Plant Science, The University, Newcastle upon Tyne Received may 11, June 1, 1970
Summary. The distribution of nitrate and nitrite rcductase, glutamic dehydrogcnasc, cytochrome oxidase, fumarase, peroxidas3 and catalasc in particular fractions of barley roots, separated by differential and density gradient centrifugation, has been determined. Evidence obtained suggests that there are three separate groups of particles, one, the mitochondria, containing cytochrome oxidase, fumarase and glutamic dehydrogenase, one containing catalase, and one containing nitrate and nitrite reductase. The results show that, under certain conditions, the high osmotic pressures obtained in sucrose density gradients may cause artefacts due to the release of enzymes, especially nitrite reductase, from the particles. Introduction
A proportion of the nitrate and nitrite reducing enzymes of barley roots is associated with a particulate fraction (Miflin, 1967). This association does not appear to be the result of adsorption during the isolation of the particles (Miflin, 1968a), or contamination b y bacteria (Miflin, 1970). On the basis of differential centrifugation, this fraction was originally termed the mitochondria. However, there is much evidence to suggest t h a t this fraction is a n y t h i n g but pure. Briedenbach and Beevers (1967) showed t h a t the enzymes of the glyoxylate cycle in the castor bean endosperm, previously believed to be associated with the mitochondria were, in fact, located in particles more dense t h a n mitochondria, termed the glyoxysomes. Preliminary experiments showed t h a t barley root particulate nitrate and nitrate reductase were also not associated with the main b a n d of mitochondria separated b y density gradient centrifugation (Miflin, 1968 b). This paper reports further studies of the distribution of particulate enzymes from root tissue, especially of nitrate and nitrite reductase.
Location of Nitrate and Nitrite Reductase
161
Methods Barley (oar Proctor) was grown by a modification (Miflin, 1970) of the method Epstein a n d Hagen (1952) over a nitrate free n u t r i e n t solution. The enzymes were induced by adding nitrate to 0.01 M 20--44 hours before harvesting the roots. The n u t r i e n t solutions were aerated and changed daily. The roots were 4 - - 6 days old a t harvest. They were ground with a chilled pestle a n d mortar in ice cold isolation medium (0.2 M sucrose, 0.3 5I mannitol, 0.05 3I phosphate (pH 7.8), 0.005 M EDTA, 1% PVP, 0.005 M 31gC1~, 0.001 31 glutathione) without sand. The homogenate was then filtered through 4 layers of nylon gauze (300/400 mesh) and centrifuged at 500 • g for 5 minutes and the pellet discarded. The supernatant was then centrifuged a t 18,000 • g for 10 minutes. In some experiments the preliminary spin was omitted. The 18,000 • g pellet was resuspended in 0.4 M sucrose, 0.001 31 phosphate p H 7.5 0.001 M MgCle and layered on top of the density gradients. The original composition of the gradients is given in the figures relating to the individual experiments. All gradients stood at least 1 hr before use. The gradients were formed in 60 ml Spinco cellulose acetate tubes and centrifuged in a SW 25 head in an L2-50 Spinco (Beckm a n Instruments Ltd., Glenrothes, Scotland) centrifuge at 2 ~ C. At the end of the r u n the b o t t o m of the tubes were pierced and fractions collected by drop counting, or b y volume. All assays were done as soon as possible, nitrate and nitrite reductase were always done on the same day, b u t occasionally it was necessary to determine cytochrome oxidase and glutamic dehydrogenase activity the following day.
Enzyme Assays Nitrate and nitrite reductase were determined as described previously (Miflin, 1967, 1970). Succinate was used as the electron donor for nitrate reductase and the assay carried out under vacuum. Glutamic dehydrogenase (GDH) was assayed by determining the initial rate of decrease in absorption at 340 m~ in the presence of 50 ~ moles (NI-Ia)eSOa 0.6 ~ moles N A D H (or NADPH), 1.5 ~ moles KCN, 250 moles phosphate p i t 7.4 15 ~ moles c~-oxoglutarate and a suitable aliquot of particles made 1% with respect to Triton X-100. The final volume was 3 mls and the rate in the absence of a-oxoglutarate was used as the control. Fumarase was assayed by the method of Racker (1950). Peroxidase was measured b y a modification of the method of Gregory (1966) in which the H202 concentration was decreased to 0.02 3I. This resulted in a 5-fold increase in activity. Catalase was assayed by following the decrease in OD at 240 m ~ after Beers and Sizer (1952). Cytochrome oxidase was determined from the initial rate of decrease in OD at 550 m~ in the presence of 1% Triton X-100. An abitrary unit of activity for the various enzymes is defined as follows in order to allow the results to be presented on the same graphical scale: - cytochrome oxidase 20, nitrite reductase 1, nitrate reductase 0.1, glutamic dehydrogenase 10, Fumarase 10, catalase 2,000 and peroxidase 4,000, all in n moles/ min/ml of gradient.
