Effect of lysophosphatidylcholine on the NADH-ferricyanide reductase activity associated with the plasma membranes of corn roots

Plant Science, 70 (1990) 143-153 Elsevier Scientific Publishers Ireland Ltd.

143

EFFECT OF LYSOPHOSPHATIDYLCHOLINE ON THE NADH-FERRICYANIDE REDUCTASE ACTIVITY ASSOCIATED WITH THE PLASMA MEMBRANES OF CORN ROOTS

ISABELLE BOURDIL, MARIE-LOUISE MILAT and JEAN PIERRE BLEIN* Laboratoire des Herbicides, I.N.R.A., B. V. 15~0, 2103~ Dijon cedex (France)

(Received July 24th, 1989) (Revision received January 29th, 1990) (Accepted April 10th, 1990) Plasma membranes from corn roots (Zea mays L.) were isolated by aqueous two, phase partitioning. A fraction enriched in a vanadate-sensitive ATPase showed characteristics of a plasma membrane ATPase. The sidedness of these vesicles was 89% right-side~ut, as evaluated by the ATPase latency. A NADH-ferricyanide reductase was associated with these plasma membrane vesicles. The rate of ferricyanide reduction was 1.3 lanol • min-1. mg-1 protein and was strongly enhanced by the addition of lysophosphatidylcholine (LPC). The effect of this detergent on membrane solubflization and reductase activity was particularly studied. This type of detergent treatment revealed two pH optima (7.0 and 5.0) for the reductase activity, which exhibited biphasic kinetics in the absence or presence of the detergent. These data suggest that two or more reductases could be involved. In addition, membrane vesicle solubilization and determination of ATPase and reductase latency were simultanously studied. From these experiments, it is postulated that the reductase, which exhibits an optimum pH at 7.0 and is slightly stimulated by LPC, could be located on the external side of the plasmalemma. In contrast, the reductase at pH 5.0 strongly stimulated by the detergent treatment, is probably located on the internal side of the membrane, such as the catalytic site of ATPase. Finally, a possible direct action of LPC on the enzymes, is discussed. Key words: plasma membrane; NADH-ferricyanide reductase; latency; Zea mays

Introduction Previous studies have demonstrated the p r e s e n c e of e l e c t r o n t r a n s f e r s y s t e m ( s ) a t t h e p l a s m a m e m b r a n e of h i g h e r p l a n t s [1,2]. T h e p h y s i o l o g i c a l r e d u c t a n t a p p e a r s t o b e N A D H or N A D P H [3--5], a n d o x i d a t i o n of e x o g e n o u s N A D ( P ) H i n cells a n d t i s s u e s h a s b e e n r e p o r t e d [6,7]. I s o l a t e d p l a s m a m e m b r a n e p r e p a r a t i o n s are able to r e d u c e artificial electron acceptors [ 5 , 8 - 1 1 ] , a s c o r b a t e f r e e r a d i c a l s [12,13] a n d , t o *To whom correspondence should be sent. Abbreviations: CMC, critical micellar concentration; DTT, dithiothreitol; LPC, lysophosphatidylcholine; NFR, NADHferricyanide reductase; Mes, 2-(N-morpholino)ethanesulfonic acid; PMSF, phenylmethansulfonylfluoride; SOD, superoxide dismutase; SW26, 2,2,2-trichloroethyl~3,4dichlorocarbanilate; Tris, tris(hydroxy-methyl)-aminomethane.

a lesser extent, oxygen [6,8,14].These various activitieshave been characterized, but the precise mechanism of the electron transfer is still unclear. Luster and Buckhout have partiallypurified several electron transport systems from corn root plasma membranes, and suggested the presence of separate enzymatic redox components [13]. Moreover, kinetic studies have raised the possibility of there being two or three pyridine nucleotide-dependent dehydrogenases. These dehydrogenases which are stimulated by duroquinone [15,16],have been separated by chromatography and identifiedby SDS-gel electrophoresis [17,18]. As we previously reported [19], corn root plasma membranes contain a N F R which exhibits biphasic kinetics and is strongly stimulated by LPC. In order to evaluate this stimulation,

0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

144

we have studied the effect of LPC on the solubilization of membrane vesicles and on NFR activity. The results lead us to hypothesize that the redox system could be localized on both plasmalemma faces, and suggest that LPC could affect directly the enzyme activities.

