ARCHIVES OF BIOCHEMISTRY Vol. 191, No. 2, December,
AND BIOPHYSICS pp. 452-465, 1978
Purification and Characterization of a Wound-Induced o-Hydroxyfatty Acid:NADP Oxidoreductase from Potato Tuber Disks ( Solanum tuberosum L.)’ VISHWANATH Department
of
Agricultural
P. AGRAWAL2
AND
P.E. KOLATTUKUDY3
Chemistry and Program in Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164 Received
June
19,1978;
revised
August
3,1978
w-Hydroxyfatty acid dehydrogenase (w-hydroxyfatty acid:NADP oxidoreductase) catalyzes the reaction w-hydroxyfatty acid -I- NADP + w-oxofatty acid + NADPH +H’. In wound-healing potato tuber disks, the w-oxofatty acid generated by this enzyme is further oxidized to the corresponding dicarboxylic acid by a separate enzyme, w-oxofatty acid dehydrogenase. w-Hydroxy acid dehydrogenase, but not w-0x0 acid dehydrogenase, was found to be induced by wounding potato tubers. w-Hydroxy acid dehydrogenase has been purified 600-fold to near homogeneity from wound-healing potato tuber disks by a combination of gel filtration, anion-exchange, and hydroxylapatite chromatography followed by NADP-Sepharose affinity chromatography, in about 1% yield. The molecular weight and Stokes radius of this enzyme as determined by gel exclusion chromatography are 60,090 and 31 A, respectively. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 31,000, indicating that the deydrogenase is a dimer with subunits of similar molecular weight. The pH optima for the reaction in the forward and reverse directions are 9.5 and 8.5, respectively, and V in the forward and reverse directions are 140 and 3200 nmol/min/mg, respectively. Apparent K, values for NADP, 16-hydroxyhexadecanoic acid, NADPH, and 16-oxohexadecanoic acid are 100, 20, 5, and 7 PM respectively. The equilibrium constant of the reaction at pH 9.5 and 30°C is 1.4 x 10m9 M. The enzyme preparation did not show any stereospecificity for hydride transfer from NADPH to 16-oxohexadecanoic acid.
Suberin is a polymer depositied just inside and most probably covalently attached to the cell wall of the outermost layers (one to three) of cells in the underground parts of plants (1, 2). Recent evidence that suberin deposition is also involved in wound healing of aerial parts of plants suggested that suberization is a fundamental process involved in wound healing of plant tissues irrespective of nature of natural covering material of the tissue (3). Even though the structure of this polymer is poorly understood, during the past few years, it has 1 Scientific Paper 5121, College of Agriculture Research Center, Washington State University, Pulhnan, Washington 99164. This work was supported in part by Grants PCM 74-09351 and PCM 77-00927 from the National Science Foundation. * Fulbright-Hays fellow from Tribhuvan University, Kathmandu, Nepal. ’ To whom all correspondence should be addressed.
become apparent that esterified aliphatic compounds with long carbon chains constitute a significant component of this polymer (2). w-Hydroxyfatty acids and dicarboxylic acids are the major classes of aliphatic monomers of suberin. Among them, w-hydroxypahnitic acid, w-hydroxyoleic acid, and the corresponding dicarboxylic acids are the major components in all suberin samples thus far examined (4-7). Biosynthesis of suberin is not well understood. Since the composition of the aliphatic components of suberin deposited in the wound periderm of potato tubers is identical to that of the natural skin of the tuber (8), wound-healing potato tissue appeared to be a suitable experimental material for biosynthetic studies on suberin. On the basis of the structure of the monomers and from precursor feeding studies, a biosynthetic pathway has been proposed (2).
452 0003-9861/78/1912-0452$0290/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
WOUND-INDUCED
POTATO
TUBER
However, little is known about the enzymes involved in suberixation, except for a demonstration that a cell-free preparation from wound-healing potato tuber slices catalyzed conversion of 16-hydroxyhexadecanoic acid to the corresponding dicarboxylic acid (9). In the present paper, it is demonstrated that this conversion occurs in two steps and that the enzyme which catalyzes the first step, but not that which catalyzes the second step, is induced by wounding. The purification and some properties of this wound-induced o-hydroxyfatty acid dehydrogenase are described. MATERIALS
AND
METHODS
Materials White Rose potatoes grown in California were obtained from a local grocery store and stored at 4’C. NADP, NADPH, NAD, NADH, mercaptoethanol, Sepharose, malic enzyme from chicken liver (EC 1.1.1.40, 13.5 units/mg), hexokinase from yeast (EC 2.7.1.1, 310 units/mg), glucose g-phosphate dehydrogenase from torula yeast (EC 1.1.1.49, 340 units/mg), glutamic dehydrogenase from bovine liver (EC 1.4.1.3, 56 units/mg), DEAE-Sephadex, nitroblue tetrazolium, and phenazine methosulfate were obtained from Sigma Chemical Co. Hydroxylapatite and Coomassie brilliant blue G-256 used for protein assay were obtained from Bio-Rad. Sodium boro[‘H]hydride and D[l-3H]glucose were purchased from Amersham. Pyridinium chlorochromate and 16-hydroxyhexadecanoic acid were purchased from Aldrich Chemical Co.
Preparation of Methyl Oxohexadecanoate
16-
One n-mole of methyl 16-hydroxyhexadecanoate in 1.5 ml of CHzClz was added dropwise to 1.5 mmol of pyridinium chlorochromate suspended in 2 ml of CH&L. After stirring the reaction mixture for 1 h, it was diluted with 15 ml of ether and was passed through a column (1.3 x 4 cm) of Silica Gel G. After evaporating the ether, the product was purified by TLC4 on Silica Gel G with hexanediethyl ether:formic acid (65:35:2, v/v) as the developing solvent (Rf, 0.51; yield, 80%).
Preparation
of 16-Oxohexadecanoic
16-Oxohexadecanoic droxyhexadecanoic acid for the preparation of except in this case, the for 30 min. The product ’ Abbreviation pk.
used
Acid
acid was prepared from 16-hyaccording to the method used methyl-16-oxohexadecanoate, reaction mixture was refluxed after purification by TLC on TLC, thin-layer cbromatogra-
w-HYDROXYACID
453
DEHYDROGENASE
Silica Gel G with diethyl ether:hexane:formic acid (30:2&l, v/v) as the developing solvent (RI, 0.47) was identified by combined gas-liquid chromatographymass spectrometry. The diagnostic fragments of the trimethylsilyl ester derivative were M+-CHa, m/e 327, 9% of the base peak, M+-CHs-HzO, m/e 309,42%; M+CHICO, m/e 299, 1995; M+-CHa-CHzO, m/e 297, 9% M’-CH&HO, m/e 295, 4%; and M+-CH&H&HO, m/e 283, 13%. The metastable for M+-CH3 + M+CH3-Hz0 transition was slso observed.
