An NADH-diaphorase is located at the cell plasma membrane in a mouse neuroblastoma cell line NB41A3

Biochimica et Biophysica Acta, 1183 (1994) 513-520

513

© 1994 Elsevier Science B.V. All rights reserved 0005-2728/94/$07.00

BBABIO 43947

An NADH-diaphorase is located at the cell plasma membrane in a mouse neuroblastoma cell line NB41A3 R.

Zurbriggen and J.-L. Dreyer

*

Department of Biochemistry, University of Fribourg, CH-1700 Fribourg (Switzerland)

(Received 13 April 1993)

Key words: Transplasma membrane redox; Cellular activation; Diaphorase; Plasma membrane; Neuroblastoma celt line Plasma membranes from most mammalian cells display significant transplasma membrane oxidoreductase (PMO) activity. The enzymes use an extracellular, impermeant electron acceptor as substrate and intracellular reduced pyridine nucleotide as electron donor. The plasma membrane from a neuroblastoma cell line, NB41A3, has been biotinylated and purified by immunoprecipitation with avidin and antiavidin-antibodies. The protein recovery of an immunopurified membrane preparation was < 0.15% of the protein content in the cell extract. The preparation displays an increase in the specific activity of PMO's of 15- to 20-fold compared to the activity in whole cells. With this approach the presence of a NADH-diaphorase within the cell plasma membrane can be demonstrated. This activity accounts for about one third of the total cellular diaphorase activity. The PMO activity cannot be attributed to an increased permeabilization of the plasma membrane induced upon biotinylation nor to intracellular activity from lysed cells. Activation of basal metabolism (glycolysis) stimulates PMO activity up to approx. 54%, presumably through a raise of the intracellular NADH store. PMO also promotes cell growth at low substrate concentrations (0.1-1 p.M). Native gel electrophoresis of iminobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several PMO activities at the plasma membrane.

Introduction Plasma m e m b r a n e dehydrogenases (PMO's) represent a poorly defined goup of oxidoreductases that have been demonstrated in all cells tested [1], but their real function have not been established so far. The enzymes were initially observed in studies related to oxygen stress but subsequent investigations also showed a possible implication in the control of cell growth and differentiation [1]. Cell proliferation is generally associated with a more oxidizing state in the cytosol, e.g., a higher N A D + / N A D H ratio, in most mammalian cell types investigated [2,3]. In previous studies we have described the presence of a variety of oxidoreductases with distinct properties in purified synaptic plasma m e m b r a n e s from rat or beef brain. Depending upon the source material from five to seven distinct transplasma m e m b r a n e oxidoreductase activities were described [4] and their subunit composition has been characterized in more detail. Nevertheless, it was un-

* Corresponding author. Fax:+41 37 826341. Abbreviations: DCIP, dichlorophenol-indophenol: DMEM, Dulbecco's modification of Eagle's medium. SSDI 0005-2728(93)E0143-E

clear whether the observed heterogeneity should be attributed to multiple isoenzymes in the plasma membrane of a single neuronal cell or rather to the heterogeneity of the plasma m e m b r a n e preparations used, which was m a d e from a mixture of pre- and post-synaptic and glial membranes. The putative involvement of P M O ' s in the modulation of important processes within the cell m e m b r a n e has been described, particularly on the Ca 2+ effiux mechanisms, on the Mg 2÷A T P - d e p e n d e n t Ca 2+ pump, on the N a + - C a 2÷ exchanger [5,6] and on intracellular protein phosphorylation or associated mechanisms [7]. This stresses the potential roles of t r a n s m e m b r a n e redox in plasma m e m b r a n e activation. P M O ' s transfer electrons from the intracellular pool of reduced nicotinamide nucleotide to an extracellular electron acceptor, but the nature of the physiological acceptor substrates are not known at present. The enzyme activity is usually tested as a NAD(P)H-diaphorase using artificial impermeant electron acceptors as oxidized substrates (dichlorophenol-indophenol, juglone or ferricyanide) in the presence of pyridine nucleotides as electron donors. Attempts at isolating and purifying the enzymes have failed however, due to the difficulty of producing highly purified plasma mem-