Solubilization of membrane vesicles by detergent Solubilization was followed by turbidity determinations at 400 nm. LPC aliquots were added and solubilization was achieved when further additions of detergent no longer changed the absorbance.

Materials and Methods

Membrane preparation Corn seeds (Zea mays var. Mona) were surface sterilized with calcium hypochlorite, soaked in water for one day and germinated on stainless steel screens above water for 6 - 7 days in the dark. All steps were done at 4 °C. Corn root microsomes were prepared and clarified by centrifugation through a 30% (w/w) sucrose layer according to described procedures [20]. Microsomes, resuspended in 5 mM phosphate buffer (pH 6.8) containing 250 mM sucrose and 1 mM dithiothreitol (DTT), were further purified by aqueous two-phase partitioning as described by Kjellbom and Larsson [21] and Sandelius et al. [9], with some modifications. A 16 g two-phase polymer system was used (1 g of microsomes and 15 g of the polymer mixture), with a final composition of 6.40/o (w/w) Dextran T500 and 6.4% (w/w) polyethylene glycol 4000, in a 5 mM phosphate buffer (pH 6.81 containing 250 mM sucrose and 1 mM DTT. After mixing the system by 40 inversions, the two phases were separated by centrifugation (1500 x g, 5 min). The upper phase was collected and rewashed by a fresh lower phase. This step was repeated 5 times, the lower phases being discarded each time. Finally, the upper phase was diluted (5-fold) with resuspension buffer containing 10 mM Tris--Mes (pH 7.3), 250 mM sucrose, 1 mM ATP, 2 mM EDTA, 1 mM DTT and 1 mM PMSF, and centrifuged 60 min at 80 000 × g. The membrane pellet was washed once with the same buffer and then resuspended (ml/ 100 g of roots, i.e. 1 mg/ml protein) in 10 mM Tris--Mes buffer (pH 7.3) containing 1 mM ATP, 2 mM EDTA, 1 mM DTT, I mM PMSF and 20O/o glycerol (w/v). This membrane fraction was stored at - 80 °C.

Enzyme activities Phosphohydrolase activity was measured after 30 rain incubation at 38 °C in 1 ml of T r i s Mes buffer (pH 6.5), containing 100 mM KC1, 3 mM MgS04, 3 mM ATP (Na salt) and 5 - 1 0 pg membrane protein. For The IDPase assay, ATP was replaced by IDP. Inorganic phosphate was estimated as described by Pullman and Penefsky [22]. NADH-cytochrome c oxidoreductase activity was measured in 3 ml reaction mixture containing 50 mM phosphate buffer (pH 7.5), 1.66 mM sodium cyanide, 30 ~M cytochrome c, 0.1 mM NADH and 10--20 pg membrane protein, [23]. Cytochrome c oxidase activity was measured in 3 ml reaction medium containing 50 mM phosphate buffer (pH 7.5), 30 ~M reduced cytochrome c and 10--20 pg membrane protein [24]. Both were spectrophotometrically assayed at 550 nm. Cytochrome c concentration was calculated using a molecular extinction coefficient of 18.5 mM -1. cm- 1. Except where otherwise stated, NFR assays were carried out at 25 °C in 2 ml of 50 mM Tris - M e s buffer (pH 7.0), containing 0.25 mM ferricyanide and 1 0 - 2 0 ~g membrane protein. The reaction was initiated by addition of NADH (final conc., 0.25 mM) and the reduction of ferricyanide was measured at 420 nm. The sum of the rates obtained without enzymes and without NADH was subtracted from the initial rate of NFR activity. Ferricyanide concentration was calculated using a molecular extinction coefficient of 1.0 mM -1. cm- 1. Pro rein de termination Protein concentration was determined by the method of Bradford [25] as modified by Fanger [26] with bovine serum albumin as standard.

145 Table I.

Distribution of enzymatic activities during plasma membrane enrichment. Activities were estimated as described in Materials and Methods, in the presence of LPC (LPC/protein = 5). Results are expressed as specific activity (SA) and as percentage of the total activity, measured in crude microsomes. Activities of NADH~ytochrome c reductase and cytochrome c oxidase, are given in pmol cyt c reduced or oxidized, min-1. mg -~ protein, respectively. IDPase and ATPase activities are in ~mol P~liberated, rain-~ • mg(-1 protein. NFR activity is expressed in ~mol ferricyanide reduced-~ •mg -~ protein. Protein recovery corresponds to 100 g fresh weight.

