The distribution in sucrose density gradients of nitrate reductase from leaves of Hordeum vulgare

The distribution in sucrose density gradients of nitrate reductase from leaves of Hordeum vulgare M.I.M. Soares and C.F. Cresswell C.S.I.R./ University Photosynthetic Nitrogen Metabolism Unit, Department of Botany, University of the Witwatersrand, Johannesburg

The activity and distribution of barley (Hordeum vulgare L.) leaf nitrate reductase in sucrose density gradients varied with the composition of the extraction medium. Removal of Dithiothreitol Cleland's Reagent (DTI) resulted in an increase of nitrate reductase activity throughout the gradient (especially at the low density fractions) but decreased the percentage of the total activity associated with the organelles. Nitrate reductase activity was scattered in floating gradients and in non-floating gradients which were subjected to either long or short centrifugation. This pattern was not affected by the addition of bovine serum albumin (BSA). In crude extracts DTI preserved the activity of nitrate reductase. Possible explanations for the observed effect of DTI on the activity and distribution of nitrate reductase in sucrose density gradients are discussed and include: a) dissociation of a nitrate reductase complex system, b) release of nitrate reductase from a membranous system and c) interference with the nitrite accumulating properties of the enzyme in the in vitro assay system . S. Afr. J. Bot 1984,3: 146-152

Die aktiwiteit en verspreiding van gars (Hordeum vulgare L.) blare se nitraatreduktase in sukrose digtheidsgradiente varieer volgens die samestelling van die ekstraheringsmedium . Verwydering van DTI het 'n verhoogde nitraatreduktase-aktiwiteit dwarsdeur die gradient tot gevolg gehad (veral in die lae digtheidsfraksies), maar die persentasie van die totale aktiwiteit wat met die organelle geassosieer is, is verlaag. Nitraatreduktase-aktiwiteit het in die drywende gradiente en in die nie-drywende gradiente wat aan lang of kart sentrifugering onderwerp is, versprei. Hierdie patroon is nie beinvloed deur die byvoeging van BSA nie. In ru-ekstrakte het DTI die aktiwiteit van nitraatreduktase bewaar. Moontlike verklarings vir die bevindings van die invloed van DTI op die aktiwiteit en verspreiding van nitraatreduktase op sukrose digtheidsgradiente word bespreek en sluit die volgende in : a) ontbinding van 'n nitraatreduktase komplekssisteem, b) die vrystelling van nitraatreduktase van 'n membraanstelsel en c) in vitro versteuring van die ensiem se nitriet-ophopingseienskappe. S.·Afr. Tydskr. Plantk. 1984,3: 146- 152

Keywords: Hordeum vulgare leaf, nitrate reductase distribution, sucrose density gradients

This work forms part of a Ph.D. thesis submitted to the University of the Witwatersrand

M.I.M. Soares* Present address: Department of Botany, University of Cape Town , Rondebosch , 7700 C.F. Cresswell C.S.I.R./University Photosynthetic Nitrogen Metabolism Unit , Department of Botany, University of the Witwatersrand , 1 Jan Smuts Ave. , Johannesburg, 2001 Republic of South Africa *To whom correspondence should be addressed Accepted 4 January 1984