Preparation of 16-Hydroxy[l Hexadecanoic Acid
6-3H]
To 209 mg of methyl-16-oxohexadecanoate in 3 ml of tetrahydrofuran, 25 mCi of sodium boro[3H]hydride (293 mCi/mmol) was added. After stirring for 16 h, the reaction mixture was acidified and extracted with chloroform. Purification of the hydroxyacid methyl ester by repeated TLC followed by hydrolysis and purification of the hydroxyacid by TLC with ethyl ether:hexane:formic acid (30:2&l, v/v) gave chemically and radiochemically pure (>99%) 16-hydroxy[l63H]hexadecanoic acid.
Preparation
of Suberizing
Potato
Disks
Potato tubers were immersed in 2% (w/v) hypochlorite (30%, v/v, chlorox) solution for 5 min and thoroughly washed with water. They were cut into two halves and tissue disks (2 x 10 mm) were cut from tissue cores prepared with a cork borer. The disks were transferred to layers of rubberized mesh in lgallon jars and tive layers of disks, each separated by a rubberized mesh, were placed in each jar (400 g tissue/jar). Humid air was passed (0.6 l/h) through the jars for 120 h at 22’C and the wound-healing disks were taken out and used for preparation of acetone powder. All materials used were sterilized prior to use and all operations from cutting the potatoes to placing disks in glass jars were performed under sterile conditions in a glove box.
Preparation
of Acetone Powder
Suberized potato disks were homogenized with cold acetone (5 ml/g tissue) at -20°C for 1 min in an explosion-proof Waring blendor, the suspension was filtered under suction, the insoluble material was washed with cold acetone and air-dried. The acetone powder could be stored in airtight containers at -20°C for 4 weeks without significant loss of enzymatic activity. The yield of acetone powder from suberized potato disks was 12-18% of the fresh weight.
Assay for w-hydroxyfatty acid dekydrogenase activity The w-hydroxy acid dehydrogenase was assayed three different methods. Tracer assay I. This assay was done essentially
by as
454
AGFtAWAL
AND
described elsewhere (9). 16-Hydroxy[G-3H]hexadecanoic acid and NADP were incubated with the enzyme preparation and the product isolated by TLC was assayed for 3H with a liquid scintillation spectrometer. Tracer assay II. In this assay, 2- to lo-p1 aliquota of the enzyme preparation were incubated with 0.1 mM 16-hydroxy[16-3H]hexadecanoic acid (1.5 x lo6 cpm) and 1 mu NADP in 0.2 ml of 0.2 M glycineNaOH buffer, pH 9.5, for 30 min at 30°C. The reaction was terminated by adding 1.8 ml of 0.5 M HCl and the mixture was extracted four times by vigorously shaking with chloroform (5 ml each time) using a vortex mixer. One milliliter of the aqueous layer was transferred into scintillation vials, the residual chloroform was removed by heating and passage of NB through the vials, and subsequently assayed for 3H with 10 ml of ScintiVerse.
Spectrophotometric
Assay
In this assay, the rate of reduction of w-0x0 acid was determined by measuring the decrease in absorbance at 340 nm using a Beckman model 25K spectrophotometer. The reaction mixture contained 40 pM 16oxohexadecanoic acid, 80 PM NADPH, and 0.2 M glytine-NaOH buffer, pH 8.5, in a total volume of 0.25 ml and the reaction was initiated by addition of the enzyme.
Assay for o-Oxofatty
Acid Dehydrogenase
o-Oxofatty acid dehydrogenase activity was measured according to the method described elsewhere (9). An aliquot of enzyme solution was incubated at 30°C for 20 min with 1 mM NADP and 0.1 mM 16oxo[G-3H]hexadecanoic acid (2.86 x lo6 cpm) in a total volume of 1 ml of 0.1 M glycine-NaOH buffer, pH 9.5. The reaction was terminated by adding sodium borohydride, and after 15 min, the mixture was acidified and the lipids extracted with chloroform were subjected to TLC after mixing it with unlabeled 16hydroxyhexadecanoic acid and hexadecan-1,16dioic acid. The plates were sprayed with 0.1% ethanolic solution of dichlorofluorescein, the lipids were located under UV light, and the silica gel from the region of hexadecan-1,16-dioic acid was scraped and assayed for 3H directly by liquid scintillation spectrometery.
Assay for Alcohol Activity
Dehydrogenase
The reaction mixture contained 1 mM NAD, 0.1 M ethanol, and 0.2 M glycine-NaOH buffer, pH 9.5, in a total volume of 0.25 ml. The reaction was started by addition of the enzyme solution and increase in absorbance at 340 nm was measured in a Beckman model 25K spectrophotometer.
Determination
of Protein
The widely be used
used method of Lowry due to its sensitivity
not
Concentration et al. (10) could to glycerol and
KOLA’M’UKUDY mercaptoethanol present in the fractions obtained during enzyme purification. Therefore, a dye-binding method was used for protein determination (11). The commercially available concentrated solution of Coomassie blue dye was diluted B-fold with water, 5 ml of the diluted dye solution was mixed thoroughly with 0.1 ml of the protein solution, and absorbance at 595 nm was measured after 5 min. Bovine serum albumin was used as standard.
Preparation
of NADP-Sepharose
NADP-Sepharose was prepared by the method described elsewhere (12). Sepharose 4B was activated with CNBr and then treated with the dihydrazide derivative of adipic acid. This dihydrazide-Sepharose 4B gel was coupled to periodate-oxidized-NADPSepharose. The amount of NADP bound to the gel, calculated from the phosphorus content (I3), was 2 to 3 eole/mI of bed volume.
Enzyme Purification Extraction of theprotein from acetone-powder and ammonium sulfate precipitation. Acetone powder (400 g) was slowly added with stirring to 4 liter 0.1 M Na-phosphate buffer, pH 6.7, containing 0.05 M mercaptoethanol over a period of 4 h. The mixture was squeezed through eight layers of cheesecloth and the volume of the filtrate was adjusted to 4 liters. To this solution, 704 g of granular ammonium sulfate was slowly added with stirring to obtain a 30% saturation and the resulting precipitate was removed by centrifuging the mixture at 10,006 rpm for 10 min. To the supernatant, 856 g of ammonium sulfate was added to attain a 60% saturation, and the resulting precipitate was collected as above and dissolved in a minimum volume of 0.05 M Tris-HCl, pH 8.3, containing 10 mM mercaptoethanol and 20% glycerol (buffer A). This solution was dialyzed against buffer A for 48 h and the volume was adjusted to 500 ml (11 g protein). Treatment with DEAE-cellulose. The protein solution from the previous step was stirred for 30 min with 75 g DE-52 which was previously equilibrated with buffer A and centrifuged at 10,000 rpm for 10 min. The supernatant was discarded and the pellet was resuspended in 500 ml of buffer A, and again centrifuged. The pellet was stirred with 300 ml of buffer A containing 0.2 M KC1 for 1 h, centrifuged, and the supernatant was saved. The pellet was resuspended in 200 ml of buffer A containing 0.2 M KC1 and centrifuged. Both supernatants were combined and concentrated to 40 to 45 ml by ultrafiltration using UM 10 membrane. Sepharose 6B gel filtration. About lo-ml portions (1.6 g protein) of the protein solution from the above step were applied to Sepharose 6B columns (3.9 X 100 cm) previously equilibrated with 0.05 M Tris-HCl buffer, pH 7.3, containing 10 mM mercaptoethanol and 20% glycerol (buffer B). The proteins were eluted with
WOUND-INDUCED
POTATO
TUBER
the same buffer at a flow rate of 30 to 40 ml/h and the effluent was monitored for absorbance at 280 nm using an ISCO model UA-5 monitor. The fractions (9-10 ml) containing enzyme activity greater than half of the maximum activity were pooled (70-75 ml from each column).