Results Initial experiments, using a discontinuous multistep sucrose gradient, suggested that nitrite reductase was located in a band around a density e q u i v a l e n t t o 1.8 M s u c r o s e (Fig. 1). T h e m a i n b a n d of m i t o c h o n d r i a l a c t i v i t y as m a r k e d b y c y t o c h r o m e o x i d a s e , g l u t a m i c d e h y d r o g e n a s e a n d f u m a r a s e w a s l o c a t e d a t a d e n s i t y a p p r o x i m a t e l y e q u i v a l e n t t o 1.5 M
162
B.J. Miflin: ~
2"0
w
1"6 1.4 b2
o
~
L__
I'0 20 16
5~
12
ag ~)
>--
E~
8
N55
p I 2 3 4 5 6 7 8 9 I0 II 12 13 fraction number
Fig. 1. Distribution of particulate enzymes on a multistep discontinuous gradient. The gradient was centrifuged for 4 hr at 23,000 r.p.m, fraetionated and nitrite reductase ( e - - o ) , nitrate reductase (A--A), glutamie dehydrogenase ( A - - A ) and cytochrome oxidase (~7--v) assayed. The pellet (p) was resuspended in 3 mls of resuspension buffer and also assayed. The original sucrose concentration of the gradient in relation to the fraction number is given
sucrose, with a denser s u b - b a n d . All of the above activities also occurred i n the pellet. N i t r a t e reductase was f o u n d only i n the pellet. Differential centrifugation, i n which the h o m o g e n a t e was centrifuged a t successively increasing g / m i n values was used to give a series of pellets which were t h e n assayed. The results i n d i c a t e d t h a t the n i t r a t e a n d n i t r i t e reducing enzymes, which co-sediment a t lower g values t h a n g l u t a m i c dehydrogenase a n d fumarase, reside i n a particle separate from the m i t o c h o n d r i a (Table 1). T h e y differ from those shown i n Fig. 1 i n t h a t , Table 1. Distribution of various barley root enzymes in the particulate fractions obtained by differential centri/ugation. Distribution in each/raction given in terms of percent of total particulate activity. Assays as described in the text Maximum centrifugal force (g)
Time (rain)
Nitrite reductase
Nitrate reductase
NADKGDH
Fumarase
Catalase
1,500 4,700 8,400 21,500
5 7 7 15
25 46 21 8
25 51 18 6
9 40 36 15
19 28 27 26
3 14 31 52
Location of Nitrate aad Nitrite Reductase
163
under differential eentrifugation, nitrite and nitrate reductase behave in exactly the same manner, suggesting that the two enzymes reside in the same particle. The latter conclusion appears more likely on teleological grounds. I t seems advantageous to the plant to have the two enzymes located in the same place and to reduce NOs through to NI-Ia without the need to release and transfer nitrite. This viewpoint has recently been supported by experimental evidence showing that nitrate can be reduced through to ammonia b y the particles (Bourne and Miflin, 1970). Results from the sucrose density studies (Fig. 1) also seemed peculiar in t h a t the nitrite reductase activity in the gradient was not associated with any particle band and only with a small protein peak. The location of nitrate and nitrite reductase activities in the pellet does not allow any conclusions to be made because material can arrive at the bottom of the gradient either by passing through the gradient or around it (Anderson, 1955). Those activities in the pellet that are also represented in the gradient above could have reached the pellet by the particles impacting on the side of the tube and passing down outside the gradient. However, since nitrate reduetase was not found in the gradient, it is most likely that it is present in very dense particles that have sedimented through the gradient. To a t t e m p t to overcome these difficulties a variety of techniques have been tried. Continuous sucrose gradients up to 2.5 M sucrose were used, but the results did not differ materially from Fig. 1. Self-generating silica sol gradients (Pertoft, 