Introduction

The intracellular site of nitrate reductase in green leaves is still controversial. A variety of techniques used by numerous workers have yielded conflicting results . Del Campo et al. (1963) found that isolated illuminated chloroplasts were able to reduce nitrate; location of nitrate reductase in the chloroplasts was also proposed by Coupe et al. (1967) using barley leaf extracts separated in non-aqueous density gradients. However , Grant et al. (1970) reported that in spinach and sunflower leaf extracts separated by differential centrifugation the main site of nitrate reductase was external to the chloroplast and only 25% of the enzyme was inside the chloroplasts. Association of nitrate reductase with the chloroplast envelope was envisaged by Beevers & Hageman (1969) and Eaglesham & Hewitt (1971) and later found in chloroplasts of maize and foxtail isolated by aqueous and non-aqueous techniques (Ritenour et al. 1967) , in chloroplasts ofWolffia isolated by the method of Jensen & Bassham (1966) (Swader & Stocking 1971) and in isolated envelope membranes from millet (Rathnam & Das 1974) . Location of nitrate reductase in the cytoplasm was proposed by Dalling et al. (1972) and Wallsgrove et al. (1979) using spinach and tobacco leaf extracts separated in sucrose density gradients. Lips & Avissar (1972) observed that nitrate reductase was associated with the peroxisome fraction of tobacco leaf extracts separated in a large zonal rotor. Inconsistent results have also been obtained in studies with inhibitors of protein synthesis (Schrader et al. 1967; Stewart 1968; Sluiters-Scholten 1973) and the site of synthesis of nitrate reductase may differ from the ultimate location of the enzyme (Hewitt 1975). In addition one has to consider problems inherent to studies with isolated organelles such as the differential release of enzymes from organelles and the adsorption of soluble enzymes by organelles (Stocking 1959; Leech & Murphy 1976). Sucrose density gradients were employed in the study of the intracellular location of nitrate reductase in barley leaf extracts. The effect of some isolation conditions on the distribution of the enzyme was analysed. Materials and Methods

Seeds of Hordeum vulgare L. (var. Gars Clipper ex Napier) were soaked overnight in aerated distilled water. The seeds were then spread on a layer of cheesecloth supported by a stainless steel screen and suspended over the surface of aerated full strength Long Ashton solution (Hewitt 1952) .

147

S. Afr. J. Bot., 1984, 3(3)

Water was added to the nutrient solution daily to maintain the original volume (5 I) and the solution was completely changed on the 8th and 15th day after sowing. Plants were grown in a phytotron chamber, under controlled conditions of 14 h light at 250 p,E m - 2 s- 1 at 27°C and 10 h dark at 22°C. The relative humidity in the chamber was 70±5% during the light period and 80±5% during the dark period. Leaves were harvested 4 h after the beginning of the light period. Mature second leaves of 16-day-old plants were excised and one-fifth of each leaf length discarded from each end. The middle three-fifths of several leaves were used in all experiments. Leaves were homogenized in a chilled medium (1 g/cm 3 ) with a VirTis 45 homogenizer and the extract filtered through one layer of Miracloth, dampened with cold extraction medium. A modified Honda medium (Honda et al. 1966) was used in which mercaptoethanol was substituted by 10 mmol dm - 3 OTT (Oithiothreitol- Cleland's Reagent) (purchased from Sigma) and the concentration of BSA (bovine serum albumin, fraction V purchased from Sigma) was 0,1% (w/v). In some experiments, BSA and/or OTT were omitted. A volume of 25 cm 3 of crude extract was placed onto the sucrose density gradient. In floating gradients, the leaf extract was placed at the bottom of the centrifuge tube, followed by layered cold sucrose solutions of the gradient. Linear sucrose density gradients were prepared in Beckman 65 cm3 cellulose nitrate tubes by sequentially layering 2,5 cm 3 each of 60,0; 57,5; 55,0 ... 30,0% sucrose solutions on each other. The 60% solution was prepared by dissolving solid sucrose (obtained from BOH) in 100 mmol dm- 3 glycylglycine buffer, pH 7 ,5. Solutions of decreasing concentrations