DEAE-Sephadex anion exchange chromatography. The protein obtained from the previous step (300 ml, 2.2 g protein) was passed through a DEAE-Sephadex column (bed volume, 200 ml) equilibrated with buffer B, at a flow rate of 40 ml/h. The column was washed with 300 ml of buffer B at the same flow rate and the absorbed proteins were eluted at a flow rate of 30 ml/h, with a linear gradient of 0 to 0.2 M KC1 in a total volume of 1000 ml of buffer B. The effluents were monitored for absorbance at 280 nm. Fractions (10 ml) containing enzyme activity and eluting in the range of 0.09 to 0.14 M KC1 were pooled (200 ml). This solution was concentrated by ultrafiltration and diluted with 5 mu Na-phosphate buffer, pH 6.8, containing 5% glycerol, and 10 mu mercaptoethanol (buffer C). This process was repeated 4 times in order to change the medium. Hydroxylapatite chromatography. The protein obtained from the anion exchange chromatography step (40 ml, 600 mg protein) was applied to a hydroxylapatite column (inner diameter, 2.6 cm, bed volume, 25 ml) equilibrated with buffer C. After removing unabsorbed proteins with 50 ml of buffer C, the absorbed proteins were eluted with a gradient prepared by mixing 125 ml of buffer C and 125 ml of 0.2 M Na-phosphate buffer, pH 6.8, containing 5% glycerol and 10 mM mercaptoethanol at a flow rate of 15-20 ml/h. Fractions containing w-hydroxy acid dehydrogenase activity were pooled, the buffer was changed to 0.01 M Tris-HCl buffer, pH 7.5, containing 20% glycerol and 10 mru mercaptoethanol (buffer D) by ultrafiltration as above, and the volume was adjusted to 20 ml with buffer D.
NADP-Sepharose affinity chromatography I (with KCZ as eluant). The enzyme preparation (115 mg protein) obtained from the above step was chromatographed on a NADP-Sepharose column (inner diameter, 1 cm, bed volume, 20 ml) equilibrated with buffer D. The column was washed with 40 ml of buffer D and the absorbed proteins were eluted with a linear gradient of 0 to 2 M KC1 in a total volume of 200 ml of buffer D at 10 to 12 ml/h. Fractions containing the enzyme activity were pooled, KC1 was removed by ultrafiltration, and the volume was adjusted to 5 ml with buffer D.
NADP-Sepharose affinity chromatography II (with NADP as eluant). The protein fraction (2 mg) obtained from the previous step was passed through a NADP-Sepharose column (inner diameter, 1 cm, bed volume, 5 ml) equilibrated with buffer D and unabsorbed proteins were removed by washing the column with 15 ml of the same buffer. The absorbed proteins were eluted with a linear gradient of 0 to 0.1 mM
o-HYDROXYACID
DEHYDROGENASE
455
NADP in a total volume of 40 ml of buffer D and 1.4ml fractions were collected at a flow rate of 5 to 6 ml/h. Fractions containing enzyme activity were pooled, concentrated to 1 ml by ultrafiltration, diluted with 10 ml of buffer D, and again concentrated to 1 ml. This process was repeated 3 to 4 times to remove NADP and the resulting enzyme solution was stored at 0°C. Electrophoresis. Polyacrylamide gel electrophoresis was performed in an analytical electrophoresis apparatus from Hoefer Scientific Instruments, according to the method described elsewhere (14). The gel system consisted of a 7.5% polyacrylamide resolving gel (pH 8.9) and 2.5% stacking gel (pH 7.2). The lengths of resolving and stacking gels were 7.5 and 2.5 cm, respectively, with a gel tube inner diameter of 0.45 cm. Protein samples, containing glycerol and bromphenol dye, were applied to the gels in a volume of 0.1 to 0.2 ml. Electrophoresis was performed at 4°C at a constant current of 2 mA/tube until the dye migrated 7 cm into the resolving gel. Protein bands were fixed by immersing the gels in 12.5% trichloroacetic acid solution for 30 min and then stained overnight in a solution containing 0.2% Coomassie blue, 7.5% acetic acid, and 50% methanol. The excess dye was removed from the gels electrophoretically by an Ames model 1801 quick gel destainer with a destaining solution containing 7.5% acetic acid and 5% methanol. The gels were scanned for absorbance at 600 nm in a Beckmann model 25K spectrophotometer with a gel scanner accessory. Sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis was done according to the method of Maize1 (15). The gel system of the same dimensions as above, containing 0.1% dodecyl sulfate, consisted of a 10% polyacrylamide resolving gel (pH 8.9) and 2.5% stacking gel (pH 7.2). The proteins were heated with 1% SDS and 0.1 M dithioerythritol in a boiling water bath for 3 min. Electrophoresis was done at 2.5 mA/tube until the dye migrated 7 cm into the resolving gel and the protein bands in the gel were located as described earlier.
Location of w-hydroxyfatty activity in the gel. w-Hydroxy
acid dehydrogenase
acid dehydrogenase activity was located in the polyacrylamide gels either by staining the gel for enzymic activity or by recovering the enzyme from gel slices. To stain for enzymic activity, the gels were incubated at 37°C in dark with 10 ml of a solution which contained 0.1 mM of 16hydroxyhexadecanoic acid, 1 m$NADP, 2.5 mg nitroblue tetrazolium, and 0.25 ml of phenazine methosulfate in 10 ml of 0.2 M glycine-NaOH btiffer, pH 9.5. The progress of staining was checked frequently because incubation for excessively long periods resulted in a dark background. For control, incubation mixtures containing no substrate were used. To recover the enzyme, the gel was cut into 2-mm slices with a gel slicer obtained from Bio-Rad. The gel slices were soaked overnight at 4°C in 0.1 ml of 0.2 M glycineNaOH, pH 9.5, containing 20% glycerol and 10 mM
456 mercaptoethanol enzyme activity tometric assay.