1966) were also tried but, although giving very good density ranges, the particulate fraction coagulated into clumps within the gradient and did not separate into discrete bands. Ficoll, when used in a continuous gradient (18 to 36% (w/w) in 0.4 M sucrose), layered over 2.0 M sucrose, allowed a certain amount of separation, but further resolution using higher concentrations of Ficoll was not practical due to its high viscosity. Ficoll gradients also did not give consistent results (cf. Beaufay etal., 1964). However, with the Fieoll/sucrose gradients a proportion of the nitrate reductase activity stayed on top of the 2.0 M sucrose whereas, with all previous sucrose gradients (both continuous and discontinuous) it went into the pellet. This suggests that the particles are shrunk by passing through the sucrose, which has a higher osmotic pressure, and their density increased. The effect of the high osmotic pressure in sucrose gradients might also cause the release of enzymes as well as the contraction of the particles, explaining the presence of a nitrite reductase band in Fig. 1 in the absence of visible particles. To test this a particulate fraction was prepared b y differential centrifugation and then resuspended in sucrose concentrations of different molarity. After standing for 30 min all the suspensions were made to
165
B. J. YIiflin: 50 40 3O
b I0
~.
0
I
'-'~'/I
2 3 4 5 6 7 8 q I0 II 12 13 14 15 Ib
~ 2s
I
fraction number
E
~5
I
2
3
5
6
8
7
fraction number
N
!
visible particle bandinq
I
2,7
I-5
1
sucrose c o n c ,
I
I0
1.0(N )
I
20
N I
homoqenate
I
30
qradient volume (ml)
Fig. 2. Distribution of particulate enzymes on a three step sucrose gradient. The gradient was centrifuged for 30 min at 23,000 r.p.m, fractionated and nitrite reductase ( e - - e ) , nitrate reductase (a--A), fumarase ( o - - - o ) , glutamic dehydrogenase ( A - - A) and peroxidase ( I - - I ) assayed
0.4 M a n d t h e n a d j u s t e d to equal v o l u m e with 0.4 M sucrose. The particles were centrifuged off a n d resnspended, a n d t h e y a n d t h e supern a t a n t , assayed. I t was f o u n d (Table 2) t h a t a t sucrose c o n c e n t r a t i o n s a b o v e 1.4 M t h e r e is considerable increase in t h e release of n i t r i t e r e d u c t ase a n d a t 2.0 M 60% of t h e a c t i v i t y is lost from t h e particles. The results strongly support the contention that the nitrite reductase band around 1.8 M sucrose in Fig. 1 is due to t h e release of t h e e n z y m e from t h e particles due to osmotic shock as t h e y pass down t h e gradient. Since t h e m i t o c h o n d r i a l fraction a p p e a r s to b a n d a t densities less t h a n 1.7 M a n d n i t r i t e r e d u c t a s e release is r e l a t i v e l y low a t c o n c e n t r a t i o n s less t h a n 1.5 M, two large step, discrete gradients, b a s e d on these observations, were c o n s t r u c t e d a n d t h e particles b a n d e d according to r a t e
Location of Nitrate and Nitrite Reductase
165
50
2 40
._>
20 >-. r
0
-~ I ~ I 2
1 v--4"-"v~ I 4 5 6
3
N
I
I 7
fraction number
9
m
visible particle banding
I
I
2-7
I
Iq
1.5
I'0
sucrose cone. (M)
I 3
I 8
I
I
I
6
9
12
I [5
I
18
gradient volume [ml)
I 21
I 24
27
Fig. 3. Distribution of particulate enzymes on a four step sucrose gradient. The gradient was centrifuged for 30 rain at 23,000 r.p.m, fractionated and nitrite reductase ( e - - e ) , catalase (9169 and eytoehrome oxidase ( v - - v ) assayed Table 2. Osmotic release o] nitrite reductase /tom particles. Particles isolated by differential centri]ugation and washed once in resuspending bu/]er and treated as described in the text. The percent o/ the total recovered activity [ound in the supernatant alter treatment is termed as the percent released. Virtually 100% recovery o/ the initial activity was obtained with all treatments Sucrose cone.