514 branes, devoid of contaminants by NADH-dehydrogenases from intracellular membranes. Finally, most studies pertaining to transplasma membrane oxidoreductases have been merely phenomenological in nature so far. For these reasons the very occurrence of these enzymes at the level of the plasma membrane has long been a matter of controversy. In the present study we have developed a different approach which demonstrates the presence of an oxidoreductase at the level of the plasma membrane and eventually introduces methodologies for the successful enzyme purification from limited amounts of source material. The plasma membrane from a neuroblastoma cell line, NB41A3, has been biotinylated and affinity purified with avidin and anti-avidin antibodies. This report unambiguously describes the presence of a NADH-diaphorase activity in the cell plasma membrane which accounts for more than one-third of total cellular diaphorase. Material and Methods

Chemicals Aprotinin, avidin, biotin, pepstatin, sodium nitrite, insoluble Protein-A, Triton X-100, ferricyanide and NAD(P)(H) were all purchased from FlukaTM; Antiavidin-Ab, DCIP, DT-diaphorase (EC 1.8.1.4), 2-iminobiotin hydrazide, oxirane and PMSF were from SigmaTM, growth media including DMEM, fetal calf serum, trypsin and PBS were from Gibco; [3H]thymidine was from American Radiolabeled Chemicals. Growth of neuroblastoma cells NB41A3 NB41A3 cells were grown according to Ref. 8 under an atmosphere of 5% CO 2 and 95% air at 37°C. The growth medium was Dulbecco's modification of Eagle's medium (DMEM) containing 3.7 g / l NaHCO3, 4.5 g/1 D-glucose, 0.11 g / l sodium pyruvate and 0.58 g/1 glutamine, 10% fetal calf serum, 50 units/l of penicillin and 50/zg/1 of streptomycin, pH 7.4 and the cells were maintained in a similar medium containing 10% DMSO and 20% fetal calf serum. Confluent monolayer cultures were then trypsinized in the presence of 0.25% trypsin and 0.1% EDTA and the cell suspension was pelleted at 500 × g for 10 min. The pellet was washed with PBS, recentrifuged and diluted in the same buffer to the appropriate final concentration. For activation studies cells washed in PBS were incubated for 10 rain with either 1 mM glucose or deoxyglucose in PBS together with the indicated agents. The effects of transplasma membrane dehydrogenases on cell growth were investigated by incubating the cells for 24 h in the presence of various concentrations of ferricyanide and 0.1 /~Ci in 1 ml of [3H]thymidine. Fetal calf serum and pyruvate were omitted in the growth medium. In some studies cells were preincubated for 24 h with substrates alone prior to addition

of [3H]thymidine for 8 h. After 24 h the growth medium was removed. Cells were washed three times with cold PBS and treated for 1 h at 37°C with 0.25% Trypsin and 2% Triton X-100 and the radioactivity was measured. NADH-DCIP reductase activity measurements in NB 41,43 NADH-DCIP reductase activity was tested according to [9]. Extracts or membranes from 106 cells in 800 ~1 PBS buffer were incubated for 1 rain before addition of 100/xl of 1.3 mM NADH and 100 /~1 of 0.51 mM DCIP. After further 90 s incubation at room temperature the absorption change at 600 nm was measured for 2 rain. Where necessary, appropriate modifications were performed, as mentioned in the figures. Where the activity of intact cells was measured, the addition of NADH was generally ommitted unless specified otherwise. Ferricyanide reductase was determined according to the same outlines using 0.5 mM ferricyanide as a substrate [9]. Other enzyme activities (including cytochrome c reductase, cytochrome oxydase and lactate dehydrogenase) were determined according to the methods previously described [4]. Membrane biotinylation and purification All procedures were carried out at 4°C unless otherwise specified. NB41A3 cells washed twice in PBS and resuspended in 1 ml PBS were added to freshly prepared 2-iminobiotin-ac~jl-azide according to [10], incubated for 30 min at 4°C and washed three times with PBS. After addition of 0.1 /xg/ml aprotinin, 1 mM EDTA, 0.3 ~M PMSF and 0.7 t~g/ml pepstatin, the labeled cells were homogenized 5 times for 10 s with a Polytron PT 1200 and centrifuged for 1 h at 100000 × g. Membrane proteins were solubilized for 2 h in 50 mM borate (pH 8.0) containing 2% Triton X-100, 1 mM EDTA, 0.1 ~ g / m l aprotinin, 0.3 mM PMSF and 0.7 /~g/ml pepstatin and the unsolubilized material was sedimented at 16000 × g for 15 min. Immunoprecipitation was achieved in 50 mM borate buffer (pH 9.5), containing 0.3 M NaCI, 1 m g / m l bovine serum albumin and 0.5% Triton X-100. Solubilized extracts were incubated for 2 h in the presence of an excess of avidin. Anti-avidin antibodies (diluted 1:200) were added and incubated for 2 h. The immuno-complex was then precipitated with 25 /xl insoluble protein A, incubated for 2 h, centrifuged and washed twice with the incubation buffer, then twice with 10 mM Tris-HC1 (pH 8.0). Proteins were then released by incubating the immunoprecipitate with 1 mM biotin for 3 h. Other assays Protein concentrations were determined according to the micromethod of Pierce TM.The effects of biotiny-