Determination
AGHAWAL
AND
and the solution was assayed for the using tracer assay II and spectropho-
of Isoelectric
Point
The enzyme preparation from the second NADPSepharose affinity chromatography was electrofocused in a LKB 8100 Ampholine electrofocusing apparatus for 72 h at 1.5 mA current and 809 V. Sucrose was used as the stabilizing medium and 0.9% of pH 3-10 Ampholine was used.
Determination of Molecular Weight and Stokes’ Radius of w-Hydroxyfatty Acid Dehydrogenase A Sephadex G-150 column (1.3 x 84 cm), equilibrated with 0.05 M phosphate buffer, pH 7.6, was calibrated with standard proteins by measuring their elution volumes (V.) from the column. Protein standards used, their molecular weight, and Stokes’ radii in A (16) were as follows: A, immunoglobulin G, 150,000 51; B, bovine serum albumin, 68,000, 35; C, ovalbumin 43,000,30; D, chymotrypsinogen, 25,700,22; E, myoglobin, 17,200, 19; and F, cytochrome c, 11,700,17. The void volume (VO) was measured by using blue dextran and Vt was calculated from the column dimensions. The protein obtained from last purification step was used to measure the elution volume of whydroxyfatty acid dehydrogenase. Molecular weight of w-hydroxy acid dehydrogenase was determined from the linear plot of log of molecular weight vs. partition coefficient (&). For the determination of Stokes’ radius, a linear plot of Stokes’ radius, R, us erf’ (1 &) was used (17). For the determination of molecular weight of the enzyme under denaturing conditions, the enzyme preparation and protein standards were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described earlier. The standards were 1) bovine serum albumin (68,000), 2) heavy chain of y-globulin (50,000), 3) alcohol dehydrogenase (37,500), 4) chymotrypsinogen (25,700), and 5) light chain of y-globulin (23,000), and the molecular weight of whydroxy acid dehydrogenase was determined from the linear plot of log of molecular weight us mobility.
Preparation
of [A-3H] @ro-R) NADPH.
D,L-[2-%]
Malate was prepared by stirring 106 mg of oxaloacetate in 1.5 ml HxO, pH 8.5 (adjusted by 1 N NaOH), with 25 mCi of NaB3H4 (specific activity, 661 mCi/mmol) for 16 h. The reaction mixture was aciditied with dilute HCl, exchangeable 3H was removed by lyophilization, and the residue (in 5 ml water) was applied to a Dowex anion exchange column, Cl- form (bed volume, 75 ml). Labeled malate was eluted with 0.05 M HCl, the radioactive fractions were pooled and lyophilized (4.7 x 10’ cpm). Malic enzyme was used to transfer 3H from the labeled malate to the A side of NADPH (18). The reaction mixture contained 0.8 mu NADP, 0.8 mM D,L-[3H]malate (2.35 X lo9 cpm), 0.2
KOLATTUKUDY mM MnClz, and 0.07 M triethanolamine buffer, pH 7.4, in a total volume of 30 ml. The reaction was initiated by adding 0.1 unit of malic enzyme (0.01 ml) and 2 ml of the reaction mixture was monitored for absorbance change at 340 nm. After the reaction rate leveled off (about 15 min), the reaction mixture was frozen and lyophilized. The lyophilized material was dissolved in 10 ml of Hz0 and chromatographed on a DEAEHC03column (1 X 15 cm) and the products were eluted with 0 to 0.5 M linear gradient of NHdHC03 in a total volume of 1060 ml (19). The fractions were monitored for AzK,, As%, and radioactivity and those with A&A339 between 2.3 to 2.6 and a constant ratio of cprn/Aasg were pooled and lyophilized. The content of NADPH in the preparation was measured by converting NADPH to NADP using glutamate dehydrogenase (20). The value obtained was 10 to 15% lower than that calculated from A~X+ Unlabeled NADPH was added to adjust the specific activity to 1.49 x 10’ dpm/pmol. Preparation of [B-3H] bra-S) NADPH. [B-3H] NADPH was prepared by transferring 3H from [1-3H] glucose to NADP using a combination of hexokinase and glucose g-phosphate dehydrogenase. The reaction mixture contained 20 pM [l-3H]glucose (0.5 mCi), 0.55 mM ATP, 0.33 mM NADP, 7.5 mu MgClz, 0.03 M TrisHCl, pH 8 buffer, 0.2 unit of glucose g-phosphate dehydrogenase, and 0.31 unit of hexokinase in a total volume of 3 ml. After 15 min of incubation at 30°C, 2 mg glucose and 1.5 units of hexokinase were added and incubated for 1 h. The reaction mixture was diluted to 10 ml and NADPH was purified and quantitated as described in the previous section. Unlabeled NADPH was added to adjust the specific activity to 1.49 x 10’ cpm/amol. A and B side-labeled [3H] NADPH were stored in 0.01 M carbonate buffer, pH 10.
Determination fer. The required
of stereospecificity of hydride trans-
amount of enzyme (5 to 10 al) was incubated with 0.34 mu [3H]NADPH (2.5 X lo7 cpm), and 0.4 mM 16-oxohexadecanoic acid in a total volume of 0.5 ml of 0.1 M glycine-NaOH buffer, pH 8.5, at 30°C for the required period of time. At the end of the incubation, 1 ml of 1 M HCl was added and the reaction mixture was extracted with chloroform. The chloroform layer was washed with water and evaporated to dryness in a rotary evaporator. From the residue, 16hydroxyhexadecanoic acid was isolated by TLC as indicated in a previous section and assayed for 3H.
Determination of the Equilibrium Constant of the Reaction Catalyzed by w-Hydroxyfatty Acid Dehydrogenase The equilibrium constant was calculated from the expression [16-oxohexadecanoic acid][NADPH][H+] /[16-hydroxyhexadecanoic acid][NADP]. The equilibrium of the reaction was approached from both directions in 0.2 M glycine-NaOH buffer, pH 9.5, at 30°C.
