ARCHIVES

OF BIOCHEMISTRY

Vol. 284, No. 1, January,

AND

BIOPHYSICS

pp. 127-132,

1991

Purification and Characterization Dehydrogenase from Cabbage Atsuko

Nagai

International

Received

July

and Alfred

of Histidinol

Scheidegger’

Research Laboratories,

24, 1990, and in revised

CIBA-GEIGY

form

August

(Japan)

Ltd., P.O. Box 1, Takarazuka,

26, 1990.

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quarternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 j&M, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni’+, Ca2+ Zn’+, or Cu2+. Histidinol dehydrogenase is the first hiitidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms. o 1991 Academic

Press,

Inc.

The histidine biosynthesis in higher plants has not been studied well. Some evidence for the existence in plants of a biosynthetic pathway similar to that in microorganisms has been obtained from in uivo experiments using various different plants and a blue-green algae (l-4). Only few attempts were made to study the enzymes involved in His biosynthesis in higher plants. The impossibility of purifying any His biosynthetic enzyme to homogeneity from

plants so far seemsto be the limiting step in biochemical investigations. In crude extracts from shoots of barley, oats, and peas, the activities of ATP-phosphoribosyl transferase, imidazoleglycerol phosphate dehydratase, and histidinol phosphate phosphatase were detected (5). Hist&no1 dehydrogenase activity was found in crude extracts of different plant species (6). These data suggest that His biosynthesis in plants follows the same pathway as in microorganisms, although no protein has ever been isolated from plants and identified as an enzyme involved directly in His biosynthesis. For the elucidation of the His biosynthetic pathway in plants, we investigated the role of histidinol dehydrogenase [L-histidinol:NAD oxidoreductase (EC 1.1.1.23)] in higher plants. The enzyme in microorganisms catalyzes the two final reaction steps, leading from histidinol over histidinal to the endproduct His, thereby reducing 2 mol of NAD’ for 1 mol of formed His: Histidinol + NAD+ + histidinal + NADH histidinal + NAD+ + histidine + NADH. Histidinol dehydrogenase has been studied extensively in Salmonella typhimurium (7-9) and to a much lesserextent in Escherichia coli (10). In Saccharomyces cerevisiae (11, 12) and Neurospora crassa (13), there is some evidence that the dehydrogenase activity is associated with two other His biosynthetic activities in a single protein. The lo-fold purification of the enzyme from wheat germ was not sufficient to identify the protein (6). Here we describe the presence of histidinol dehydrogenase in several plant species and its purification from cabbage by a new affinity chromatography. Some properties of the enzyme are also reported. EXPERIMENTAL

1 To whom correspondance should be addressed search Laboratories, CIBA-GEIGY (Japan) Ltd., zuka, 665 Japan. FAX: 0797-74-2325. 0003.9861/91 Copyright All rights

$3.00 0 1991 by Academic Press, of reproduction in any form

665 Japan

at International ReP.O. Box 1, Takara-

PROCEDURES

Materials and strains. Sepharose 4B was purchased from Pharmacia LKB. Histidinol dihydrochloride was from Aldrich, and NAD+, sodium metaperiodate, epichlorohydrin, and nitroblue tetrazolium were from 127

Inc. reserved.