500

were made up by adding buffer until the required density values were obtained. Centrifugation of the gradients was carried out at 4°C in a Beckman L3-50 ultracentrifuge using an SW 25.1 rotor. Three main methods of centrifugation were used: 'long' centrifugation, 'short' centrifugation and 'floating' gradient centrifugation. In the short centrifugation, gradients were centrifuged for 5 min at 4 000 rpm, followed by 10 min at 10 000 rpm (Miflin & Beevers 1974). The long centrifugation lasted 3 h at 24 000 rpm (Schnarrenberger & Burkhard 1977). Floating gradients were centrifuged for 23 h at 16 000 rpm. The gradients were fractionated from the top into 1,2 cm 3 fractions by means of an Isco model640 density gradient fractionator and the fractions were kept in ice. Catalase (EC 1.11.1.6) was assayed according to Luck (1963), cytochrome c oxidase (EC 1.9.3.1) and nitrate reductase (EC 1.6.6.1) according to Soares (1982) and chlorophyll according to Arnon (1949). Sucrose concentrations were measured in an Abbe refractometer. All reagents used in the current studies were of analytical grade. Results

The following organelle markers were used: chlorophyll for chloroplasts, catalase for peroxisomes and cytochrome c oxidase for mitochondria. A distinct separation between chloroplasts and mitochondria was not obtained and a certain degree of cross-contamination occurred between these organelles and the peroxisomes. Centrifugation of the crude extract prior to loading onto the gradient, as described by Schnarrenberger & Burkhard (1977) did not eliminate organelle contamination (results not shown). It is common procedure to add OTT to nitrate reductase

60

30.0

0

c: 2375

50

u

~ 0

.r:

0

u

0"'

••

~

s:

z

Q)

(i250

e40

E

u

Q)

en

.s

::J

#

(/)

u"'::J

••

"~

•

Q)

~ 125

z

•

••

•

•

•

••

•

•

•

•

••

•

• ••

60

•

22

5~

45

'c: 0

u ~

'c:

-~ >

(_) ~

~

"'

Ol

15.0-

3oS

~

>.r:

0

g

c:

a.

::J

~

Q)

0

(/)

.r:

"'

0

«i (ij

0 7.5

1

15

1

J 0

0

Figure 1 Distribution of nitrate reductase in a linear sucrose density gradient after long centrifugation. The extraction medium contained BSA and DTT.

S.-Afr. Tydskr. Plantk., 1984, 3(3)

148 extraction media (Hageman & Hucklesby 1971) and DTI has been used in studies on the intracellular distribution of nitrate reductase (Dalling et al. 1972). This substance protects the enzyme by maintaining its thiol groups in the reduced state (Cleland 1964). However, DTI has been reported to have other effects such as the solubilization of coat proteins from bacterial spores (Aronson & Fitz-James 1968; Spudich & Kornberg 1969), the maintenance of the subunits of glycinin dissociated (Catsimpoolas & Wang 1971) and the release of enzymes from the cell wall of yeast (Smith & Ballou 1974) . Should DTI have similar effects in the homogenates of barley leaves, the distribution of nitrate reductase in the density gradients could be affected. This was tested. After long centrifugation, nitrate reductase activity in extracts containing BSA and DTI (Figure 1) was low at the top of the gradient (23% sucrose), showed a peak at the low density section of the gradient (23-30% sucrose) and was scattered in the chloroplast-mitochondria fractions , decreasing towards the peroxisome band. When DTI was excluded from the extraction medium (Figure 2) , the total activity of nitrate reductase increased markedly , the top and low density fractions of the gradient accounting for most of the enhancement. As observed in the presence of DTI (Figure 1), the activity along the gradient was scattered and decreased in the peroxisome peak (Figure 2) . At the concentration used, DTI did not interfere with the colorimetric determination of nitrite and therefore the lower nitrate reductase activity could not be ascribed to the underestimation of nitrite. There was no marked effect of DTI on the activity of the freshly extracted enzyme but in the long term it contributed to better preservation of enzyme

500

actiVIty (Table 1). The lower nitrate reductase activity observed in the density gradients did not therefore appear to be due to enhanced decay of the enzyme caused by DTI. Table 1 Effect of DTT on the activity and stability of

nitrate reductase in crude extracts. The extraction medium contained BSA in the presence or in the absence of DTT. Nitrate reductase activity was assayed immediately after tissue homogenization and 2 h later. Enzyme rate: p,mol NO:Z accumulated h- 1 g - 1 fr. wt. Extraction media