% release of nitrite reductase
0.7 M 1.4 M 1.7 M 2.0 M
27 34 47 60
zonal s e p a r a t i o n (30 rain) r a t h e r t h a n equilibrium d e n s i t y g r a d i e n t eentrifugation. The g r a d i e n t s a n d the results o b t a i n e d from t h e m are shown in Figs. 2 a n d 3. A f u r t h e r modification used in these e x p e r i m e n t s was the omission of the p r e l i m i n a r y s e p a r a t i o n of t h e particles b y differential eentrifugation. A n y damage, clumping or o t h e r a r t e f a c t s caused b y such a e e n t r i f u g a t i o n should t h u s be eliminated. A f t e r grinding, t h e h o m o g e n a t e was filtered t h r o u g h n y l o n mesh a n d l a y e r e d over t h e gradients.
166
B.J. Miflin:
Using this technique 2 major bands and a very small pellet were obtained. A further band at the bottom of the original homogenate was also observed and presumably consisted of small membrane fragments. I n Fig. 2 all of the nitrate reductase activity and most of the nitrite reductase were located in a band resting on the 2.7 M sucrose. Some nitrite reductase remained in the 1.5 M sucrose slightly below the peak of fumarase and N A D H glutamic dehydrogenase activity. Peroxidase activity was almost entirely located in the supernatant fraction of the homogenate. Fumarase and g]utamic dehydrogenase have a small peak of activity corresponding to the nitrite and nitrate reduetase. However, these activities are completely separated from the main nitrite rednctase band by the inclusion of a 1.7 M sucrose band in the gradient (Fig. 3). This band increases the release of nitrite reduetase from the particles. Table 3. Distribution o] pea root nitrite reductase and /umarase on a discontinuous density gradient. Pea roots were homogenized as described in the method section /or barley. The homogenate was ]iltered and layered over a discontinuous gradient of 8 ml 2 . 7 M , 8 ml 1 . 7 M , 6 ml 1 . 5 M and 8 ml 1 . 0 M sucrose. The gradient was centrifuged at 23,000 r.p.m. /or 30 minutes Fraction number
Sucrose cone. (M)
Enzyme activity (units) Nitrite Reductase
Fumarase
2 3 4 5 6 7 8
].7 1.7 1.5 1.5 1.0 1.0 1.0
89 59 40 26 29 27 75
6.0 4.4 25.5 27.4 13.3 10.9 6.1
In this experiment it can be seen that catalase co-sediments with cytochrome oxidase, but there is no evidence of any activity associated with nitrite and nitrate reductase activity. I t is not suggested from these results that cata]ase is a mitochondrial enzyme since several observations exist to show that it is not (Plesniear e t a l . , 1967; Breidenbach e t a l . , ]968). Particularly the results in Table 1 indicate that catalase does not co-sediment with the mitochondrial enzymes under differential centrifugation. I t is probable that catalase containing particles and mitochondria from barley are both too close in density to be resolved on this gradient. Besides the enzymes shown in figures 4 and 5 N A D P H glutamie dehydrogenase has been assayed and found to be correlated with cytochrome oxidase activity.
Location of Nitrate and Nitrite Reductase
167
J o y (personal communication) has also found that particulate nitrite reduetase from pea roots can be separated by differential centrifugation from N A D H glutamie dehydrogenase. The behaviour of pea root nitrite reductase on sucrose density gradients has also been investigated and the results are shown in Table 3. Again there is a clean separation between nitrite reductase and fumarase. The pea particles do not seem to be so sensitive to osmotic pressure as there is no release of nitrite reduetase at the 1.5--1.7 M sucrose interface.