515 cytochrome c - oxidase

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Fig. 1 Effects of cell homogenization on plasma membrane dehydrogenase activity. ( A - D ) Cells and cell homogenates were mixed at different ratio as indicated and the activity measured. (A) Ferricyanide reductase activity; (B) cytochrome oxydase activity; (C) ferricyanide reductase after mild trypsinization. 5' 10 6 cells were incubated with 1 m g / m l trypsin for 30 min in PBS buffer; (D) DCIP reductase activity; (E) 5.10 6 cells were homogenized for several seconds, as indicated, and the activity was measured in the whole cell homogenate; (F) ferricyanide activity in relation to cell concentration using non-homogenized cells.

516 lation on the cells were tested by electron microscopy by the gold-streptavidin procedure described by Bush et al. [11]. Non-denaturing gel electrophoresis was performed at 4°C in 7.5 to 15% (w/v) polyacrylamide gels containing 0.1% Lubrol, 0.03% deoxycholate and 0.375 M Tris-HCl buffer (pH 8.8). approx. 80 /zg of solubilized membranes (equivalent to immunoprecipated membranes from a batch of approximately 500-106 iminobiotinylated cells) were applied per slot. Specific assays of diaphorase on PAGE-gels were performed under anaerobic conditions by overnight incubation of the gel in a degased dessicator with a solution of 1 m g / m l NADH and 0.75 m g / m l Tetranitroblue-tetrazolium in PBS buffer in the presence of 5 /xM FAD and 1.5 /xM bovine serum albumin. Iminobiotinylated membrane proteins were also revealed after Westernblotting with avidin-peroxidase by chemiluminescence (System ECL, AmershamTM using Kodak X-O-Mat films). Results

and Discussion

Plasma membrane oxidoreductase activity in NB41A3 Plasma membranes from most mammalian cells investigated so far display significant transplasma membrane oxidoreductase activity. In NB41A3 neuroblastoma cells a high activity was found in intact cells incubated with an extracellular, impermeant electron acceptor such as DCIP or ferricyanide. The activity was directly proportional to the amount of cells (Fig. 1F). In order to test whether this activity is localized in the plasma membrane, the plasma membranes of intact

TABLE I

Plasma membrane dehydrogenase activity in NB41A3 Protein and enzyme activities were measured on intact cells from 5.106 cells ('Cells'), on cells homogenized 5 times for 10 s with a Polytron PT 1200 ('Extract') and on purified plasma m e m b r a n e from 5.106 cells, iminobiotinylated and affinity purifed ('Plasma membranes'). See the Material and Methods section for the detailed procedure. DCIP reductase was measured in presence of 0.13 m M N A D ( P ) H in all cases, including intact cells, for a better comparison. The data for DCIP reductase activity with N A D H , expressed in m U per mg protein, are average value from 9 sets of experiments, each one performed in duplicate. Other data are average values of at least two sets of experiments, each one in triplicate, n.d. = no activity could be detected in the fraction. DR: DCIP-reductase activity (plasma m e m b r a n e dehydrogenase measured as DCIP reductase).