Activity atOtime

Activity at2h

% Activity after2 h

5,019 5,118

4,119 3,435

82,1 67,1

+DTI

-orr

After long centrifugation, nitrate reductase activity in gradients of extracts containing DTI in the absence of BSA (Figure 3) was low at the top of the gradient and was not preferentially associated with any type of organelle. The distribution of nitrate reductase was very similar to that observed in the presence of both BSA and DTI (Figure 1), except for the absence of the low density peak. When both BSA and DTI were omitted from the extraction medium (Figure 4), nitrate reductase activity was high at the top and in the low density fractions of the gradient and lower and scattered in the organelle fractions . The exclusion of BSA from a medium without DTI resulted in lower activity in the organelle fraction (compare Figures 2 & 4). The effects of BSA and DTI on the activity and distribution

60

30.0

•• 0 ,

c 0 ;; 375 u ~

'.r::

u u

"' 0z

3:

~

E

.s

40

u

•

:::>

(f)

Q) (/)


u"':::>

•

•

•

• 22 5;:'c

45

0

u

'c

~

0

u ~

(Jl

E

30-

c

:::>

•

••

'0

~

Q)

~

e Q)

o25u

•

••

•

•

••

••

•

60

•

125

z

J

•••••••••••••• 0

••

• 0

Figure 2 Distribution of nitrate reductase in a linear sucrose density gradient after long centrifugation. The extraction medium contained BSA (DTI excluded).

S. Afr. J. Bot., 1984, 3(3)

149

of nitrate reductase are also shown in Table 2. The total nitrate reductase activity was markedly higher in the absence of OTT and more than 50% of the enzyme was at the top of the gradient. Addition of BSA in the absence of OTT

500

100

resulted in a higher association of the enzyme with the organelle fraction (28% as compared with 12% ), without having a distinct effect on the activity of the low density fractions of the gradient. In the presence of OTT, the total

30.0

60

•• 'c 0

1

c

g()

••

75f- ;3 375 C1l

i .;:

~

'c

'c

(.) ()

E

• • •

C1l

0

E

••

•

20

•

• 22 .5::-

c0

•

~

1:0

~

~

~

ti

C1l

15.00

C1l (j)

0

rJ)

C1l

(j)

ti ..,

E

[I! (j)

(.)

~25

"§

0

z

:;, c.

125

~

0

.c 0

rJ)

~ Cll

<;;

I

0 30

5

1 I 0

.c

(j)

•• •

D

~

.c

CJ)

E 10-

-~ c 2.

D

()

ti

.~

~ 50 X

15

• 0

0

Figure 3 Distribution of nitrate reductase activity in a linear sucrose density gradient after long centrifugation . The extraction medium contained OTT (BSA excluded).

100

'c: 0 :;:o 75 (.)

500

'c

0 :;:o 375

••

(.)

~

~

'c

~

E 0 E

60

• ••

•

•

•

•

30.0

20

22 .5'c

15

0

ti

"'

0

~

z

5l 50 0

"'

E

:2

250

.s

X

0

(.)

rJ)

Q)

ti

~ .c

[I! Q)

">.25 0

0

0

Q)

.c 0

Cll

co

<;;

0 7.5

5

z

1 1 ••••••••••••••• 0

>-

c.

rJ)

"§ 125

0

•

.s .r::.

c

2.

•••

"0

10

-~

#

()

0

150c;

40

::::>

::::>

CJ)

Cll

Q)

(.)

"'

E

~

ti

e (/)

Q)

ti

~ >

-...~

'N

c:

'c0

~

c.) (.)

I

• 0

Figure 4 Distribution of nitrate reductase activity in a linear sucrose density gradient after long centrifugation. Both BSA and OTT were excluded from the extraction medium.