Crude microsomes 30% sucrose pellet Lower phase Upper phase

NADH-cytochrome c reductase

IDPase pH 6.5

SA

%

SA

0.59 0.24 0.10 0.17

100 12 4 0.8

0.61 0.44 0.39 0.11

Cytochrome c oxidase

NFR

%

SA

%

SA

%

SA

%

mg

%

100 21 16 0.5

0.91 0.90 0.77 0.05

100 29 21 0.2

2.4 3.9 3.0 5.9

100 47 31 7

1.0 1.5 0.7 4.5

100 44 17 13

31.5 9.2 7.8 0.9

100 29 25 3

Results

Isolation and characterization of plasma membranes The microsomal fraction was washed through a 30% sucrose cushion before laying o n t o a n a q u e o u s t w o - p h a s e s y s t e m , e x a c t l y as indicated in M a t e r i a l s and Methods. All the enzymatic activities studied, strongly d e c r e a s e d d u r i n g t h e f i r s t s t e p of w a s h i n g . F o r e x a m p l e , 8 8 % of t h e a n t i m y c i n A - i n s e n s i t i v e NADH-cytochrome c reductase was discarded, w h i l e 4 4 % of t h e m i c r o s o m a l A T P a s e w a s r e c o v e r e d ( T a b l e I). After partitioning, the different activities d e t e c t e d w e r e e s s e n t i a l l y p r e s e n t in t h e l o w e r p h a s e , e x c e p t for A T P a s e w h i c h w a s p r e s e n t i n b o t h p h a s e s ( T a b l e I). I n a d d i t i o n , o n l y t h e A T P a s e specific a c t i v i t y a s s o c i a t e d w i t h t h e upper phase increased during the membrane preparation (4.5-fold). T h e NFR activity d e c r e a s e d d u r i n g t h e p r e p a r a t i o n , s i n c e ferric y a n i d e c a n a c c e p t e l e c t r o n s f r o m b o t h endoplasmic reticulum and mitochondria redox s y s t e m s [2]. T h e r e f o r e , t h e m a i n a c t i v i t y of N F R w a s d e t e c t e d i n t h e l o w e r p h a s e (31%, T a b l e I), w h i l e 7 % of t h e t o t a l a c t i v i t y r e m a i n e d in t h e u p p e r p h a s e . A t t h i s s t a g e of t h e p u r i f i c a t i o n , o n l y 3 % of t h e m i c r o s o m a l

ATPase pH 6.5

Protein

Table Ii. Characterization of ATPase and NFR activities. Assays were carried out at 38°C for ATPase activity and at 25 °C for NFR activity, as described in Materials and Methods in the presence of LPC (LPC/protein = 5). Results are expressed as either specific activity (SA, i~molPi liberated or ferricyanide reduced, min-1. mg-1 protein, respectively) or as percentage of the activity in the complete medium.

ATPase activity

Complete medium - LPC - MgSO~ - KC1 pH 9.0 NaN s (150 ~M) Oligomycin (5 ~g/ml) Molybdate (100 ~M) - KC1 + KNOs (100 raM) Vanadate (100 ~M) SW26 (100 ~M)

SA

%

9.2 1.0 1.1 6.9 0.1 8.8 9.6 8.7 8.4 0.9 0.2

•100 11 12 75 1 96 104 95 91 10 2

NFR activity

Complete medium - LPC AntimycinA (20 ~M) KCN (500 ~Mt SOD (few crystals) Mn 2÷(500 ~M)