128

NAGAI

AND

Sigma. Sodium cyanoborohydride was purchased from Nakarai, and phenazine methosulfate from Wako Pure Chemical Industries. All other chemicals used were of analytical grade. Cabbage (Brassica oleracea), cucumber (Cucumis satiuus), asparagus (Asparagus oficinalis), eggplant (Solonurn melongem), lettuce (Lactuca satiua), and pimento (Cupsicum annum) were cultivated in a growth chamber with a 16 h/8 h light/dark cycle at 25°C during the illumination period and 15’C during the dark period. The relative humidity was constant at 80%. Cell cultures of Rosa “Paul’s Scarlet” were a gift of Andre Strauss, CIBA-GEIGY Ltd., Switzerland, and cultured in suspension as previously described (14). Cell cultures of tobacco (Nicotiana tabacum L. cv Samsun NN) and suspension cultures of wheat (Triticum acstiuum var Chinese Spring) were kindly provided by Yasuyuki Yamada, Kyoto University, Japan, and grown as described elsewhere (15, 16). Mature spring cabbage (Brussicu oleruceu L. var cupitutu L.) was purchased from local grocer. Wheat germ was obtained from Sigma. Preparation of enzyme extract. Two-week-old plants, whole cabbage heads, or well grown cell tissues were used as the enzyme sources. The plant material was homogenized with a Polytron blender in cold 100 mM sodium phosphate buffer, pH 7.2 (buffer A), and the homogenate passed through a filtration cloth. After centrifugation at 30,OOOg for 20 min, the supernatant was saturated to 80% with ammonium sulfate on ice. The precipitate was collected by centrifugation and dissolved in buffer A and the solution desalted on Sephadex G-25. This proteinenriched extract was used for the determination of histidinol dehydrogenase activity in various plants. For the purification of the enzyme from cabbage, the same procedure was applied except that the supernatant was fractionated with 45560% saturation of ammonium sulfate. Purification of histidinol dehydrogenuse. Spring cabbage heads were homogenized using a kitchen mixer and the suspension processed as described above, except that desalting was performed by extensively dialyzing against 50 mM Tris/HCl buffer, pH 7.4, (buffer B). All subsequent procedures were carried out at 4’C. Dialyzed protein extract was applied to a DEAE-Toyopearl column equilibrated with buffer B. After the column was washed with buffer B, protein was eluted with a linear gradient of buffer B containing sodium chloride (O-500 mM). Fractions containing enzyme activity were pooled and directly applied to a histidinol-Sepharose 4B column (2.5 X 8 cm) at a flow rate of 36 ml/h. Unadsorbed protein was eluted by extensively washing with 10 mM Tris/HCl buffer, pH 7.3, containing 140 mM NaCl (buffer C). Bound histidinol dehydrogenase was eluted with 50 mM Tris/ HCl buffer, pH 7.3, containing 700 mM imidazole. After desalting all fractions on Sephadex G-25, the enzyme activity in each fraction was determined. Active fractions were pooled and concentrated on an Amicon ultrafiltration membrane (YM-10). This enzyme preparation was stored at ~80°C and used for all experiments, except for the activity screening in plants. Gel filtration. A Superose 12 column (Pharmacia LKB) was equilibrated with purification buffer C at room temperature. Samples of purified enzyme were applied to the column and eluted with buffer C at a flow rate of 0.5 ml/min. The molecular mass was calculated using a molecular weight marker kit (Sigma). Polyucrylumide gel electrophoresis. SDS-polyacrylamide gel electrophoresis’ was performed as described elsewhere (17). using lo20% acrylamide. Proteins in sample buffer were put in a heating block at 100°C for 10 min. For the calculation of the molecular mass of denatured and reduced protein, an electrophoresis calibration kit for molecular weight determination (Pharmacia) was used. Isoelectric focusing. PhastGel (Pharmacia LKB) with a pH range of 4-6.5 was used for isoelectric focusing, performed on a Phast System (Pharmacia LKB) according to the instruction manual of the supplier.

‘Abbreviations thiohydantoin;

used: SDS, sodium dodecyl Ches, 2(cyclohexylamino)ethanesulfonic

sulfate;

PTH, acid.

phenyl-

SCHEIDEGGER The isoelectric point calibration

point of the protein was determined kit (Pharmacia LKB).

