cotyls of etiolated soybean seedlings by two-phase partition was stimulated. 2- to 3-fold by auxins, indole-3-acetic add, 2,4-dichlorophenoxy acetic acid (2,4-D), ...
Plant Physiol. (1988) 86, 1264-1269 0032-0889/88/86/1264/06/$O1.0O/O
Auxin-Stimulated NADH Oxidase Purified from Plasma Membrane of Soybean' Received for publication July 21, 1987 and in revised form January 4, 1988
ANDREW 0. BRIGHTMAN, RITA BARR, FREDERICK L. CRANE, AND D. JAMES MORlu* Department of Biological Sciences and Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907 ABSTRACT NADH oxidation by plasma membrane vesicles purified from hypocotyls of etiolated soybean seedlings by two-phase partition was stimulated 2- to 3-fold by auxins, indole-3-acetic add, 2,4-dichlorophenoxy acetic acid (2,4-D), and a-nphthaleneacetc acid. The stimulation was concentration dependent in the presence or absence of detergent with a maximum for 2,4-D at 1 micromolar. The NADH oxidation activity was solubilized with the zwitterionic detergent CHAPS and purified by ion exchange chromatography and gel fitration approximately 2000-fold over the total homogenate. Both the partially purified fraction and an active band from nondenaturing gel electrophoresis revealed the same three bands when analyzed by denaturing gel electrophoresis. When obtained from plasma membrane vesicles from the region of rapid cell elongation, the NADH oxidase complex retained auxin responsiveness throughout purification
(3- to 5-fold stimulation by 1 micromolar 2,4-D).
A role for plasma membrane NAD(P)H oxidoreductases has been implicated in numerous cellular processes in plants, including proton efflux, potassium uptake, iron reduction, and membrane polarization (see reviews, Refs. 5 and 16). Evidence of a role for plasma membrane redox in growth has come from studies with animal cells where cell proliferation is stimulated by external oxidants, such as ferricyanide (6). However, with cultured carrot cells, externally added ferricyanide inhibited growth (1). Redox enzymes of plasma membrane vesicles from mammalian cells are responsive to hormones and growth factors. Examples include stimulation by glucagon (14) and inhibition by insulin (8) of NADH dehydrogenase activity and the stimulation of NADH oxidase by the thyroid hormone T3 (7). Recently, Morre et al. (18) reported the stimulation by auxin of an NADH oxidase in plasma membrane vesicles isolated from soybean hypocotyls. This report concerns the solubilization and purification of an NADH oxidase from plasma membrane vesicles isolated from soybean hypocotyls by aqueous two-phase partition. The NADH oxidase retains responsiveness to auxin throughout purification. MATERIALS AND METHODS Iolation of Plasma Membrane. Soybean seeds (Glycine max L. var Williams) were soaked for 4 h in water and grown in the dark (20-22°C) in moist vermiculite. After 4 to 6 d, 2 cm hypocotyl segments, cut just below the cotyledon, were harvested under diminished room light and placed in cold water. Segments
(40 g) were chopped with razor blades in 40 ml of homogenization buffer (0.3 M sucrose, 50 mm Tris-Mes [pH 7.5], 10 mM KCl, 1 mM MgCl2, 1 mm PMSF); 0.1% BSA (0.1% w/v) was included in the homogenization medium in initial trials and was not found to be beneficial. The homogenate was filtered through one layer of Miracloth (Chicopee Mills) and centrifuged for 10 min at 6,000g (HB-4 rotor). The supernatant was recentrifuged at 60,000g (Beckman SW 28 rotor) for 30 min and the pellets were resuspended in 0.25 M sucrose with 5 mm potassium phosphate (pH 6.8). Plasma membrane vesicles were prepared using a 16 g aqueous two-phase partitioning system (10). Resuspended 60,000g pellets were mixed with 6.4% (w/w) polyethylene glycol 3350 (Fisher), 6.4% (w/w) Dextran T500 (Pharmacia), 0.25 M sucrose, and 5 mm potassium phosphate (pH 6.8). After mixing the tubes by 40 inversions, the phases