Catalase (few crystals}

SA

%

6.4 1.3 6.5 6.1 5.7 6.0 6.1

100 20 102 95 89 94 95

146 protein was r e c o v e r e d in the u p p e r phase (Table I). The u p p e r phase was f u r t h e r c h a r a c t e r i z e d (Table II). A T P a s e a c t i v i t y was Mg2+-ATPdependent, w e a k l y s t i m u l a t e d by potassium, and inhibited by v a n a d a t e or SW26 (both known to be specific inhibitors of the plasma m e m b r a n e A T P a s e [27,28]). The absence of activity at pH 9.0 and the v e r y faint inhibition by azide, olygomycin, n i t r a t e and m o l y b d a t e demonstrc.ted the low levels of mitochondria, vacuolar and cytoplasmic contamination. The characterization of this p r e p a r a t i o n was similar to those of plant plasma m e m b r a n e of high p u r i t y [ 2 9 - 31]. N F R activity was s t r o n g l y increased by L P C (Table II). I t was not inhibited either by mitochondrial inhibitors (antimycin A or KCN), or by catalase and SOD, and was not stimulated by Mn ~÷. The p r e s e n c e of mitochondrial contamination and the implication of superoxide radicals [32], in the reduction of ferricyanide, could thus be discounted. The membrane fraction, enriched in a v a n a d a t e - s e n s i t i v e A T P a s e and identified as plasmalemma, s h o w e d also N F R activity. In the absence of LPC, m a x i m u m N F R activity was obtained at pH 7.0 (Fig. 1). In the presence of d e t e r g e n t , this activity was s t r o n g l y

6

.>

ni.i. z 2

5

6

7

8

9

pH Fig. 1. Effect of pH on NFR activity in the absence (n) or in the presence (11)of LPC (LPC/protein = 5). Assays were carried out as described in Materials and Methods in 2 ml of 50 mM Tris- Mes buffer at the indicated pH. Specific activity is expressed as/~mol ferricyanide reduced • rain-~ •mg-1 protein. 1.s

•~"

1.o

~

0.5

0

2

B

1

-0.1

1/ferricyanide

0

0.1

concentration

0. .1 (t~M -1)

0 1/NADH

concentration

• (~LM1

Fig. 2. Doublereciprocal plot of NFR activity versus (A) ferricyanide and (B) NADH concentration in the absence (n) or in the presence (11) of LPC (LPC/protein = 5). Kinetics were followed at 25°C in 2 ml of 50 mM Tris-Mes buffer (pH 7.0) containing (A) 0.25 mM NADH and (B) 0.25 mM ferricyanide and 10 t~g protein. Various concentrations of (A) ferricyanide and (B) NADH were added. Specific activity is expressed as ~mol ferricyanide reduced • rain-1 • mg-~protein.

147

Table III. Kinetic parameters of the NFR activity as evaluated from Lineweaver-Burk representation. K m values were expressed as ~M and V values as ~anol ferricyanide reduced, rain -1. mg -1 protein, data from 3 independent experiments. -LPC

+LPC

Km

Vma,

Km

~ax

Ferricyanide pH 7.0

92.0 ± 6.4 14.3 ± 3.3

2.3 ± 0.5 1.2 +_ 0.3

69.0 ± 5.3 10.6 _+ 0.9

5.9 _+ 1.1 2.6 _+ 0.3

NADH pH 7.0

93.3 ± 9.9 17.0 ± 0.5

2.0 +_ 0.3 1.0 _ 0,1

64.0 _ 6.7 13.3 _ 0.9

5.3 _+ 0.3 2.7 ± 0.2

Ferricyanide pH 5.0

92.3 _+ 6.9 17.3 +_ 0.9

3.8 - 0.2 1.8 _ 0.1

NADH pH 5.0

75.0 _+ 9.9 18.3 _+ 2.4

3.8 -+ 0.1 2.1 + 0.1

enhanced and a second peak of activity was observed at p H 5.0. Therefore, the N F R activity showed two p H optima (Fig. 1). Similar results were obtained with Triton X-100 (data not shown). The dependence of the N F R activity on

NADH and investigated

ferricyanide concentrations was b y l i n e a r r e g r e s s i o n a n a l y s i s of

Lineweaver Burk plots. In the absence or prese n c e of L P C , s u b s t r a t e saturation curves clearly revealed biphasic kinetics. Thus, two K m a n d t w o Vmax w e r e d e t e r m i n e d .

Figure 2 repre-

0.250,

"~"",.= (1) 0 C t~ j~ !_ 0

O~

j~

(1)

0.200

0.150

0.100

.......

0.050

0

0

50

100

i;:::;=

..... t

150

20O

LPC concentration (l g/ml) Fig. 3.