WOUND-INDUCED
POTATO
TUBER
For the approach from the forward direction, the initial concentrations of NADPH and 16-hydroxyhexadecanoic acid were 20 pM each. For the reverse direction, the initial concentrations of NADPH and 16oxohexadecanoic acid were 50 and 40 ELM, respectively. Measurement of radioactivity. Lipid samples and TLC fractions were mixed with either 15 ml of a scintillation fluid consisting of 30% ethanol in toluene containing 0.4% (w/v) Omnifluor or 10 ml of ScintiVerse and assayed for tritium in a Packard Model 3255 Tri-Carb liquid scintillation spectrometer. Internal standard of [3H]toluene was used to determine counting efficiency, which was usually 27% for 3H in Omnifluor and 48% in ScintiVerse. Each sample was checked for quenching by the external standard ratio method, appropriate corrections were made for counting efficiency, and all counting was done with a standard deviation
AND BIOPHYSICS pp. 452-465, 1978
Purification and Characterization of a Wound-Induced o-Hydroxyfatty Acid:NADP Oxidoreductase from Potato Tuber Disks ( Solanum tuberosum L.)’ VISHWANATH Department
of
Agricultural
P. AGRAWAL2
AND
P.E. KOLATTUKUDY3
Chemistry and Program in Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164 Received
June
19,1978;
revised
August
3,1978
w-Hydroxyfatty acid dehydrogenase (w-hydroxyfatty acid:NADP oxidoreductase) catalyzes the reaction w-hydroxyfatty acid -I- NADP + w-oxofatty acid + NADPH +H’. In wound-healing potato tuber disks, the w-oxofatty acid generated by this enzyme is further oxidized to the corresponding dicarboxylic acid by a separate enzyme, w-oxofatty acid dehydrogenase. w-Hydroxy acid dehydrogenase, but not w-0x0 acid dehydrogenase, was found to be induced by wounding potato tubers. w-Hydroxy acid dehydrogenase has been purified 600-fold to near homogeneity from wound-healing potato tuber disks by a combination of gel filtration, anion-exchange, and hydroxylapatite chromatography followed by NADP-Sepharose affinity chromatography, in about 1% yield. The molecular weight and Stokes radius of this enzyme as determined by gel exclusion chromatography are 60,090 and 31 A, respectively. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 31,000, indicating that the deydrogenase is a dimer with subunits of similar molecular weight. The pH optima for the reaction in the forward and reverse directions are 9.5 and 8.5, respectively, and V in the forward and reverse directions are 140 and 3200 nmol/min/mg, respectively. Apparent K, values for NADP, 16-hydroxyhexadecanoic acid, NADPH, and 16-oxohexadecanoic acid are 100, 20, 5, and 7 PM respectively. The equilibrium constant of the reaction at pH 9.5 and 30°C is 1.4 x 10m9 M. The enzyme preparation did not show any stereospecificity for hydride transfer from NADPH to 16-oxohexadecanoic acid.
Suberin is a polymer depositied just inside and most probably covalently attached to the cell wall of the outermost layers (one to three) of cells in the underground parts of plants (1, 2). Recent evidence that suberin deposition is also involved in wound healing of aerial parts of plants suggested that suberization is a fundamental process involved in wound healing of plant tissues irrespective of nature of natural covering material of the tissue (3). Even though the structure of this polymer is poorly understood, during the past few years, it has 1 Scientific Paper 5121, College of Agriculture Research Center, Washington State University, Pulhnan, Washington 99164. This work was supported in part by Grants PCM 74-09351 and PCM 77-00927 from the National Science Foundation. * Fulbright-Hays fellow from Tribhuvan University, Kathmandu, Nepal. ’ To whom all correspondence should be addressed.
become apparent that esterified aliphatic compounds with long carbon chains constitute a significant component of this polymer (2). w-Hydroxyfatty acids and dicarboxylic acids are the major classes of aliphatic monomers of suberin. Among them, w-hydroxypahnitic acid, w-hydroxyoleic acid, and the corresponding dicarboxylic acids are the major components in all suberin samples thus far examined (4-7). Biosynthesis of suberin is not well understood. Since the composition of the aliphatic components of suberin deposited in the wound periderm of potato tubers is identical to that of the natural skin of the tuber (8), wound-healing potato tissue appeared to be a suitable experimental material for biosynthetic studies on suberin. On the basis of the structure of the monomers and from precursor feeding studies, a biosynthetic pathway has been proposed (2).
452 0003-9861/78/1912-0452$0290/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
WOUND-INDUCED
POTATO
TUBER
However, little is known about the enzymes involved in suberixation, except for a demonstration that a cell-free preparation from wound-healing potato tuber slices catalyzed conversion of 16-hydroxyhexadecanoic acid to the corresponding dicarboxylic acid (9). In the present paper, it is demonstrated that this conversion occurs in two steps and that the enzyme which catalyzes the first step, but not that which catalyzes the second step, is induced by wounding. The purification and some properties of this wound-induced o-hydroxyfatty acid dehydrogenase are described. MATERIALS
AND
METHODS
Materials White Rose potatoes grown in California were obtained from a local grocery store and stored at 4’C. NADP, NADPH, NAD, NADH, mercaptoethanol, Sepharose, malic enzyme from chicken liver (EC 1.1.1.40, 13.5 units/mg), hexokinase from yeast (EC 2.7.1.1, 310 units/mg), glucose g-phosphate dehydrogenase from torula yeast (EC 1.1.1.49, 340 units/mg), glutamic dehydrogenase from bovine liver (EC 1.4.1.3, 56 units/mg), DEAE-Sephadex, nitroblue tetrazolium, and phenazine methosulfate were obtained from Sigma Chemical Co. Hydroxylapatite and Coomassie brilliant blue G-256 used for protein assay were obtained from Bio-Rad. Sodium boro[‘H]hydride and D[l-3H]glucose were purchased from Amersham. Pyridinium chlorochromate and 16-hydroxyhexadecanoic acid were purchased from Aldrich Chemical Co.
Preparation of Methyl Oxohexadecanoate
16-
One n-mole of methyl 16-hydroxyhexadecanoate in 1.5 ml of CHzClz was added dropwise to 1.5 mmol of pyridinium chlorochromate suspended in 2 ml of CH&L. After stirring the reaction mixture for 1 h, it was diluted with 15 ml of ether and was passed through a column (1.3 x 4 cm) of Silica Gel G. After evaporating the ether, the product was purified by TLC4 on Silica Gel G with hexanediethyl ether:formic acid (65:35:2, v/v) as the developing solvent (Rf, 0.51; yield, 80%).
Preparation
of 16-Oxohexadecanoic
16-Oxohexadecanoic droxyhexadecanoic acid for the preparation of except in this case, the for 30 min. The product ’ Abbreviation pk.
used
Acid
acid was prepared from 16-hyaccording to the method used methyl-16-oxohexadecanoate, reaction mixture was refluxed after purification by TLC on TLC, thin-layer cbromatogra-
w-HYDROXYACID
453
DEHYDROGENASE
Silica Gel G with diethyl ether:hexane:formic acid (30:2&l, v/v) as the developing solvent (RI, 0.47) was identified by combined gas-liquid chromatographymass spectrometry. The diagnostic fragments of the trimethylsilyl ester derivative were M+-CHa, m/e 327, 9% of the base peak, M+-CHs-HzO, m/e 309,42%; M+CHICO, m/e 299, 1995; M+-CHa-CHzO, m/e 297, 9% M’-CH&HO, m/e 295, 4%; and M+-CH&H&HO, m/e 283, 13%. The metastable for M+-CH3 + M+CH3-Hz0 transition was slso observed.