were separated by centrifugation at 750g for 5 min. The lower phase was repartitioned with a fresh upper phase, and the two upper phases were repartitioned twice with fresh lower phases. The upper phases were diluted approximately fourfold with buffer and collected by centrifugation at 100,OOOg for 30 min. The yield was 1 to 2 mg of purified plasma membrane. Solubilization of NADH Oxidase. Purified plasma membrane vesicles were incubated in buffer A (10 mM Tris-HCl [pH 8], 1 mM EDTA, 12 mm 8-mercaptoethanol, 10% [v/v] glycerin, 3 mM NaN3, and 1 mm PMSF) at 4°C for 30 min, and then centrifuged at 50,000g for 20 min. The NADH oxidase was solubilized by incubating the washed plasma membrane pellet resuspended to 1 to 2 mg/ml in buffer B (buffer A at pH 7, minus EDTA) with the zwitterionic detergent, CHAPS (Boehringer), at a ratio of 2:1, CHAPS:protein (w/w). The solution was stirred gently for 2 h at 4°C, and then centrifuged at 50,000g for 20 min. The supernatant was collected for further purification. Ion Exchange Chromatography and Gel Filtration. Solubilized proteins were fractionated on a 1 x 15 cm DEAE-cellulose column (DE52, Whatman), which had been equilibrated with buffer C (buffer B including 0.03% CHAPS). The loaded column was washed with 50 ml buffer C and eluted stepwise with 50 ml volumes each of 0.1, 0.2, and 0.3 M NaCl. Fractions of 10 ml were collected. The chromatography fractions with highest NADH oxidase activity were further purified on a 1 x 15 cm Ultrogel AcA 44 column (LKB, France) with an exclusion limit of 200 kD. Fractions of 1 ml were collected at a flow rate of 0.5 ml/ min. The void volume was determined with blue dextran 2000. The DE52 fraction also was chromatographed on a 2.5 x 18 cm agarose gel filtration column (Bio-Gel A-0.5 m, Bio-Rad) 2Abbreviations: PMSF, a-phenylmethylsulfonyl fluoride; CHAPS, 3-
([3-cholamidopropyljdimethylammonio)-l-propane-sulfonate; PCMB,
Work supported in part by a grant from the National Institutes of acetone. Health, P01 CA36761. 1264 I
p-
chloromercuribenzoic acid; DBMIB, 2,5-dibromo-3-methyl-6-isopropylp-benzoquinone; SOD, superoxide dismutase; ITFA, thenoyltrifluoro-
AUXIN-STIMULATED NADH OXIDASE with an exclusion limit of 500 kD. The protein sample (1 ml) was loaded onto the column, followed by 100 ml of equilibration buffer consisting of 50 mM Tris HCl (pH 7.5), 10% glycerol, 1 mM PMSF, 0.1 M KCl. Fractions of 5 ml were collected. The column was calibrated by separating a protein sample containing blue dextran, catalase, and Cyt c. Gel Electrophoresis. Samples (1-10 ,ug protein) from various stages of purification were analyzed for protein composition with the gel electrophoresis system of Laemmli (11) with 4% (wlv) SDS or 0.1% (w/v) CHAPS (native gel). Electrophoresis was at a constant current of 20 mA for 4 to 6 h. Samples for SDS gel electrophoresis were precipitated with ice-cold acetone (2 h), collected by centrifugation at 10,000g (10 min), and solubilized. Nondenaturing gels of solubilized proteins were sliced into 3 mm sections. Proteins were removed from the gel by soaking the slices overnight in buffer C. Eluted proteins were assayed directly. Polyacrylamide gels were silver stained according to Oakley et al. (20). Protein was determined by the method of Bradford (3) with BSA as a standard. Enzyme Assays. Redox enzymes were assayed with a DW-2a (Aminco) spectrophotometer in the dual-wavelength mode. NADH oxidase was assayed by decrease in absorbance at 340 nm (430 nm reference) in 25 mm Tris-Mes (pH 7), 0.1 M sucrose, and 10 mm of each CaCl2, NaCl, KCl. NADH-ferricyanide reductase was assayed at 420 nm (500 nm reference) in 25 mM Tris-Mes (pH 7), with 100 a.M potassium ferricyanide. Assay temperature was 25° and NADH was added to a final concentration of 100 ,uM to start the reactions. Auxin and auxin-related compounds were obtained from Sigma Chemical Co.