Solubilization of membrane vesicles by LPC. Assays were carried out at 25°C in 1.5 ml T r i s - Mes buffer (pH 7.0) at the following membrane protein concentrations: (1) 38 pg/ml, (2) 32 ~g/ml, (3) 25 ~g/ml and (4) 19 pg/ml. Solubilization was followed by turbidity determinations at 400 nm and was judged complete when further addition of LPC no longer led to a decrease in absorbance.

148

E 1so :::k v

o

100

, m

f

~o eo

5O

.I

f •

0

(3 _3

0 0

I

!

I

I

10

20

30

40

protein

concentration

50

(gg/ml)

Fig. 4. Phase diagram for complete m e m b r a n e vesicle solubilization by LPC. Linear r e g r e s s i o n is [DT]t = 2.4 x [protein] + 26, rc = 0.98.

sents the results of a typical experiment, and Table III summarizes the values obtained from ferricyanide and NADH kinetic analysis. Effect of L P C on membrane solubilization and N F R activity Stepwise addition of LPC to the plasma membrane preparation led to a progressive

'J"

1500

decrease in absorbance (Fig. 3). In order to determine the LPC concentration needed for complete membrane solubilization, we used the intercept of the straight lines relating to the absorbance decrease resulting from the detergent addition, and the constant level reached when further LPC addition no longer changed absorbance (Fig. 3). These experiments clearly show that membrane solubilization and detergent concentration are linearly related. Thus, a phase diagram was obtained by plotting detergent concentrations versus the corresponding membrane protein concentrations (Fig. 4). This relation is described by the following equation [DT]t = Re × [protein] [DT]f, where [DT]t is the total detergent concentration, Re the effective solubilization ratio and [DT]f the free detergent concentration (CMC). In our experimental conditions, Re was 2.4. Re and CMC values were constant in the pH range (5.0 and 7.0), and insensitive to temperature between 25 ° and 38°C. Simultaneously to membrane solubilization, the stimulation of NFR and ATPase activities by detergent was measured at different detergent/protein ratios (R).

B

I I Y

i

1000

- -

r'l

I

rt

•

l w

0L~,

I

I,

I

- 5

0

Re

5

R

r

, 1

-20

-10

0 Re

0

R

Fig. 5. Stimulation of N F R and A T P a s e activities as a function o f R (R = ([DT]t - CMC)/[protein]): ( • ), N F R at pH 7.0; (m), N F R at pH 5.0; ([::]), A T P a s e . All assays w e r e carried out at 25 °C, (A) LPC t r e a t m e n t : maxima specific activities were 6.4 and 4,9/~nol ferricyanide r e d u c e d • rain -1 • m g -1 for N F R activities at pH 7.0 and 5.0, respectively, and 3.1 ~ n o l Pi liberated" rain -~ • m g -~ protein for A T P a s e . (B) Triton X-100 t r e a t m e n t : Re = 0.95 and maxima specific activities were 5.1 and 3.6 pmol ferricyanide r e d u c e d " rain -~ " m g -~ protein for N F R activities at pH 7.0 and 5.0, respectively, and 1.7 ~mol P~ liberated" rain -1" m g -~ protein for A T P a s e .

149

At low detergent concentrations, NFR activity at pH 7.0 was stimulated and reached a plateau for R < 0 (Fig. 5A). In contrast, this activity was strongly enhanced by LPC at pH 5.0 and like the ATPase activity, reached a plateau at a detergent/protein ratio close to Re (Fig. 5A). In addition, study of the membrane solubilization by Triton X-100 ([Triton]t = 0.95 [protein] + 163, concentrations expressed in ~g/ml) and the corresponding stimulation of ATPase and NFR activities, showed similar results (Fig. 5B). However, inhibition of ATPase activity could be observed at R-values higher than -7.5, i.e. at Triton X-100 concentrations higher than 0.0125% (w/v), whereas NFR activities did not seem to be inhibited.