150

S.-Afr. T ydskr. Plantk., 1984, 3(3)

Table 2 Effects of OTT and BSA on the activity and distribution of nitrate reductase on linear sucrose gradients after long centrifugation . Top of grad.: fractions from the top of the gradient to 25% sucrose; Low dens.: fractions between 25- 30% sucrose; Chl.+mit.+per.: fractions containing chloroplasts, mitochondria and peroxisomes a) Activity (p,mol NO; accumulated h- 1 ) b) Percentage of total activity in gradient +DTI +BSA

-DTI +BSA

+DTI -BSA

-DTT -BSA

Top of grad .

0,454 (a) (15 ,6)(b)

4,882 (51 ,0)

0,571 (25 ,2)

5,441 (63 ,9)

Low dens.

0,905 (31 ,2)

2,016 (21 ,0)

0,438 (19,3)

2,019 (23 ,7)

Top of grad. +Low dens.

1,360 (46,8)

6,899 (72 ,0)

1,010 (44,5)

7,459 (87,6)

Chl. + mit.+per.

1,548 (53 ,2)

2,675 (28,0)

1,259 (55 ,5)

1,055 (12,4)

Treatment

nitrate reductase activity decreased to about 30% of that measured in the absence of DTI; the decline being more noticeable at the top of the gradient. Addition of BSA to the medium containing DTI resulted in a different distribution of the enzyme activity at the top and in the low density fractions of the gradient, without affecting the sum of both: BSA brought about an increase of the activity found in the low density section , apparently at the expense of the activity at the top of the gradient. In terms of total activity in each

500

gradient , the percentage of activity associated with the organelle fractions varied from 12% in the medium lacking both BSA and DTI, to more than 50% in media containing DTI, either in the presence or in the absence ofBSA . In the medium containing BSA and no DTI, 28% of the total activity was found in the organelle fractions . In the short centrifugation method described by Miflin & Beevers (1974) the chloroplasts reach their equilibrium position in the gradient while peroxisomes and mitochondria , having lower sedimentation rates , appear at a density position lower than that of equilibrium. According to Miflin & Beevers (1974) , a less contaminated chloroplast fraction is then obtained. In the present study, however , an appreciable amount of catalase activity was associated with the chlorophyll peak from extracts in a medium containing BSA in the absence of DTI (Figure 5). The total nitrate reductase activity was high with a very small amount present in the chloroplast fractions . A higher percentage of nitrate reductase activity was associated with the organelle fractions in floating gradients (Figure 6) when compared with the distribution of the enzyme extracted in an identical medium but subject to long (Figure 2) or short (Figure 5) centrifugation. The lower activity in the top and low density fractions of the floating gradient (Figure 6) was probably due to the decay of the enzyme during the very long centrifugation period (23 h) . A nitrate reductase peak coincided with the high density shoulder of the chloroplast fraction (intact chloroplasts). Discussion

The sucrose density gradient centrifugation technique has yielded conflicting results on the distribution of leaf nitrate

60

•• ' 0c

;; 3 75

••

u

~

-:.:: u u

~

..... "' 0 z

•

l

0 250

•

•

30.0

70.0

22.5--.. c

52.5

•• '0

13

•

"'

350-

!!.!

r:

~

>a.

::J

0

0

Q>

r:

Vl

~

()

"'

iii

()

Q>

"§ z

E

c

• ••

"0

0>

15.00

;I?.

u"'::J

~

>

"<3

E Vl

Q

u

.£

.s Q>

'c

~

125

7.5

1 0

I

17.5

1

0

Fraction number

Figure 5 Distribution of nitrate reductase activity in a linear sucrose density gradient after short centrifugation . The extraction medium contained BSA (DTI excluded).

S. Afr. 1. Bot., 1984, 3(3)

151

70.0

40

500

• 'c

••

0

73

37 5

~

'r:: u u

...0"'

•

0 250 E c

••

(lJ

0

u

u ~

>

u

"'

20-

O'l

35

0

o.S >-

-~

£

c

Q.

2

2.

.