Proteins (mg) D R w, N A D H ( m U / m g ) D R w. N A D P H ( m U / m g ) Cyt c Oxidase ( m U / m g ) Cyt.c reductase ( m U / m g ) LDH (mU/mg)

Cells

Extract

Plasma membranes

6.2 8.7+05 5.8 + 0.4 3.5 + 0.9 4.9 + 0.8 53.6 + 8.6

6.6 25.5:1:0.2 11.3+0.8 27.3+0.7 13.3+1.8 1115.0+9.6

0.01 136.0+41 n.d. n.d. n.d. n.d.

NB41A3 cells from confluent cell cultures were labeled with iminobiotin-acylazide. The cells were then homogenized, membrane proteins were extracted with Triton X-100, and proteins from the plasma membrane were finally affinity precipitated with avidin and anti-avidin antibodies. This procedure yielded a 15.6-fold increase in the specific activity of plasma membrane dehydrogenase compared to the activity in intact cells, or a 5.3-fold increase over the crude cell extract (Table I). This purification step yielded less than approx. 0.15% of the total protein content from the cell extract. Assuming that plasma membranes roughly account for < 5% of total cellular membranes, this purification factor is very satisfactory. The high purification factor - associated both with the low protein recovery and the high specific activity - which has been achieved in this single step is a good indication for the specific location of DCIP reductase at the plasma membrane level. As controls, neither cytochrome oxidase, a typical mitochondrial marker enzyme nor NADPH-cytochrome c reductase, a marker of microsomal membranes, could be detected in preparations of purified plasma membranes. Lactate dehydrogenase, a cytoplasmic enzyme loosely associated with the plasma membrane, is also undetectable in these preparations. Cell homogenization displays an increase in the activity measured as DCIP reductase (25.5 mU in the extract compared to 8.7 mU in intact cells), because it makes the DCIP accessible to intracellular oxidoreductases (e.g., mitochondrial NADH-dehydrogenase or microsomal DT-diaphorase) which also reduce DCIP unspecifically. From this increase, one can infer that 16.8 mU accounts for intracellular DCIP reductase activity, i.e., 65.8% of the total measured activity. Then 34% of the activity appears to be associated with the plasma membrane. In vitro studies with extract show that both NADH and NADPH can serve as electron donors, but NADH yields 2.3-fold higher specific activities (Table I). In purified plasma membranes no DCIP reductase activity could be detected with NADPH as a substrate, in contrast to what is observed for most intracellular DT-diaphorases which usually are more active with NADPH. Specific inhibitors of intracellular dehydrogenases such as rotenone, antimycin, cyanide have no effects on DCIP- or ferricyanide reductase activity, indicating that the activity associated to the plasma membrane is distinct from mitochondrial dehydrogenases. Neither has dicoumarol, a specific inhibitor for DT-diaphorases, any effect in purified membrane preparations, even at high concentrations (up to 30 /xM). On the basis of these data, a number of control experiments were performed to exclude that artefacts might account for the high DCIP reductase activity observed in the purified plasma membrane preparations. First we excluded that the observed activity was