Preparation of 16-Hydroxy[l Hexadecanoic Acid
6-3H]
To 209 mg of methyl-16-oxohexadecanoate in 3 ml of tetrahydrofuran, 25 mCi of sodium boro[3H]hydride (293 mCi/mmol) was added. After stirring for 16 h, the reaction mixture was acidified and extracted with chloroform. Purification of the hydroxyacid methyl ester by repeated TLC followed by hydrolysis and purification of the hydroxyacid by TLC with ethyl ether:hexane:formic acid (30:2&l, v/v) gave chemically and radiochemically pure (>99%) 16-hydroxy[l63H]hexadecanoic acid.
Preparation
of Suberizing
Potato
Disks
Potato tubers were immersed in 2% (w/v) hypochlorite (30%, v/v, chlorox) solution for 5 min and thoroughly washed with water. They were cut into two halves and tissue disks (2 x 10 mm) were cut from tissue cores prepared with a cork borer. The disks were transferred to layers of rubberized mesh in lgallon jars and tive layers of disks, each separated by a rubberized mesh, were placed in each jar (400 g tissue/jar). Humid air was passed (0.6 l/h) through the jars for 120 h at 22’C and the wound-healing disks were taken out and used for preparation of acetone powder. All materials used were sterilized prior to use and all operations from cutting the potatoes to placing disks in glass jars were performed under sterile conditions in a glove box.
Preparation
of Acetone Powder
Suberized potato disks were homogenized with cold acetone (5 ml/g tissue) at -20°C for 1 min in an explosion-proof Waring blendor, the suspension was filtered under suction, the insoluble material was washed with cold acetone and air-dried. The acetone powder could be stored in airtight containers at -20°C for 4 weeks without significant loss of enzymatic activity. The yield of acetone powder from suberized potato disks was 12-18% of the fresh weight.
Assay for w-hydroxyfatty acid dekydrogenase activity The w-hydroxy acid dehydrogenase was assayed three different methods. Tracer assay I. This assay was done essentially
by as
454
AGFtAWAL
AND
described elsewhere (9). 16-Hydroxy[G-3H]hexadecanoic acid and NADP were incubated with the enzyme preparation and the product isolated by TLC was assayed for 3H with a liquid scintillation spectrometer. Tracer assay II. In this assay, 2- to lo-p1 aliquota of the enzyme preparation were incubated with 0.1 mM 16-hydroxy[16-3H]hexadecanoic acid (1.5 x lo6 cpm) and 1 mu NADP in 0.2 ml of 0.2 M glycineNaOH buffer, pH 9.5, for 30 min at 30°C. The reaction was terminated by adding 1.8 ml of 0.5 M HCl and the mixture was extracted four times by vigorously shaking with chloroform (5 ml each time) using a vortex mixer. One milliliter of the aqueous layer was transferred into scintillation vials, the residual chloroform was removed by heating and passage of NB through the vials, and subsequently assayed for 3H with 10 ml of ScintiVerse.
Spectrophotometric
Assay
In this assay, the rate of reduction of w-0x0 acid was determined by measuring the decrease in absorbance at 340 nm using a Beckman model 25K spectrophotometer. The reaction mixture contained 40 pM 16oxohexadecanoic acid, 80 PM NADPH, and 0.2 M glytine-NaOH buffer, pH 8.5, in a total volume of 0.25 ml and the reaction was initiated by addition of the enzyme.
Assay for o-Oxofatty
Acid Dehydrogenase
o-Oxofatty acid dehydrogenase activity was measured according to the method described elsewhere (9). An aliquot of enzyme solution was incubated at 30°C for 20 min with 1 mM NADP and 0.1 mM 16oxo[G-3H]hexadecanoic acid (2.86 x lo6 cpm) in a total volume of 1 ml of 0.1 M glycine-NaOH buffer, pH 9.5. The reaction was terminated by adding sodium borohydride, and after 15 min, the mixture was acidified and the lipids extracted with chloroform were subjected to TLC after mixing it with unlabeled 16hydroxyhexadecanoic acid and hexadecan-1,16dioic acid. The plates were sprayed with 0.1% ethanolic solution of dichlorofluorescein, the lipids were located under UV light, and the silica gel from the region of hexadecan-1,16-dioic acid was scraped and assayed for 3H directly by liquid scintillation spectrometery.
Assay for Alcohol Activity
Dehydrogenase
The reaction mixture contained 1 mM NAD, 0.1 M ethanol, and 0.2 M glycine-NaOH buffer, pH 9.5, in a total volume of 0.25 ml. The reaction was started by addition of the enzyme solution and increase in absorbance at 340 nm was measured in a Beckman model 25K spectrophotometer.
Determination
of Protein
The widely be used
used method of Lowry due to its sensitivity
not
Concentration et al. (10) could to glycerol and
KOLA’M’UKUDY mercaptoethanol present in the fractions obtained during enzyme purification. Therefore, a dye-binding method was used for protein determination (11). The commercially available concentrated solution of Coomassie blue dye was diluted B-fold with water, 5 ml of the diluted dye solution was mixed thoroughly with 0.1 ml of the protein solution, and absorbance at 595 nm was measured after 5 min. Bovine serum albumin was used as standard.
Preparation
of NADP-Sepharose
NADP-Sepharose was prepared by the method described elsewhere (12). Sepharose 4B was activated with CNBr and then treated with the dihydrazide derivative of adipic acid. This dihydrazide-Sepharose 4B gel was coupled to periodate-oxidized-NADPSepharose. The amount of NADP bound to the gel, calculated from the phosphorus content (I3), was 2 to 3 eole/mI of bed volume.
Enzyme Purification Extraction of theprotein from acetone-powder and ammonium sulfate precipitation. Acetone powder (400 g) was slowly added with stirring to 4 liter 0.1 M Na-phosphate buffer, pH 6.7, containing 0.05 M mercaptoethanol over a period of 4 h. The mixture was squeezed through eight layers of cheesecloth and the volume of the filtrate was adjusted to 4 liters. To this solution, 704 g of granular ammonium sulfate was slowly added with stirring to obtain a 30% saturation and the resulting precipitate was removed by centrifuging the mixture at 10,006 rpm for 10 min. To the supernatant, 856 g of ammonium sulfate was added to attain a 60% saturation, and the resulting precipitate was collected as above and dissolved in a minimum volume of 0.05 M Tris-HCl, pH 8.3, containing 10 mM mercaptoethanol and 20% glycerol (buffer A). This solution was dialyzed against buffer A for 48 h and the volume was adjusted to 500 ml (11 g protein). Treatment with DEAE-cellulose. The protein solution from the previous step was stirred for 30 min with 75 g DE-52 which was previously equilibrated with buffer A and centrifuged at 10,000 rpm for 10 min. The supernatant was discarded and the pellet was resuspended in 500 ml of buffer A, and again centrifuged. The pellet was stirred with 300 ml of buffer A containing 0.2 M KC1 for 1 h, centrifuged, and the supernatant was saved. The pellet was resuspended in 200 ml of buffer A containing 0.2 M KC1 and centrifuged. Both supernatants were combined and concentrated to 40 to 45 ml by ultrafiltration using UM 10 membrane. Sepharose 6B gel filtration. About lo-ml portions (1.6 g protein) of the protein solution from the above step were applied to Sepharose 6B columns (3.9 X 100 cm) previously equilibrated with 0.05 M Tris-HCl buffer, pH 7.3, containing 10 mM mercaptoethanol and 20% glycerol (buffer B). The proteins were eluted with
WOUND-INDUCED
POTATO
TUBER
the same buffer at a flow rate of 30 to 40 ml/h and the effluent was monitored for absorbance at 280 nm using an ISCO model UA-5 monitor. The fractions (9-10 ml) containing enzyme activity greater than half of the maximum activity were pooled (70-75 ml from each column).