~

Q

'2:

0

(lJ

£

VJ

•

(lJ

'c

~

•

'0

52.5

'c

??

u:::J ~ z

•

30

•

•

z

~

••

••

~

0

-;"'

I

0

125

•••••

••••••••• ••••

•• •

0

••

10

17.5

I

0

Figure 6 Distribution of nitrate reductase activity in ·a floating linear sucrose density gradient. The extraction medium contained BSA (OTT excluded).

reductase. Dalling et al. (1972) observed that when extracted in a medium without BSA, nitrate reductase was present throughout the organelle fractions and that addition of BSA to the extraction medium resulted in the appearance of the enzyme in the supernatant. Dalling et al. (1972) concluded that nitrate reductase was soluble and BSA eliminated the indiscriminate adsorption of the enzyme to organelles. Other factors may also affect the distribution of nitrate reductase in sucrose gradients. Lips (1975) observed that the composition of the homogenization media, the ratio of homogenization medium volume to tissue weight, the type of gradient and the rate of acceleration of the centrifuge rotor affected the nitrate reductase content of the microbody fraction. On the other hand, the distribution of the organelles in the sucrose gradients is also affected by the experimental conditions. Phosphate in the sucrose density gradients causes attachment of chloroplasts and peroxisomes, resulting in the sedimentation of the peroxisomal fraction together with intact chloroplasts (Schnarrenberger & Burkard 1977) while light promotes the attachment of microbodies to chloroplasts (Kagan-Zur & Lips 1975). This association between organelles is probably more than a separation artefact and evidence of in situ association of organelles has been obtained in electron microscopy (Frederick & Newcomb 1969) and cinephotomicrography (Wildman et al. 1962) studies and in counter-current separation of chloroplasts in a two phase system (Larsson et al. 1971). The results obtained in the present study did not allow any conclusion on the intracellular location of nitrate reductase. The results obtained using BSA differ from those of Dalling et al. (1972) who observed a marked increase of nitrate

reductase activity in the supernatant and the disappearance of the scattered activity in the organelle fractions. In their studies, the extraction medium lacking BSA contained DTT (10 mmol dm - J , as in the present study) but it is unclear whether DTT was present in the medium containing BSA. Contrary to results obtained in the density gradients, OTT preserved the activity of nitrate reductase in crude extracts (Table 1) . Possible explanations for this differential effect of DTT on the activity of nitrate reductase could be envisaged as follows: (a) Nitrate reductase may be part of a complex system which remained relatively intact in the crude extracts. In the presence of DTT, this system could dissociate but its components remained spatially close , the sulfhydryl groups of the enzyme were protected and , as a consequence, the activity ofthe enzyme was preserved. However, when OTTcontaining extracts were centrifuged in density gradients, the dissociated components of the enzyme complex were separated and the activity declined drastically. (b) In the presence of DTT, nitrate reductase could be more susceptible to an endogenous inhibitor owing to the dissociation and/or removal of the enzyme complex from the membranes. The supernatant and low density fractions of the gradients were rich in membranes (electron microscopy observations not shown). Dalling et al. (1972) reported an inhibitor of nitrate reductase in the supernatant of density gradients from tobacco leaves; the inhibitor did not adhere to the organelles and was removed by Sephadex G-25. (c) Comparative studies on the accumulation of nitrite and disappearance of nitrate in the nitrate reductase in vitro assay system have shown that the amount of nitrate lost exceeds the amount of nitrite accumulated (Soares 1982).