RESULTS NADH oxidase activity of isolated plasma membrane, prepared from soybean hypocotyls, transfers electrons from NADH to oxygen with a optimum pH of 7 and a Km of approximately 200 tM NADH (2). The activity required 02. It was not due to mitochondrial contamination based on the insensitivity to antimycin A and sodium azide and the extremely low levels of succinic dehydrogenase and cytochrome oxidase of the plasma membrane preparations. NADH oxidase was also insensitive to KCN (1 mM) and SOD (0.2 mg/ml). TFFA, an iron chelator, inhibited NADH oxidase approximately 70% and PCMB (10 /M), a sulfhydryl reagent, inhibited the enzyme about 30% (Table I). The NADH oxidase activity also was inhibited by actinomycin D and quinacrine (Table II), compounds known to block electron transport in the plasma membrane of animal cells (4, 22). The specificity of the auxin response was determined by comparing active and, chemically related, inactive auxin analogs. The NADH oxidase of plasma membrane isolated by aqueous twophase partition from regions of actively growing tissue was stimulated two- to threefold by both IAA and the synthetic auxins 2,4-D and a-NAA (Table III). Benzoic acid, 2,3-D and /3-NAA, Table I. Inhibitor Effects on NADH Oxidase Activity of Isolated Plasma Membrane Vesicles Plasma membrane vesciles were prepared from soybean hypocotyls. Vesicles were incubated with inhibitors for 3 min and then assayed for NADH oxidation Inhibitor None KCN NaN3
TTFA PCMB
Concentration
Specific Activity
zmM
m nmollmg protein * min 5.5 ± 0.9 5.3 ± 0.3 5.5 ± 0.1 1.8 ± 0.2 3.7 + 0.1
1.00 0.05 0.15 0.01
Control 100 96 100
33 68
1265
Table II. Inhibition of Partially Purified NADH Oxidase by Actinomycin D and Quinacrine NADH oxidase was solubilized and then partially purified by ion exchange chromatography on a DEAE-cellulose column as described in "Materials and Methods." The fraction with peak NADH oxidase activity was incubated (3 min) with the inhibitors prior to assay for NADH oxidation.
Inhibitor
Concentration
Specific Activity
Control
AM Actinomycin D
None 0.01 0.02 0.03 0.04 None 1 10 100
nmollmg protein min 34 28 8 1 0 80 51 46 2
% 100 82 21 3 0 100 64 58 3
Quinacrine
Table III. Specificity of the Response to Auxins by NADH Oxidase of Isolated Plasma Membrane Vesicles Plasma membrane vesicles were isolated by aqueous two-phase partition from the region of cell elongation of etiolated soybean hypocotyls (see "Materials and Methods"). Active (2,4-D and a-NAA) and inactive (2,3-D and -NAA) synthetic auxins were tested along with indole-3acetic acid (IAA) and benzoic acid for their ability to stimulate the NADH oxidase. Growth active means the ability to stimulate elongation growth of hypocotyl segments. a- and,8-NAA were tested at 0.1 .LM, 24-D, 2,3-D, and benzoic acid at 1 ,u M, and IAA at 10 AM. Specific Activity Growth Active Compound nmollmg protein min 18 ± 2 None + 45 ± 15 2,4-D 20 2 2,3-D + 36 3 a-NAA /3-NAA 21 3 + IAA 32 7 15 ± 5 Benzoic acid -