Discussion The separation techniques used in this study suggest the presence of at least two, and probably three, types of particle in pea and barley roots. The first are the mitochondria which have been identified on the basis that they contain cytochrome oxidase and fumarase. This identification has been confirmed by observation of their structure under the electron microscope. The second group are those particles containing nitrite and nitrate reductase activity. The nature and the origin of these particles is not positively known. Preliminary electron microscopic evidence shows that the fraction is rich in membranous vesicles. These vesicles may exist in vivo, or could result from cell membranes, such as the endoplasmie reticulum rolling up into spheres upon rupture of the cell. Should the isolated particles represent in vivo particles they could be nitrosomes. The term nitrosome might cause some confusion since it has already been used in a slightly different context by Sims et al. (1968) to refer to the physical association of the two enzymes in yeast which are coordinately induced and repressed. In yeast there is no evidence that the enzymes are in a membrane bound particle sedimenting in a centrifuge. In barley, although both of the enzymes are inducible, the term nitrosome is not meant to imply that they are coordinately controlled. I n fact, the evidence suggests that the two enzymes are not so controlled in higher plants (Ingle etal., 1966; Schrader, 1969) and in barley particulate nitrite and nitrate reductase are differentially affected by oxygen tension (Miflin, 1970). The one point of similarity between the yeast and barley nitrosome is that in both eases it is envisaged that the two enzymes are in a close physical association forming a functionally interactive system catalysing the reduction of nitrate to ammonia. Another possibility is that since nitrite reductase is associated with chloroplasts in the leaves (l~itenour et al., 1967) the activities could be associated with proplastids in the root. This has not been eliminated, partly because of the difficulty of finding a known marker enzyme for the proplastids, however, Breiden-
168
B.J. Miflin:
baeh et al. (1968) report the density of castor bean endosperm proplastids as less than glyoxysomes (eatalase containing bodies) and, if barley root proplastids have similar density, they would not be found at the 1.7, 2.7 M sucrose interface. Further researeh is needed to identify the particles definitely. The next enzyme in the nitrogen ehain-glutamie dehydrogenase is not in the "nitrosome", but in the mitochondria. Although this has long been postulated on the evidence of differential eentrifugation, this is not in itself sufficient and the present results with density gradient eentrifugation provide the necessary confirmation t h a t this is so. I t has been speculated t h a t N A D H and NADPI-I dependent glutamie dehydrogenases m a y act in different directions in vivo, however, our results suggest t h a t they have the same distribution in the cell. The third group of particles postulated are those containing eata.lase. As suggested previously, it is considered on the basis of Table 1 and Fig. 3 t h a t barley root tissue contain eatalase containing particles (peroxisomes) separate from, but of a similar density, to mitoehondria. Breidenbaeh et al. (1968) mention t h a t maize roots have eatalase containing particles intermediate in density between the catalase containing glyoxysomes of the castor bean endosperm and mitoehondria. Peroxidase has been shown to be a particulate enzyme on the basis of histochemieal evidence, but only a small proportion of it can be sedimented in higher plant preparations (Plesnicar et al., 1967). In barley only a small minority of the activity is particle bound and it is distributed in a similar manner to eatalase in the large step density gradients. There is no evidence of any association of peroxidase with the nitrosomes. The extent of the particulate bound enzymes in the overall nitrogen economy of the cell is not known. I t nmst be re-emphasized that the majority of both the nitrate and nitrite reduetase is found in the supernatant fraction after homogenisation and eentrifugation. The relationship of the nitrosomal enzymes to the rest of the nitrate and nitrite reductase in the cell is not absolutely elear. One possibility is that the particle is an artefact caused by accidental enclosure of cytoplasmic enzymes in membranous vesicles formed upon rupturing the eelI. This is unlikely since the particulate nitrate reduetase can use sneeinate as an electron donor and the supernatant one cannot. Further, nltrasonieation and repeated freezing and thawing are not able to completely release nitrate reduetase from the particle (Miflin, 1970). A converse hypothesis is that the supernatant enzymes originate from the particles due to damage during extraction. A parallel for this is the ease of peroxidase which is considered in vivo to be located in the peroxisomes and yet, upon fraetionation, is found largely in the particle-free supernatant. Although