DEAE-Sephadex anion exchange chromatography. The protein obtained from the previous step (300 ml, 2.2 g protein) was passed through a DEAE-Sephadex column (bed volume, 200 ml) equilibrated with buffer B, at a flow rate of 40 ml/h. The column was washed with 300 ml of buffer B at the same flow rate and the absorbed proteins were eluted at a flow rate of 30 ml/h, with a linear gradient of 0 to 0.2 M KC1 in a total volume of 1000 ml of buffer B. The effluents were monitored for absorbance at 280 nm. Fractions (10 ml) containing enzyme activity and eluting in the range of 0.09 to 0.14 M KC1 were pooled (200 ml). This solution was concentrated by ultrafiltration and diluted with 5 mu Na-phosphate buffer, pH 6.8, containing 5% glycerol, and 10 mu mercaptoethanol (buffer C). This process was repeated 4 times in order to change the medium. Hydroxylapatite chromatography. The protein obtained from the anion exchange chromatography step (40 ml, 600 mg protein) was applied to a hydroxylapatite column (inner diameter, 2.6 cm, bed volume, 25 ml) equilibrated with buffer C. After removing unabsorbed proteins with 50 ml of buffer C, the absorbed proteins were eluted with a gradient prepared by mixing 125 ml of buffer C and 125 ml of 0.2 M Na-phosphate buffer, pH 6.8, containing 5% glycerol and 10 mM mercaptoethanol at a flow rate of 15-20 ml/h. Fractions containing w-hydroxy acid dehydrogenase activity were pooled, the buffer was changed to 0.01 M Tris-HCl buffer, pH 7.5, containing 20% glycerol and 10 mru mercaptoethanol (buffer D) by ultrafiltration as above, and the volume was adjusted to 20 ml with buffer D.
NADP-Sepharose affinity chromatography I (with KCZ as eluant). The enzyme preparation (115 mg protein) obtained from the above step was chromatographed on a NADP-Sepharose column (inner diameter, 1 cm, bed volume, 20 ml) equilibrated with buffer D. The column was washed with 40 ml of buffer D and the absorbed proteins were eluted with a linear gradient of 0 to 2 M KC1 in a total volume of 200 ml of buffer D at 10 to 12 ml/h. Fractions containing the enzyme activity were pooled, KC1 was removed by ultrafiltration, and the volume was adjusted to 5 ml with buffer D.
NADP-Sepharose affinity chromatography II (with NADP as eluant). The protein fraction (2 mg) obtained from the previous step was passed through a NADP-Sepharose column (inner diameter, 1 cm, bed volume, 5 ml) equilibrated with buffer D and unabsorbed proteins were removed by washing the column with 15 ml of the same buffer. The absorbed proteins were eluted with a linear gradient of 0 to 0.1 mM
o-HYDROXYACID
DEHYDROGENASE
455
NADP in a total volume of 40 ml of buffer D and 1.4ml fractions were collected at a flow rate of 5 to 6 ml/h. Fractions containing enzyme activity were pooled, concentrated to 1 ml by ultrafiltration, diluted with 10 ml of buffer D, and again concentrated to 1 ml. This process was repeated 3 to 4 times to remove NADP and the resulting enzyme solution was stored at 0°C. Electrophoresis. Polyacrylamide gel electrophoresis was performed in an analytical electrophoresis apparatus from Hoefer Scientific Instruments, according to the method described elsewhere (14). The gel system consisted of a 7.5% polyacrylamide resolving gel (pH 8.9) and 2.5% stacking gel (pH 7.2). The lengths of resolving and stacking gels were 7.5 and 2.5 cm, respectively, with a gel tube inner diameter of 0.45 cm. Protein samples, containing glycerol and bromphenol dye, were applied to the gels in a volume of 0.1 to 0.2 ml. Electrophoresis was performed at 4°C at a constant current of 2 mA/tube until the dye migrated 7 cm into the resolving gel. Protein bands were fixed by immersing the gels in 12.5% trichloroacetic acid solution for 30 min and then stained overnight in a solution containing 0.2% Coomassie blue, 7.5% acetic acid, and 50% methanol. The excess dye was removed from the gels electrophoretically by an Ames model 1801 quick gel destainer with a destaining solution containing 7.5% acetic acid and 5% methanol. The gels were scanned for absorbance at 600 nm in a Beckmann model 25K spectrophotometer with a gel scanner accessory. Sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis was done according to the method of Maize1 (15). The gel system of the same dimensions as above, containing 0.1% dodecyl sulfate, consisted of a 10% polyacrylamide resolving gel (pH 8.9) and 2.5% stacking gel (pH 7.2). The proteins were heated with 1% SDS and 0.1 M dithioerythritol in a boiling water bath for 3 min. Electrophoresis was done at 2.5 mA/tube until the dye migrated 7 cm into the resolving gel and the protein bands in the gel were located as described earlier.
Location of w-hydroxyfatty activity in the gel. w-Hydroxy
acid dehydrogenase
acid dehydrogenase activity was located in the polyacrylamide gels either by staining the gel for enzymic activity or by recovering the enzyme from gel slices. To stain for enzymic activity, the gels were incubated at 37°C in dark with 10 ml of a solution which contained 0.1 mM of 16hydroxyhexadecanoic acid, 1 m$NADP, 2.5 mg nitroblue tetrazolium, and 0.25 ml of phenazine methosulfate in 10 ml of 0.2 M glycine-NaOH btiffer, pH 9.5. The progress of staining was checked frequently because incubation for excessively long periods resulted in a dark background. For control, incubation mixtures containing no substrate were used. To recover the enzyme, the gel was cut into 2-mm slices with a gel slicer obtained from Bio-Rad. The gel slices were soaked overnight at 4°C in 0.1 ml of 0.2 M glycineNaOH, pH 9.5, containing 20% glycerol and 10 mM
456 mercaptoethanol enzyme activity tometric assay.