517 not merely due to an interaction between the various reagents, e.g., biotin and DCIP. But neither the free reagents together nor iminobiotinylation of e.g., albumin or heat-inactivated membranes would display any DCIP reductase activity. This made sure that iminobiotinylation does not interfere in any manner with the enzyme test for DCIP- or ferricyanide reductase. Second, a number of intracellular NADH-dehydrogenases would react unspecifically with the electron acceptors used in our assay. Therefore we further checked the possibility that the activity is not due to the labeling of intracellular membranes - e.g., from permeabilized, unviable cells in the culture dish which would co-precipitate upon affinity purification. However this possibility could be excluded from the following controls: (1) Trypan blue stains less than 2% of cells in viability tests undertaken prior to biotinylation. Trypan blue tests performed after biotinylation did not show any increase of non-viable cells, indicating that the biotinylation procedure did not damage the cells. Furthermore the DCIP reductase activity is not increased in intact cells after biotinylation. This excludes that biotinylation would permeabilize cells and make intracellular dehydrogenases accessible to the impermeant electron acceptor. (2) The total diaphorase activity in cell extracts was approx. 168 mU. If this activity comes from the < 2% permeabilized cells which would be labeled intracelullarly, a total activity of 3.3 mU is expected in purified plasma membranes. Then, however, the activities of marker enzymes for intracellular organites should increase also, which is not the case. The total activity for PMO's was 1.36 mU in purified membranes and we found no detectable activity for cytochrome c reductase and for cytochrome oxidase, against what is anticipated if intracelullar labeling occurs and despite of the fact that the anticipated activities would then be easily measured under our experimental conditions. In our assays the detection limits for the marker enzymes are 2-10-fold lower than the enzyme activity from extracts of 2% of labelled cells. In addition the properties of the PMO's do not correspond to those of common intracellular diaphorases with respect to inhibition by dicoumarol and to pyridine nucleotide requirements. (3) Electron microscopy (not shown) from intact iminobiotinylated cells stained with avidin-peroxidase exhibits labeling only on the cell plasma membrane and on endosomes located very close to the plasma membrane. Intracellular membranes, mitochondria and microsomes display no labeling whatsoever. The slight labeling of endosomes can be due to the fact that in these experiments the biotinylation was carried out at room temperature and in the absence of inhibitors for endocytosis. All further biochemical studies reported

below were always performed at 4°C, to exclude unspecific effects and endocytosis. (4) Unsolubilized iminobiotinylated membranes could be affinity purified on an avidin-Sepharose column. Marker enzymes for intracellular oxidoreductases lactate dehydrogenase and cytochrome oxidase - were found in the flow-through, but absent in the plasma membrane fractions. As a control biotin-saturated Avidin-Sepharose column did not retain DCIP reductase activity, excluding an unspecific binding of labeled membranes. Together, these controls indicate that the DCIP reductase observed in purified plasma membranes is not related to intracellular membranes. Permeabilization studies These data were also corroborated by controlled permeabilization experiments summarized in Fig. 1. In this set of experiments the enzyme activity released after cell homogenization was measured under various conditions. Cell homogenates were mixed in various proportions with intact cells and the resulting activity measured. Progressive homogenization resulted in an increase of DCIP- or ferricyanide reductase activity, by making these substrates accessible to intracellular oxidoreductases, as displayed from the slopes in Fig. 1. Nevertheless, extrapolation to 0% broken cells from these plots yields 22% ferricyanide reductase activity in intact cells (Fig. 1A), or 43% DCIP reductase activity (Fig. 1D), whereas no cytochrome oxidase activity can be associated to the plasma membrane in similar extrapolation plots (Fig. 1B). The higher lipophilicity of DCIP compared to ferricyanide could account for the higher percentage of plasma membrane-associated DCIP reductase activity, assuming a better substrate accessibility. The high specific activity deduced from the extrapolation plots fully agrees with the activity measured on intact cells. For these reasons, the activity observed in intact cells cannot be attributed to lysed cells. Upon mild trypsinization of intact cells before homogenization the ferricyanide reductase activity associated to the cell plasma membrane dropped from 22% to approx. 16% (Fig. 1C), indicating that the active site for the oxidized substrate is extracellular and can be subjected to proteolytic cleavage. Similar data were also obtained with DCIP as electron acceptor. Cells were also homogenized under mild conditions as an alternative: homogenization of intact NB41A3 cells for various time periods displays similar extrapolation patterns (Fig. 1E), that associates 43% of the measured ferricyanide reductase activity to the cell plasma membrane. PMO activity and intracellular metabolism Transplasma membrane-oxidoreductases transport electron from extracellular oxidized substrates to intra-

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Fig. 2. Effect of glycolysis on plasma m e m b r a n e dehydrogenase activity 1.106 cells in 8 0 0 / z l PBS buffer were incubated for 20 rain with or without addition (1 m M glucose, deoxy-glucose or KCN) as indicated. The suspension was then transferred into a cuvette before the addition of 100/xl of DCIP 0.5 raM. After further 90 s incubation at room temperature the absorption change at 600 n m was measured for 2 min.