which do not stimulate growth, did not stimulate the NADH oxidase. Auxin stimulated NADH oxidase activity over a range of concentrations (0.1-10 ,UM) with an optimum at 1 ,xM (Fig. 1). When plasma membrane vesicles were incubated with (0.1%) nonionic (Triton X-100) or zwitterionic detergents (CHAPS) an average of 80% of the NADH oxidase activity was solubilized. Soluble NADH oxidase activity remained stimulated by auxin (Fig. 1). The optimum concentration of 2,4-D for stimulation of the solubilized NADH oxidase was 1 tlM. This result indicated the possibility of purifying an enzyme which would retain responsiveness to auxin. For purification NADH oxidase activity was solubilized from isolated plasma membrane vesicles which had been washed in a low ionic strength Tris-EDTA buffer (pH 8) to remove loosely bound proteins. At a ratio of CHAPS to protein of 2:1 (approximately 0.2%), maximum solubilization was achieved without significant loss of activity. Therefore, a ratio of 2 mg CHAPS per mg protein was selected for the purification protocol. Solubilized NADH oxidase was purified by a combination of ion exchange chromatography and gel exclusion chromatography. The solubilized proteins were loaded onto a 1 x 15 cm DEAE-cellulose ion exchange column (DE52) equilibrated with 0.03% CHAPS. Most of the NADH oxidase activity was eluted with 0.1 M NaCl (Fig. 2). Although some NADH-ferricyanide
1266
BRIGHTMAN ET AL.
Plant Physiol. Vol. 86, 1988 dei
4 z 0
180
* C.
160 _
-
*~~~~~200 ~~~~~~~~400
3 rr0Ox
a N
> 140
P v
-
I
.
o} 3
120 _
fi
x~~~~~~~~~~~~~~ ~I0 PO
a:0 -
0
2
4
6
K) 8 2 FRACTION NUMBE
14
B
V
O
CD
0
_=
0 100I _
6
I
80 _ -7I
I
-5 -6 In [2,4-D], M
I
-4
FIG. 3. Gel filtration of the chromatography (DE52) fraction with the highest NADH oxidase activity (Fig. 2) with an Ultrogel AcA 44 (LKB) column (1 x 15 cm). This column had an exclusion limit of 200 kD. The exclusion volume was determined with blue dextran 2000. Fractions of 1 ml were collected at a flow rate of 0.5 ml/min.
FIG. 1. Stimulation of NADH oxidase of plasma membrane over a range of 2,4-D concentrations. Auxin stimulation was determined by adding 2,4-D to a control reaction and measuring the increase in NADH oxidation rate. Inset, stimulation of solubilized NADH oxidase by 2,4D. A, plasma membrane activity; B, plasma membrane incubated 30 min with 0.1% CHAPS; C, supernatant of plasma membrane and 0.1% CHAPS incubation obtained after 20 min centrifugation at 50,000g; D, supernatant activity in the presence of 1 /,M 2,4-D. FRDCTON NUMBER
.IMNaCI
Q2M NaCI
03M NaCI
0'
FIG. 4. Gel filtration of NADH oxidase fraction isolated by ionic exchange chromatography (Fig. 2) on a Bio-Gel A-0.5 m (Bio-Rad) column (2.5 x 18 cm) with an exclusion limit of 500 kD. Blue dextran 2000, catalase and Cyt c were used as mol wt markers. Fractions of 5 ml were collected at a flow rate of 1 ml/min.
E C
N
0 0
0 2 4 6 8 10 12 14 1 18 20 22 24 26 28 30 32 34 36 FRACTION NUMBER
FIG. 2. Ion exchange chromatography of solubilized proteins on DEAEcellulose (DE52, Whatman). The column (1 x 15 cm) was equilibrated with buffer and 0.03% CHAPS. The column was washed with 50 ml buffer and eluted stepwise with 50 ml volumes each of 0.1, 0.2, and 0.3 M NaCl. Fractions of 10 ml were collected and assayed for NADH oxidation.
reductase activity was eluted with the NADH oxidase activity, the amount varied greatly between separations. When fractions 10-12 (Fig. 2), with the highest total activity, were combined and applied to an Ultrogel AcA 44 gel filtration column, most of the activity appeared in the exclusion volume. A second peak of similar specific activity, but much less total activity, was collected just after the major peak (Fig. 3). This step separated the NADH oxidase from the NADH-ferricyanide reductase activity.