Determination
AGHAWAL
AND
and the solution was assayed for the using tracer assay II and spectropho-
of Isoelectric
Point
The enzyme preparation from the second NADPSepharose affinity chromatography was electrofocused in a LKB 8100 Ampholine electrofocusing apparatus for 72 h at 1.5 mA current and 809 V. Sucrose was used as the stabilizing medium and 0.9% of pH 3-10 Ampholine was used.
Determination of Molecular Weight and Stokes’ Radius of w-Hydroxyfatty Acid Dehydrogenase A Sephadex G-150 column (1.3 x 84 cm), equilibrated with 0.05 M phosphate buffer, pH 7.6, was calibrated with standard proteins by measuring their elution volumes (V.) from the column. Protein standards used, their molecular weight, and Stokes’ radii in A (16) were as follows: A, immunoglobulin G, 150,000 51; B, bovine serum albumin, 68,000, 35; C, ovalbumin 43,000,30; D, chymotrypsinogen, 25,700,22; E, myoglobin, 17,200, 19; and F, cytochrome c, 11,700,17. The void volume (VO) was measured by using blue dextran and Vt was calculated from the column dimensions. The protein obtained from last purification step was used to measure the elution volume of whydroxyfatty acid dehydrogenase. Molecular weight of w-hydroxy acid dehydrogenase was determined from the linear plot of log of molecular weight vs. partition coefficient (&). For the determination of Stokes’ radius, a linear plot of Stokes’ radius, R, us erf’ (1 &) was used (17). For the determination of molecular weight of the enzyme under denaturing conditions, the enzyme preparation and protein standards were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described earlier. The standards were 1) bovine serum albumin (68,000), 2) heavy chain of y-globulin (50,000), 3) alcohol dehydrogenase (37,500), 4) chymotrypsinogen (25,700), and 5) light chain of y-globulin (23,000), and the molecular weight of whydroxy acid dehydrogenase was determined from the linear plot of log of molecular weight us mobility.
Preparation
of [A-3H] @ro-R) NADPH.
D,L-[2-%]
Malate was prepared by stirring 106 mg of oxaloacetate in 1.5 ml HxO, pH 8.5 (adjusted by 1 N NaOH), with 25 mCi of NaB3H4 (specific activity, 661 mCi/mmol) for 16 h. The reaction mixture was aciditied with dilute HCl, exchangeable 3H was removed by lyophilization, and the residue (in 5 ml water) was applied to a Dowex anion exchange column, Cl- form (bed volume, 75 ml). Labeled malate was eluted with 0.05 M HCl, the radioactive fractions were pooled and lyophilized (4.7 x 10’ cpm). Malic enzyme was used to transfer 3H from the labeled malate to the A side of NADPH (18). The reaction mixture contained 0.8 mu NADP, 0.8 mM D,L-[3H]malate (2.35 X lo9 cpm), 0.2
KOLATTUKUDY mM MnClz, and 0.07 M triethanolamine buffer, pH 7.4, in a total volume of 30 ml. The reaction was initiated by adding 0.1 unit of malic enzyme (0.01 ml) and 2 ml of the reaction mixture was monitored for absorbance change at 340 nm. After the reaction rate leveled off (about 15 min), the reaction mixture was frozen and lyophilized. The lyophilized material was dissolved in 10 ml of Hz0 and chromatographed on a DEAEHC03column (1 X 15 cm) and the products were eluted with 0 to 0.5 M linear gradient of NHdHC03 in a total volume of 1060 ml (19). The fractions were monitored for AzK,, As%, and radioactivity and those with A&A339 between 2.3 to 2.6 and a constant ratio of cprn/Aasg were pooled and lyophilized. The content of NADPH in the preparation was measured by converting NADPH to NADP using glutamate dehydrogenase (20). The value obtained was 10 to 15% lower than that calculated from A~X+ Unlabeled NADPH was added to adjust the specific activity to 1.49 x 10’ dpm/pmol. Preparation of [B-3H] bra-S) NADPH. [B-3H] NADPH was prepared by transferring 3H from [1-3H] glucose to NADP using a combination of hexokinase and glucose g-phosphate dehydrogenase. The reaction mixture contained 20 pM [l-3H]glucose (0.5 mCi), 0.55 mM ATP, 0.33 mM NADP, 7.5 mu MgClz, 0.03 M TrisHCl, pH 8 buffer, 0.2 unit of glucose g-phosphate dehydrogenase, and 0.31 unit of hexokinase in a total volume of 3 ml. After 15 min of incubation at 30°C, 2 mg glucose and 1.5 units of hexokinase were added and incubated for 1 h. The reaction mixture was diluted to 10 ml and NADPH was purified and quantitated as described in the previous section. Unlabeled NADPH was added to adjust the specific activity to 1.49 x 10’ cpm/amol. A and B side-labeled [3H] NADPH were stored in 0.01 M carbonate buffer, pH 10.
Determination fer. The required
of stereospecificity of hydride trans-
amount of enzyme (5 to 10 al) was incubated with 0.34 mu [3H]NADPH (2.5 X lo7 cpm), and 0.4 mM 16-oxohexadecanoic acid in a total volume of 0.5 ml of 0.1 M glycine-NaOH buffer, pH 8.5, at 30°C for the required period of time. At the end of the incubation, 1 ml of 1 M HCl was added and the reaction mixture was extracted with chloroform. The chloroform layer was washed with water and evaporated to dryness in a rotary evaporator. From the residue, 16hydroxyhexadecanoic acid was isolated by TLC as indicated in a previous section and assayed for 3H.
Determination of the Equilibrium Constant of the Reaction Catalyzed by w-Hydroxyfatty Acid Dehydrogenase The equilibrium constant was calculated from the expression [16-oxohexadecanoic acid][NADPH][H+] /[16-hydroxyhexadecanoic acid][NADP]. The equilibrium of the reaction was approached from both directions in 0.2 M glycine-NaOH buffer, pH 9.5, at 30°C.
WOUND-INDUCED
POTATO
TUBER
For the approach from the forward direction, the initial concentrations of NADPH and 16-hydroxyhexadecanoic acid were 20 pM each. For the reverse direction, the initial concentrations of NADPH and 16oxohexadecanoic acid were 50 and 40 ELM, respectively. Measurement of radioactivity. Lipid samples and TLC fractions were mixed with either 15 ml of a scintillation fluid consisting of 30% ethanol in toluene containing 0.4% (w/v) Omnifluor or 10 ml of ScintiVerse and assayed for tritium in a Packard Model 3255 Tri-Carb liquid scintillation spectrometer. Internal standard of [3H]toluene was used to determine counting efficiency, which was usually 27% for 3H in Omnifluor and 48% in ScintiVerse. Each sample was checked for quenching by the external standard ratio method, appropriate corrections were made for counting efficiency, and all counting was done with a standard deviation