152

Furthermore, a particulate fraction was found in barley leaf extracts separated by differential centrifugation which used nitrate at an appreciable rate while accumulating negligible levels of nitrite (Soares 1982). The effect of DTI may only have been on the capacity of the enzyme to accumulate nitrite but not on the capacity of the enzyme to use nitrate ; the particulate enzyme may not have been detected by the nitrate reductase assay used, namely the measurement of nitrite accumulation. This effect of DTI could have been mediated by the mechanisms proposed in (a) or (b) . Nitrate reductase scattering in the gradient may be due to the indiscriminate adsorption of the enzyme to several types of organelles. The sucrose density gradient method used in the study of the intracellular distribution of nitrate reductase has limitations and should be combined with other methods. A better understanding of nitrate reduction in vivo and the use of alternative methods for the estimation of nitrate reductase activity in vitro are needed for the establishment of the intracellular location of the enzyme. Acknowledgements

We wish to acknowledge research grants from the University Council, South African Council for Scientific and Industrial Research, Department of Agriculture, Nuclear Energy Corporation and the Fertilizer Society of Southern Africa, all of whom contributed towards the running of the CSIR/ University Photosynthetic Nitrogen Metabolism Unit, in which this work was undertaken . References ARNON, D.I. 1949. Copper enzymes in isolated chloroplasts . Polyphenoloxidase in Beta vulgaris. Pl. Physiol. 24: 1-15. ARONSON , A. I. & FITZ-JAMES , P.C. 1968. Biosynthesis of bacterial spore coats. J. Malec. Bioi. 33: 199-212. BEEVERS , L. & HAGEMAN, R.H. 1969. Nitrate reduction in higher plants. Ann. Rev. Pl. Physiol. 20: 495-522. CATSIMPOOLAS, N. & WANG , J. 1971. Analytical scanning isoelectrofocusing. 5. Separation of glycinin subunits in urea-dithiothreitol media. Ann. Biochem. 44: 436-444. CLELAND , W.W. 1964. Dithiothreitol , a new protective reagent for SH groups. Biochem. 3: 480-482 . COUPE, M. , CHAMPIGNY, M.L. & MOYSE, A. 1967. Sur Ia localisation intracellulaire de Ia nitrate reductase dans les feuilles et les racines d'orge. Physiol. Veg. 5:271-291. DALLING , M.J. , TOLBERT, N.E. & HAGEMAN, R.H. 1972. Intracellular location of nitrate reductase and nitrite reductase. I. Spinach and tobacco leaves. Biochim. Biophys. Acta 283: 505-512. DEL CAMPO, F. F., PANEQUE, A. , RAMIREZ, J.M. & LOSADA, M. 1963. Nitrate reduction in the light by isolated chloroplasts. Biochim.Biophys. Acta 66: 450-452. EAGLESHAM, A.R.G. & HEWITT, E.J. 1971. The regulation of nitrate reductase activity from spinach (Spinacea oleracea L.) leaves by thiol compounds in the presence of adenosine-5'diphosphate . FEBS Lett. 16: 315-318. FREDERICK, S.E. & NEWCOMB , E. H. 1969. Microbody-like organelles in leaf cells. Science, N.Y. 163: 1353-1355. GRANT, B.R. , ATKINS, C.A. & CANVIN, D.T. 1970. Intr~cellular location of nitrate reductase in spinach and sunflower leaves. Planta 94: 60-72. HAGEMAN , R.H . & HUCKLESBY, D.P. 1971. Nitrate reductase from higher plants. In: Methods in enzymology, ed. San Pietro , A., Vol. XXIII, pt. A. Academic Press, New York , London. pp. 491-503 . HEWITT, E .J. 1952. Sand and water culture methods used in the