Since the NADH oxidase activity was present in the exclusion volume of the gel, the approximate mol wt of the native enzyme could not be estimated. Therefore, the DE52 fraction also was chromatographed on a Bio-Gel A-0.5 m column which had a greater exclusion limit, 500 kD compared to 200 kD for the Ultrogel AcA 44 column. Some of the NADH oxidase activity was still present in the exclusion volume of this column as well. However, the majority of the activity was separated into two peaks. The first peak had an approximate mol wt between 200 and 240 kD based on the calibration with catalase (Fig. 4). The protein composition of Bio-Gel fraction 10 was then analyzed by SDS-PAGE (Fig. 5). The three prominent bands of approximately 36, 52, and 72 kD were seen consistently in these fractions. For comparison, if solubilized plasma membrane proteins were first separated by gel electrophoresis under nondenaturing conditions (0.03% CHAPS) and fractions eluted from the gel slices were assayed for NADH oxidase, the activity was found in a single peak of relatively high mol wt (Fig. 6). The protein composition of this peak (Fig. 6, inset) contained the same three protein bands as the partially purified NADH oxidase from the gel filtration columns, when analyzed under reducing and denaturing conditions (SDS-PAGE). The NADH oxidase of soybean plasma membrane was purified an average of 2000-fold compared to the starting homogenate with a typical yield of 24% (Table IV). The specific activity after the DE52 separation was artificially low due to a reversible in-
AUXIN-STIMULATED NADH OXIDASE
----
Ou-
ii
'! W*
T
116 97
-.
I
~%66
-
45 29
FIG. 5. SDS-polyacrylamide (10% acrylamide-4% SDS) gel electrophoretic separation of purified NADH oxidase. Fraction 10 from the Bio-Gel column (Fig. 4) was acetone precipitated and solubilized as described in "Materials and Methods." Proteins (5 ,g sample) were stained with silver.
3
60S
DYE FRONT
FE
E
a 40
66
-
E
445 x 20-
0
o0
1
J
2
4
6
8
10 SLICE
FIG.
6.
NADH oxidase
activity profile
electrophoresis. Immediately at 3 mm
intervals and
after the
slices were
at 40C. Fractions were then
12
14
18
16
on
native
gel (8% acrylamide)
electrophoresis,
the
gel
eluted with 0.5 ml buffer C
assayed
20
NUMBER
for NADH oxidase
was
sliced
overnight
activity
B. SDS-
PAGE analysis of peak fraction from native gel electrophoresis. Protein components were identified by silver staining.
hibition (about 70%) of the NADH oxidase activity by 0.1 M NaCl. The protein composition of the NADH oxidase was in four attempts consistently found to consist of three prominent protein bands of approximate mol wt 36, 52, and 72 kD. Upon incubation with 2,4-D (1 ,UM) the NADH oxidase complex of these preparations was stimulated over 500% compared to no
2,4-D (Fig. 7). DISCUSSION
Despite considerable experimental effort, little is known about the molecular details of action of growth regulators of the auxin type. A site of action at the membrane level is indicated (17, 24) but whether this site is primarily at the plasma membrane or in
1267
association with some internal endomembrane or both remains controversial. The approach followed by our laboratory has been to focus on the plasma membrane of etiolated hypocotyls of soybean. Soybean membranes, including plasma membranes, bind auxin (25) and undergo a change in conformation upon exposure to auxin (19). However, until now, we have been unable to demonstrate a major change in activity of a plasma membraneassociated enzyme. Thus, the auxin-specific stimulation of the NADH-oxidase from plasma membranes represents the first direct stimulation of a purified plasma membrane enzyme by this class of hormones. NADH oxidase, which transfers electrons from NADH to oxygen in the absence of added electron acceptors, has been linked to proton efflux and potassium uptake in protoplasts (12) and whole cells (15) as well as to membrane polarization (9) (see, however, Ref. 