cellular electron donors, generally NADH from the cytoplasm. Therefore, the enzyme activity is very much regulated by the intracellular basal metabolism that affects the N A D H / N A D balance and a modification of the intracellular N A D H / N A D ratio should modulate the activity of plasma membrane dehydrogenases. Changes in the redox state of pyrimidine nucleotides can hardly be quantified by means of direct measurements on the limited cell population available (< 10 6 cells), but such changes can be achieved qualitatively by means of activating or inhibiting glycolysis. Fig. 2 summarizes a series of experiments (each data point being performed at least twice in triplicate). Here, basal metabolism (glycolysis) has been activated in the presence of 1 mM glucose to increase the intracellular NADH concentration. This indeed stimulates the enzyme activity by approx. 54%. The effects is reversed in the presence of 1 mM deoxyglucose. Insulin, which potentiates glucose penetration into the cell, also produces an increase of the transplasma membrane oxidoreductase activity (up to 70% increase over the control). Cyanide has no effects on glucose stimulation, another indication that it is the cytoplasmic level of NADH which is involved for PMO's activation. From these data one can infer that the availability of intracellular NADH, which is modulated by the intracellular metabolism, would regulate the activity of transplasma membrane oxidoreductases. However, the NADH-pool has not been measured directly.

Effects of PMOs on cell growth Furthermore, it has been observed [1] that pyruvate in the culture medium acts as an electron acceptor for transplasma membrane oxidoreductases. This addition then yields a decrease of intracellular NADH stores

and prevents growth at higher concentrations [1]. A similar observation has been made in our studies on NB41A3. For this reason a number of experiments were performed where pyruvate was removed from the growth medium to prevent such a competition between the oxidized extracellular substrate and pyruvate as an electron acceptor. Under these experimental conditions cell growth can be affected by the substrates for DCIP reductase or ferricyanide reductase. The results of this set of experiments are displayed in Fig. 3. The cell growth was investigated under these conditions by

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mU / mg Fig. 3. Effects of plasma m e m b r a n e dehydrogenase activation on cell growth 1.0.106 cells have been grown for 24 h in the presence of various concentrations of ferricyanide or DCIP (as indicated) and with 0.1 /zCi [3H]thymidine. Fetal calf serum and pyruvate were omitted from the growth medium. After 24 h the growth m e d i u m was removed. Cells were washed three times with cold PBS, treated for 1 h at 370C with 0.25% Trypsin and 2% Triton X-100 and the radioactivity was measured.

519 two independent methods, either by measuring [3H]thymidine incorporation upon growth or by cell counting, both methods giving similar results. Ferricyanide strongly promotes growth at concentrations between 0.1-5/zM and gives an increase in cell growth of 75% of the control at 10 -6 M. DCIP with a lower redox potential is effective over a lower concentration range between 10 -8 and 10 -6 M, being optimal concentration at approx. 0.1/zM. Higher concentrations of the electron acceptors (above 10 -5 M for ferricyanide and DCIP) inhibit cell growth in both cases. It is unlikely that the observed effects are due to enhanced cell survival, since it is the total cell number counted that is increased by 54-75% of the controls at the optimal concentrations of the oxidants. So the data rather reflect true growth-promoting effects, in agreement with previous observations from the literature [1].

PAGE of plasma membranes under native conditions Plasma membranes were labeled, solubilized and immuno-purified as described. Gel electrophoresis of

"l" Fig. 4. Gel electrophoresis of purified plasma membranes from NB41A3 Gel electrophoresis under non-denaturing conditions was performed at 4°C in 7.5 to 15% (w/v) polyacrylamide gels containing 0.1% Lubrol, 0.03% deoxycholate and 0.375 M Tris-HCl buffer (pH 8.8). Immunoprecipated, solubilized membranes from approx. 500. 106 iminobiotinylated cells were applied per slot. PAGE-gels were stained for diaphorase under exclusion of air by incubation for 16 h in PBS containing 1 m g / m l NADH, 0.75 m g / m l Tetranitroblue-tetrazolium, 5/~M FAD and 1.5 ~ M bovine serum albumin.