S.-Afr. Tydskr. Plantk. , 1984, 3(3) study of plant nutrition. Commonw. Bur. Hort. Plant Crops (G.B.) Tech. Commun. 22. East Mailing, Kent. HEWITT, E.J. 1975. Assimilatory nitrate-nitrite reduction. Ann. Rev. Pl. Physiol. 26: 73-100. HONDA, S.I., HONGLADAROM, T. & LATIES, G .G. 1966. A new isolation medium for plant organelles. J. Exp. Bot. 17: 460-472. JENSEN , R.G. & BASSHAM, J.A. 1966. Photosynthesis by isolated chloroplasts. Proc. Natn. Acad. Sci. U.S.A. 56: 1095-1101. KAGAN-ZUR , V. & LIPS , S.H. 1975. Studies on the intracellular location of enzymes of the photosynthetic carbon-reduction cycle. Eur. J. Biochem . 59: 17-23. LARSSON , C., COLLIN , C. & ALBERTSSON, P.A. 1971. Characterization of three classes of chloroplasts obtained by counter-current distribution. Biochim. Biophys. Acta 245: 425-438. LEECH, R.M. & MURPHY, D.J. 1976. The cooperative function of chloroplasts in the biosynthesis of small molecules. In: The intact chloroplast, ed. Barber, J. E lsevier/North Holland Biomedical Press, Amsterdam. pp. 365-401. LIPS , S.H. 1975. Enzyme content of plant microbodies as affected by experimental procedures. Pl. Physiol. 55: 598-601. LIPS, S.H. & AVISSAR, Y. 1972. Plant-leaf microbodies as the intracellular site of nitrate reductase and nitrite reductase. Eur. J. Biochem. 29: 20-24. LUCK, H. 1963. Catalase. In: Methods of enzymatic analysis, ed. Bergmeyer, H.U. Academic Press , New York. pp. 885-894. MIFLIN, B.J. & BEEVERS , H. 1974. Isolation of intact plastids from a range of plant tissues. Pl. Physiol. 53: 870-874. RATHNAM , C.K.M. & DAS , V.S.R. 1974. Nitrate metabolism in relation to the aspartate-type c4 pathway of photosynthesis in Eleusine coracana. Can. J. Bot. 52: 2599-2605. RITENOUR, G.L. , JOY, K.W., BUNNING , J. & HAGEMAN , R.H. 1967. Intracellular localization of nitrate reductase, nitrite reductase and glutamic acid dehydrogenase in green leaf tissue. Pl. Physiol. 42: 233-237. SCHNARRENBERGER, C. & BURKHARD , CH. 1977. In vitro interaction between chloroplasts and peroxisomes as controlled by inorganic phosphate . Planta 134: 109-114. SCHRADER, L.E., BEEVERS , L. & HAGEMAN, R.H. 1967. Differential effects of chloramphenicol on the induction of nitrate and nitrite reductase in green leaf tissue. Biochem. Biophys. Res. Commun. 26: 14-17. SLUITERS-SCHOLTEN, C.M.TH. 1973. Effect of chloramphenicol and cycloheximide on the induction of nitrate reductase and nitrite reductase in bean leaves. Planta 113: 229-240. SMITH , W.L. & BALLOU, C.E. 1974. The effect of dithiothreitol on external yeast invertase. Biochem. Biophys. Res. Commun. 59: 314-321. SOARES, M.I.M. 1982. Inorganic nitrogen metabolism in the leaves of Hordeum vulgare L. Ph.D. thesis, University of the Witwatersrand, Johannesburg. SPUDICH, J .A. & KORNBERG, A. 1969. Biochemical studies of bacterial sporulation and germination . VI. Origin of spore and core coat proteins. J. Bioi. Chern. 243: 4588-4599. STEWART, G .R. 1968. The effect of cycloheximide on the induction of nitrate and nitrite reductase. Phytochemistry 7: 1139-1142. STOCKING , C.R. 1959. Chloroplast isolation in non-aqueous media . Pl. Physiol. 34: 56-61. SWADER, J .A. & STOCKING , C.R. 1971. Nitrate and nitrite reduction by Wolffia arrhiza. Pl. Physiol. 47: 189-191. WALLSGROVE, R.M., LEA, P.J . & MIFLIN , B.J. 1979. Distribution of the enzymes of nitrogen assimilation within the pea leaf cell . Pl. Physiol. 63: 232-236. WILDMAN , S.G., HONGLADAROM, T . & HONDA, S.I. 1962. Chloroplasts and mitochondria in living plant cells: cinephotomicrographic studies. Science, N.Y. 138: 434-435.