23). However, the mechanism which connects NADH oxidase to growth control is not yet known. That the auxin stimulated activity is an NADH oxidase is based on several lines of evidence. First, the enzyme requires oxygen and is inactive in an argon atmosphere (2). That the functioning involves electron flux through or within the membrane is suggested by the strong inhibition by quinacrine, an inhibitor used to interrupt transmembrane redox activities (4). Actinomycin D, a potent inhibitor of plasma membrane redox enzymes (22), also is a potent inhibitor of NADH-oxidase both before and after solubilization (Table II) again indicating an enzymic transfer of electrons in this process. The activity is not due to contaminating mitochondria or peroxidases since antimycin A and sodium azide, mitochondrial inhibitors, and KCN, a peroxidase inhibitor (90100% effective at 1 mM), were without effect on NADH oxidation by the isolated membrane vesicles. Also, NADH oxidase activity was shown previously to be stimulated by rotenone and HOQNO (2) and was stimulated by DBMIB (1 AM) and unaffected by DCMU (2 AM) and SOD (0.2 mg/ml) (our unpublished data). Inhibitors of the alternate respiration pathway, disulfiram and SHAM, stimulate, rather than inhibit, plasma membrane NADH oxidase activity (2). NADH-ferricyanide reductase, a prominent plasma membrane redox activity, was not stimulated by auxin either in whole cells or isolated plasma membrane vesicles. Moreover, the auxinstimulated complex purified from plasma membrane lacks NADHferricyanide reductase activity (Fig. 3). Although NADH oxidase may interact with the reductase in the membrane system as part of a larger redox coupled chain they are clearly different and biochemically separable activities. The specificity of the auxin response of the oxidase is demonstrated by stimulation by indole-3-acetic acid (IAA) as well as the synthetic auxins 2,4-D and ,B-NAA. The growth inactive analogs of 2,4-D and a-NAA, 2,3-D and /3-NAA were inactive in stimulating the enzyme, as was benzoic acid (Table III). The stimulation over the range 10-7 to 10-4 M 2,4-D was optimal at 10-6 M 2,4-D, paralleling the growth response of excised hypocotyl segments of soybean to the synthetic auxin. The relationship of an external NADH oxidase which promotes the flow of electrons at the plasma membrane from NADH to oxygen to the control of the growth process is less clear. In animal cells, impermeant electron acceptors, such as ferricyanide or diferric transferrin, have been utilized to promote growth (increase in cell number) presumably by the increase in the transmembrane flow of electrons (5). In plants, however, the impermeant acceptors either are without effect (growth of soybean hypocotyl sections) (our unpublished data) or inhibitory (carrot cells in culture [1]). That the plasma membrane associated NADH oxidase may be stimulated in vivo by auxin is suggested by a previously unexplained auxin stimulation of cyanide- and salicylhydroxamate-insensitive oxygen uptake with plant tissue (5, 21). Thus the NADH oxidase of the plasma membrane might
1268
BRIGHTMAN ET AL.
Plant Physiol. Vol. 86, 1988
Table IV. Values for Purification of NADH Oxidase from Plasma Membrane of Soybean Values are from a representative purification, which was selected from four repetitions of the protocol. NADH oxidase was solubilized from isolated plasma membrane by incubating plasma membrane vesicles with the zwitterionic detergent CHAPS. The activity was purified first by ion exchange chromatography on a DEAE-cellulose column (DE52). Then the NADH oxidase was purified from the chromatography fraction by gel filtration (Ultrogel AcA 44 column). Protein values were determined by the method of Bradford (3).
Total Protein
Plasma membrane Solubilized proteins DE52 fraction Ultrogel fraction a Corrected for inhibition
Total Activity
nmollmin mg 10.00 15.0 5.00 5.0 0.13 1.1 0.03 3.6 by 0.1 M NaCl.
|.OmI O.D.