the immunoprecitipate was performed under native, non-denaturating conditions and the diaphorase activity was revealed on the gel with nitroblue-tetrazolium. The preparation displayed two diaphorase-positive bands (Fig. 4). From the above-mentioned studies, these bands cannot be due to contaminants from intracellular, soluble diaphorases. It therefore appears that a homogeneous cell population can express several PMO's at the plasma membrane. The function and structure of the individual membrane-bound diaphorases remains to be elucidated. Conclusion We present some properties of a transplasma membrane redox system observed on a NB41A3, a cell line which develops as mature neurons and expresses the enzymes for the synthesis of neurotransmitters. The study was undertaken with a view to investigating the properties of transplasma membrane oxidoreductases on intact cells from a homogeneous cell population. This work describes the purification of plasma membranes in good yield and of good quality, devoid of intracellular membranes. With this procedure which overcomes many of the drawbacks previously encountered, a number of disputed issues could be settled: (1) the data unambiguously demonstrate PMO activity in the plasma membrane; (2) methods for their rapid purification (e.g., by means of gel electrophoresis under native conditions from solubilized plasma membranes) are developed; (3) plasma membranes from a homogeneous cell population display several PMOs, at least two in our preparations. Plasma membrane redox is measured as a NADHdiaphorase activity and therefore could be associated with diaphorases described in several neurons or glial cells [13] or in astrocytes primary cultures [14,15]. Recently a number of studies have shown that in certain neuronal cells the cytoplasmic DT-diaphorase is identical to nitric oxide synthase, the enzyme responsible for oxidizing arginine to citrulline and for producing the cellular messenger NO [16,17]. In brain, the NO synthase occurs in discrete neuronal populations and its distribution pattern is identical to that of DT-diaphorase [18]. Its distribution in the various brain areas is the matter of intense histochemical studies. Particulate NO synthase have also been described, but to our knowledge this type of NO-synthase has not yet been associated with the plasma membrane. It remains to establish whether the PMOs described in this study are related with DT-diaphorase/NO-synthase. The enzymes from the present study are not sensitive towards conventional inhibitors for DT-diaphorases (such as dicoumarol) or NO-synthase. The true function of the transplasma membrane oxidoreductase described in the

520 present paper remains therefore to be better established.

Acknowledgements The authors are indebted to Mrs C. Deforel-Poncet for skilled technical assistance and Dr. C. Waeber for critical comments on the manuscript. The present work was supported by a grant No. 31-27733.89 from the Swiss National Foundation.

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5 Bulliard, C., Marmy, N. and Dreyer, J.L. (1990) J. Bioenerg. Biomembr. 22, 645-662. 6 Bulliard, C. and Dreyer, J.L. (1991)J. Receptor Res. 11,653-663. 7 Dreyer, J.L. (1991) Adv. Biosci. 82, 681-684. 8 Augusti-Tacco, S., and Sato, R.T. (1969) Proc. Natl. Acad. Sci. USA, 64, 311-315. 9 Crane, F.L. and L6w, H. (1976) FEBS Lett. 68, 153-156. 10 Zeheb, R. and Orr, G.A. (1986) Methods Enzymol. 122, 87-89. 11 Bush, G., Hoder D., Reutter W. and Tauber, R. (1989) Eur. J. Cell Biol. 50, 257-262. 12 Laemmli, D.M. (1970) Nature 277, 680-684. 13 Ellison, D.W., Kowall, N.W. and Martin, J.B. (1987) J. Comp. Neurol. 260, 233-245. 14 Mersel, M., Malviya, A.N., Hindenlang, C. and Mandel, P. (1984) Biochim. Biophys. Acta 778, 144-154. 15 Malviya, A.N., Mandel, P. and Mersel, M. (1986) Biochim. Biophys. Acta 849, 288-294. 16 Knowles, R.G. and Moncada, S. (1992) Trends Biochem. Sci. 17, 399-402. 17 Hassal, C.J., Saffrey, M.J., Belai, A., Hoyle, C.H., Moules, E.W., Moss, J., Schmidt, H.H., Murad, F., Fostermann, U. and Burnstock, G. (1992) Neurosci. Lett. 143, 65-68. 18 Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H. (1991) Proc. Natl. Acad. Sci. USA 88, 7797-7801.