Table IV. Effect of thermolysin on plasma membrane vesicles. Thermolysin was used to probe polypeptides accessible from the external face of the membrane vesicles, as described by Joyard et al. [34]. 300 ~g of membrane protein was incubated for 30 rain at 4°C in the following medium: 30 mM T r i s - - M e s buffer (pH 7.0), 100 ;Ag of thermolysin and 1 mM CaCI~. The digestion was terminated by the addition of 10 mM EGTA. A control experiment was carried out under the same conditions except that the incubation medium also contained 10 mM EGTA. After digestion, membranes were centrifuged twice 1 h at 80 000 × g in order to remove thermolysine CaC12 and EGTA. Protein and enzymatic activities were measured as described in Materials and Methods in the absence (**) and in the presence of LPC (*). Results are the means of 3 separate treatments with two replicates. They are expressed as total protein (~g} and as total activities (ATPase activity in nmol Pi liberated • rain ~ and NFR activities in nmol ferricyanide reduced, min-1). Thermolysin

Discussion

Plasma membrane preparation Plasma membrane of high purity could be prepared either by sedimentation through a sucrose gradient followed by washing with Triton X-100 [20,29,30], or by two-phase partitioning [8,9,13,21,31]. In our study, these two methods were applied, i.e. microsomes were centrifuged through a 30% sucrose cushion before layered onto the two-phase system. During this preliminary sedimentation step, 70-90% of the contamination was eliminated, while 44% of the initial ATPase activity was recovered {Table I). By this method, only 29% of the crude microsomal proteins will be layered onto the two-phase system. This technique thus allows the yield of the partitioning step to be increased, since most of the contaminants are discarded. This could be useful in experiments which require a large amount of membranes, such as in the purification of inside-out vesicles by counter-current distribution of pure plasma membranes [33]. NFR activity Using the method described above, we have purified plasma membranes and studied the associated NFR activity. This NFR activity showed two pH optima and presented biphasic kinetics. This suggests the presence of at least

Protein ATPase* NFR pH 5.0* NFR pH 7.0**

% of control

Control

100 ~g

163 737 697 224

103 721 799 86

± 17 ± 4 _ 41 ± 33

__ 5 _+ 18 _+ 28 ± 20

63 98 114 38

two NADH-ferricyanide reductases, exhibiting different sensitivities to the detergents. Whatever their precise origin is, two hypotheses could explain the biphasic kinetics and the second pH optimum revealed by LPC addition. Both NFR activities could proceed either from a transmembrane protein exhibiting two catalytic sites or from two distinct enzymes. However, the first hypothesis is hardly credible since redox activities can be solubilized by Triton X-100 [13,17], in contrast with the ATPase [20]. In addition, when microsomal membranes were gently treated with thermolysin, 62% of NFR activity measured at pH 7.0 without detergent was lost. Under the same conditions, activities determined in the presence of LPC were recovered (NFR at pH 5.0 and ATPase) {Table IV). Thus the NFR activities exhibited a different sensitivity to thermolysin. Moreover, in the presence of LPC, kinetic parameters of redox activities at both

150 pH values studied were different (Table III). Therefore, it can be postulated that the observed N F R activities result from several components located on the plasmalemma. This is in agreement with the results of Luster and Buckhout [13], who reported a partial purification of multiple electron transport activities in plasma membranes from maize roots. N F R localization Fractions isolated by two-phase partitioning contained sealed right-side-out vesicles [21], and therefore the determination of total ATPase activity requires the addition of detergent [35]. Cytochemical localization of N F R has been demonstrated at the inner side of plasma membrane vesicles [36]. Askerlund et al. have also shown the existence of donor and acceptor sites on the internal face of the plasma membrane of sugar beet, but without evidence to show that similar sites are localized on the outer surface [37]. In this work, we have studied the effects of LPC on membrane solubilization and, on NFR and ATPase activities. Values for ATPase and NFR latencies were 89% and 80%, respectively (Table II). This difference obtained with the same membrane preparation, was reproducible through several experiments. This lower NFR latency could result from the presence of a NADH oxidase directly accessible to the substrates and possibly located on the external face of the plasmalemma, as reported by Lin [6] and Rubinstein et al. [7]. However, the main N F R activity remains at the internal face of the vesicles. The stimulation of the N F R at pH 7.0 was independent of the level of membrane solubilization (Fig. 5). In contrast, N F R at pH 5.0 and ATPase activities were maximum at the LPC concentration required for complete solubilization of the vesicles. In addition, NFR at pH 5.0 and ATPase activities were not sensitive to a gentle thermolysin treatment (Table IV). Under more drastic experimental conditions (higher thermolysin concentrations), both these activities were decreased by 30% while the NFR at pH 7.0 was destroyed. Thus, we can