Location of Nitrate and Nitrite Reductase
169
it is possible t h a t a proportion of the n i t r i t e a n d n i t r a t e reductase i n the s u p e r n a t a n t m a y be derived in this way, there is evidence to suggest t h a t a dissimilatory n i t r a t e reductase is present i n the particles t h a t is n o t in the s u p e r n a t a n t (Miflin, 1970). A further difficulty i n considering the roles of these enzymes in vivo is the evidence t h a t in the presence of a high level of nitrate, cells m a y over-produce n i t r a t e reductase a n d the actual in vivo reduction rate m a y be considerably less t h a n t h a t possible b y the total a m o u n t of enzymes extracted (Ferrari a n d Varner, 1969). This n o n - f u n c t i o n a l n i t r a t e reductase m a y n o t be equally dist r i b u t e d between the particulate a n d soluble phases. The author is pleased to acknowledge helpful discussions with Dr. K. Joy and the financial assistance of th~ A.R.C. in partial support of this work. References Anderson, N. G. : Studies on isolated cell components. Exp. Cell l~es. 9, 446--459 (1955). Beaufay, It., Jacques, P., Badhuin, P., Sellinger, 0. Z., Bertheret, J., Duve, C. de: Resolution of mitochondrial fraction of rat liver into three distinct populations of cytoplasmic particles by means of density gradient equilibration in various gradients. Bioehem. J. 92, 1 8 4 ~ 0 5 (1964). Beers, R. F., Jr., Sizer, I. W.: A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. biol. Chem. 195, 133 (1952). Bourne, W. F., Miflin, B. J. : ~anuscript in preparation (1970). Breidenbach, t~. W., Beevers, H. : Association of the glyoxylate cycle enzymes in a novel subeellular particle from castor bean endosperm. Biochem. biophys. Res. Commun. 27, 462--469 (1967). - - K a h n , A., Beevers, H.: Characterization of glyoxysomes from castor bean endosperm. Plant Physiol. 43, 705--713 (1968). Epstein, E., Hagen, C. E. : A kinetic study of the absorption of alkali cations by barley roots. Plant Physiol. 27, 657---474 (1952). Ferrari, T. E., Varner, J. E. : i n vivo measurement of nitrate reductase activity. Plant Physiol. 41, Abs. No 176 (1969). Gregory, tl. P. F. : A rapid assay for peroxidase activity. Biochem. J. 101, 582--584 (1966). Ingle, J., Joy, K. W., Hageman, R. H. : The regulation of activity of the enzymes involved in the assimilation of nitrate by higher plants. Biochem. J. 190, 577--588 (1966). Miflin, ]3. J. : Distribution of nitrate and nitrite reductase in barley. Nature (Lond.) 214, 1133 (1967). - - Nitrate reducing enzymes in barley. In: Recent aspects of nitrogen metabolism in plants (ed. Hewitt, E. J. and Cutting, C. V.), p. 85. London: Academic Press 1968. - - Separation of barley root mitochondria into different fractions by density gradient centrifugation ]3ioehem. J. 108, 49 (1968b). - - Nitrite and nitrate reductase systems in barley roots. P~ev. roum. Biochim. 7, 53--60 (1970). Pertoft, H. : Gradient eentrifugation in a colloidal silica polysaccharide medium. ]3iochim. biophys. Acta (Amst.) 126, 594--596 (1960). 12 ])lanta(Bcrl.), Bd. 93
170
B. J. Miflin: Location of Nitrate and Nitrite Reductase
Plesnicar, M., Bonner, W.D., Storey, B. T. : Peroxidase associated with higher plant mitochondria. Plant Physiol. 42, 366--370 (1967). Racker, E.: Spectrophotometric measurements of the enzymic formation of fumaric and eis-aconitic acids. Bioehim. biophys. Aeta (Amst.) 4, 211--214 (1950). Ritenour, G. L., Joy, K. W., Burming, J., Hageman, R. H. : Intracellularlocalization of nitrate reductase, nitrite reductase and glutamic dehydrogenase in green leaf tissue. Plant Physiol. 42, 233--237 (1967). Schrader, L. E. : Induction of nitrate and nitrite reductases in corn seedlings. Plant Physiol. 44, Abs. No 175 (1969). Sims, A. P., Folkes, B. •., Bussey, A. H. : Mechanisms involved in the regulation of nitrogen assimilation in micro-organisms and plants. In: Recent aspects of nitrogen metabolism in plants (ed. Hewitt, E. J. and Cutting, C. V.), p. 91. London: Academic Press 1968. B. J. ~iflin Department of Plant Science The University Newcastle upon Tyne NE 1 7 RU, U.K.