I min
FIG. 7. Spectrophotometric tracing of purified NADH oxidase activity. The sample was incubated with or without 1 ,UM 2,4-D for 3 min prior to addition of NADH. Control activity (no 2,4-D) was 9.4 nmol/ min-mg protein.
rate-limiting terminal oxidase to regulate a transmembrane flow of electrons somehow important to the growth procserve as a ess.
The nondenaturing zwitterionic detergent, CHAPS, was choto solubilize the membrane-bound NADH oxidase activity with the intention of purification of an active and intact auxinresponsive complex. Triton X-100 also solubilized the activity with retention of enzyme activity and auxin responsiveness. Additionally, a portion of the activity was solubilized with TrisEDTA buffer. With both the Triton X-100 and Tris-EDTA extraction procedures, the same three peptide bands were solubilized. However, CHAPS gave the best overall solubilization and greatest degree of final purification despite the reversible loss of activity and hormone responsiveness. The collection of more than one peak of NADH oxidase activity from the gel filtration columns might be explained by the formation of aggregates of the protein complex in the low ionic strength environment since, except for the relative proportions of contaminating components, the same three bands are present in both NADH oxidase peaks from the gel filtration columns. That the auxin-stimulated NADH oxidase co-purifies with at least two additional proteins may, in fact, have functional significance. For example, with plasma membrane isolated from the mature region of soybean hypocotyls, a region no longer auxin responsive, the sen
Specific Activity
Purification TotaleHomogenat Total Homogenate
nmollmg prot * min
x-fold 25 16 140 2000
1.5 1.0 8.3 120.0
% 100 33 27a 24
NADH oxidase also is no longer stimulated by auxin. This finding indicates that the auxin response of the oxidase may be mediated by a second protein, which is part of the complex in elongating tissue, but may be lacking, or not properly coupled to the oxidase in the plasma membrane of the more mature cells. If the complex of three protein bands reflects the in vivo organization of the proteins in the membrane, either the NADH oxidase is a complex composed of several subunits, at least one of which is auxin responsive, or the NADH oxidase is one protein of a complex regulatory unit of the membrane that retains auxin stimulation after detergent solubilization. With CHAPS, at concentrations greater than required to solubilize the enzyme complex, auxinresponsiveness is lost but is restored by the removal of excess CHAPS suggesting that some degree of association of the proteins of the complex is necessary for auxin stimulation. Although prosthetic groups or other potential redox carrier molecules have not yet been investigated in this system, evidence exists for such components in the plasma membrane. Both flavin and quinone dependent enzymes and b- and c-type cytochromes have been found in purified plasma membrane. In addition, sulfhydryl group inhibitors have been shown to block NADH oxidation and 02 consumption by plasma membranes (13). The inhibition by TTFA, an iron chelator, and PCMB (Table I) suggests that both iron and sulfhydryls may be involved in this redox system. Thus the presence of several possible redox constituents in the plasma membrane and the pattern of inhibition by various compounds lead us to believe that the NADH oxidase will prove to contain or interact with some redox carrier molecules. We have no information as to whether the purified NADH oxidase or one of the three peptides that co-purify in the preparation actually bind auxin. However, it has been reported that PCMB which inhibits NADH oxidase activity (Table I), also inhibits auxin binding to membranes (24). Estimates of mol wt of auxin binding proteins primarily have ranged from 40 to 47 kD (24). Our apparent mol wt of 36, 52, and 72 kD do not correspond to this range found in coleoptiles, although auxin binding proteins of soybean membranes have not been investigated. We have preliminary evidence that the 36 kD protein is phosphorylated both in the plasma membrane and purified complex. LITERATURE CITED