rule out the possibility that thermolysin will be inefficient on these proteins, and we postulate that this observation corroborates the membrane solubilization data. From all these experiments, it is suggested that the NFR with an optimum at pH 5.0 could be located on the cytoplasmic face of the plasma membrane, where the pH is about 7.0, and NFR with an optimum at pH 7.0 could be at the external surface where pH values may approach 5.0. Such a situation could seem strange and needs some explanations about the putative physiological significance of redox system associated with the plasma membrane. Firstly, Marr~ et al. reported that ferricyanide reduction depends on a plasmalemma system transporting only electrons to the extracellular aceeptor, with consequent potential depolarization and cytoplasmic acidification [38]. Secondly, the activation of an enzyme system, generating 02 and dependent on NAD(P)H oxidation, occurs on the plasma membrane upon incompatible recognition of host cells leading to hypersensitive cell death [39]. This activation could be triggered by a plasma membrane depolarization [40,41]. Moreover, the intracellular pH of elicitor-treated cells has been studied with 31p nuclear magnetic resonance spectroscopy [42]. These experiments revealed a decrease in cytoplasmic and vacuolar pH within 10 min following elicitation [42]. Therefore, it has been clearly shown that the activity of the redox system associated with the plasma membrame, which is possibly involved in the hypersensible reaction, generates cytoplasmic acidification. In addition, induction of hypersensitive reaction in plant cells proceeds through the activation of a passive plasmalemma K÷/H ÷ exchange mechanism, leading to an alkalinisation of the extracellular medium. This alkalinisation is about 0.8 pH units in tobacco cells [43] ; Atkinson and Baker concluded that the pH between the plasmalemma and the cell wall of bean cells must reach a minimum of 7 . 2 - 7.6 [44]. These pH modifications of the bulk phases on each side of the plasma membrane could be much lower than modifications occurring close

151

to the plasmamembranes faces. Therefore, the redox activity located on the cytoplasmic face of the plasmalemma should be active in a local acidic pH range, while the activity located on the external face of the plasma membrane should be active at alkaline pH range, which is in agreement with the results reported here.

Direct effect of detergents on plamalemma enzymes The enzyme activation by LPC, could be due not only to vesicle disruption and the resulting accessibility of substrates to enzyme but also to activation of the enzyme by the detergent. Assuming that the NFR activity at pH 7.0 is located at the external face of membrane vesicles, the LPC stimulation reported on Fig. 5 could result either from the permeabilization of the inside-out vesicles or from a direct effect on the enzyme. However, stimulation resulting from the addition of LPC was about 300% (much higher than the expected value from the permeabilization of the 11% inside-out vesicles). Stimulation of the NFR activity at pH 7.0 was maximum when R value was negative i.e. when detergent was present as monomers. In these conditions, the detergent only altered the lipid environment of the enzyme and thus permitted a better accessibility of the substrate. This possibility was confirmed by the change of the kinetic parameters in the presence of LPC (see K m values _+ LPC at pH 7.0, Table III). Substrate affinity increased with addition of LPC. Similar conclusions have been proposed about ATPase stimulation by LPC [45]. Additionally, Palmgren and Sommarin [46] have suggested that lysophospholipids could be part of a regulation system from plant plasma membrane H÷-ATPase activity in vivo , by an involvement of endogenous phospholipase [47]. In addition, LPC could provide an appropriate lipid environment for the enzyme activity. By contrast, Triton X-100 delipidated membraneous enzymes such as ATPase [45], which became unstable and then inactivated. Sandstrom et al. showed over the same range of concentrations (>0.01%), that Triton X-100 inhibited ATPase activity, and this was prob-

ably due to a delipidation of the enzyme [45]. This "could explain the lower stimulation induced by Triton X-100, as compared to the LPC activation (Fig. 5). The similar effects of LPC and Triton X-100 reported, leads us to the conclusion that the observations are independent of the nature of the detergent used. Finally, in latency determinations, the CMC must be taken into account. For example, experiments carried out with 25 ~g/ml LPC and 5 ~g/ml protein, lead to a detergent/protein ratio equal to 5. Therefore this value which is higher than 2.4 (i.e. the effective solubilization ratio, Re), would indicate that membranes are totally solubilized. However, if R is calculated by the equation previously described (Fig. 4), it is equal to -0.2, which indicates that total membrane solubilization is not achieved. Thus, the greatest care must be taken in the evaluation of latency using detergents. References 1

2

3

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