1. BARR R, TA CRAIG, FL CRANE 1985 Transmembrane ferricyanide reduction in carrot cells. Biochim Biophys Acta 812: 49-54 2. BARR R, AS SANDELIUS, FL CRANE, DJ MORRt 1985 Oxidation of reduced pyridine nucleotides by plasma membranes of soybean hypocotyl. Biochem Biophys Res Commun 131: 943-948 3. BRADFORD MM 1976 A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal
AUXIN-STIMULATED NADH OXIDASE Biochem 72: 248-254 4. CRANE FL, H L6w 1976 NADH oxidation in liver and fat cell plasma membranes. FEBS Lett 68: 153-156 5. CRANE FL, IL SUN, MG CLARK, C GREBING, H L6w 1985. Transplasmamembrane redox systems in growth and development. Biochim Biophys Acta 811: 233-264 6. ELLEM KAO, GF KAY 1983 Ferricyanide can replace pyruvate to stimulate growth and attachment of serum restricted human melanoma cells. Biochem Biophys Res Commun 112: 183-190 7. GAYDA DP, FL CRANE, DJ MoRRE, H Low 1977 Hormone effects on NADHoxidizing enzymes of plasma membranes of rat liver. Proc Indiana Acad Sci 86: 385-390 8. GOLDENBERG H, FL CRANE, DJ MORRE 1979 NADH oxido-reductase of mouse liver plasma membranes. J. Biol Chem 254: 2491-2498 9. IVANKINA NG, VA NovAK 1980 H+-transport across plasmalemma. H+ATPase or redox-chain? In RM Spanswick, WJ Lucas, J Dainty, eds, Plasma Membrane Transport: Current Conceptual Issues. Elsevier-North Holland Biomedical Press, Amsterdam, pp 503-504 10. KJELLBOM P, C LARSSON 1984 Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley. Physiol Plant 62: 501-509 11. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 12. LIN W 1982 Responses of corn root protoplasts to exogenous reduced nicotinamide adenine dinucleotide: oxygen consumption, ion uptake and membrane potential. Proc Natl Acad Sci USA 79: 3773-3776 13. LIN W 1984 Further characterization of the transport property of plasmalemma NADH oxidation system in isolated corn root protoplasts. Plant Physiol 74: 219-222
1269
14. Low H, FL CRANE 1976 Hormone regulated redox function in plasma membranes. FEBS Lett 68: 157-159 15. MisRA PC, TA CRAIG, FL CRANE 1984 A link between transport and plasma membrane redox systems in carrot cells. J Bioenerg Biomembr 16: 143-152 16. M0LLER IM, W LIN 1986 Membrane-bound NAD(P)H dehydrogenases in higher plant cells. Annu Rev Plant Physiol 37: 309-334 17. MoRuE DJ, CE BRACKER 1976 Ultrastructural alteration of plant plasma membranes induced by auxin and calcium ions. Plant Physiol 58: 544-547 18. MoRitE DJ, P NAVAS, C PENEL, FJ CASTILLO 1986 Auxin-stimulated NADH oxidase (semidehydroascorbate reductase) of soybean plasma membrane: role in acidification of cytoplasm? Protoplasma 133: 195-197 19. MoRRE DJ, JH CROWE, DM MoRRt, LM CROWE 1987 Infrared spectroscopic evidence for a conformational alteration of plant plasma membranes upon exposure to the growth hormone analog, 2,4-dichlorophenoxyacetic acid. Biochem Biophys Res Commun 147: 506-512 20. OAKLEY BR, DR KIRSH, NR MoRaas 1980 A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105: 361363 21. POLEVOY W, TS SALAMATOVA 1977 Auxin, proton pumping and cell trophics. In E Marre, 0 Ciferri, eds, Regulation of Cell Membrane Activities in Plants. Elsevier-North Holland, Amsterdam, pp 209-216 22. SUN I, FL CRANE 1981 Transplasmalemma NADH dehydrogenase is inhibited by actinomycin D. Biochem Biophys Res Commun 101: 68-75 23. THOM M, A MMUITzKi 1985 Evidence for a plasmalemma redox system in sugarcane. Plant Physiol 77: 873-876 24. VENIS MA 1986 Receptors for plant auxin action and auxin transport. In PM Conn, ed, The Receptors, Vol IV. Academic Press, New York, pp 275-314 25. WILLIAMSON FA, DJ MoRal, K HEss 1977 Auxin binding activities of subcellular fractions from soybean hypocotyls. Cytobiologie 16: 63-71