using

an isoelectric

Preparation of histidinol-Sephurose 4B gel. Sepharose 4B was activated with epichlorohydrin as previously described (18). Epoxyactivated Sepharose 4B gel was suspended in 0.1 N NaOH containing 0.5 g glucose per gram of gel and incubated at 4O’C on a shaker for 24 h (19). After being washed extensively with water, the gel was suspended in prechilled sodium metaperiodate and the suspension shaken for 1 h at 4OC. The gel was washed with water, suspended in 0.1 M HCl and incubated at room temperature for 1 h. Again, the gel was washed extensively with water, followed by thorough washing with 10 mM sodium phosphate buffer, pH 7.0, at room temperature. The formyl carrier gel was suspended in 10 mM sodium phosphate buffer, pH 7.0, containing 100 mM of histidinol dihydrochloride, which was previously neutralized with NaOH, and the suspension incubated at 4°C overnight. Five milligrams of sodium cyanoborohydride per gram of gel was then added to the suspension and incubated with shaking at room temperature for 6 h. The gel was extensively washed with distilled water and treated with 5 mg sodium borohydride per gram of gel for 3 h at 4°C to convert the remaining formyl groups into hydroxymethyl groups. Enzyme assay. The enzyme activity was assayed spectrophotometrically by measuring the increase in absorbance at 340 nm due to the reduction of NAD+ in a Hitachi U-3120 spectrophotometer. The reaction mixture contained 150 mM Gly/NaOH buffer, pH 9.2, 0.5 mM MnC12, 2 mM NAD+, 5 mM histidinol, and 2-5 mU of enzyme sample in a total volume of 0.5 ml. The reaction was started with the addition of histidinol, and incubated at 30°C. A reference sample containing water instead of histidinol was always run. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 2 pmol of NADH per minute under the assay conditions. In some experiments the activity was also followed by measuring the formation of His with a Hitachi L-850 amino acid analyzer. The enzyme reaction was terminated with formic acid (final concentration, 30%). The sample was then evaporated, and the precipitate dissolved in 0.2 N HCl. This preparation was used for His analysis. Isoelectric point calibration proteins and purified Activity staining. enzyme (in quadruplicate) were applied on a PhastGel (pH range 46.5) to give five lanes. After running the isoelectric focusing, the gel was cut into four pieces. The one containing marker proteins in addition to enzyme was stained with Coomassie brilliant blue R-250. Another piece of gel containing only enzyme was subjected to activity staining as described elsewhere (20), with a slight modification. The gel was immersed in 10 ml of reaction mixture containing 5 mM histidinol and 2 mM NAD+. By the addition of 150 ~1 of nitroblue tetrazolium and 150 ~1 of phenazine methosulfate to give a final concentration of 0.5 and 0.13 mM, respectively, the staining process was started. Incubation was carried out at 3O’C for 30 min. Two other pieces of gel containing only enzyme were identically incubated, but either without histidinol or without NAD+. The concentration of the protein was deterProtein determination. mined by the Bradford protein assay method using bovine serum albumin as a standard (21). N-Terminal amino acid sequencing. The purified histidinol dehydrogenase was subjected directly to automated Edman degradation with an Applied Biosystems 470A gas-liquid phase protein sequencer (22). The phenylthiohydantoin (PTH) amino acid derivatives were separated and identified with an on-line PTH analyzer (Applied Biosystems) with a PTH-Ci, column.

RESULTS

Distribution of Histidinol Dehydrogenase in Plants Eleven plant species or preparations were examined for histidinol dehydrogenase activity. Enzyme activity could not be detected in any crude extract. Activity was found in enriched extracts (040% ammonium sulfate

HISTIDINOL TABLE

Distribution

Plant

FROM

I

source

Specific activity bU/md”

300

-$

: c 2

0.9

6.1 2.1

2.8 91

0.5

0.4

6.2

0.9

0

0

FIG. tein dinol was NaCl arrow HCl

Total

Crude extract Ammonium sulfate fractionation DEAE-Toyopearl ion-exchange chromatography Histidinol-Sepharose 4B affinity chromatography

protein bd n.d.

4750 560 0.75

i

1 60

.A iv60

0 100

Number

1. Histidinol-Sepharose 4B affinity chromatography. (- * -) Proconcentration represented by the absorption at 280 nm; (0) histidehydrogenase activity expressed in mu/ml. The affinity column washed with 10 mM Tris/HCl buffer, pH 7.3, containing 140 mM until all nonadsorbed protein was eluted (at fraction 75). The at fraction 76 indicates the start of the elution with 50 mM Tris/ buffer, pH 7.3, containing 700 mM imidazole.

from Cabbage

II

of Histidinol Total

\ ‘....., ‘.,.Ul

-.-*-~-b.~-~-*-~-~-~-~~ I I 20 40

100

I

Several unsuccessful attempts were made to purify histidinol dehydrogenase from mature spring cabbage heads by conventional purification methods. The development of an affinity gel, highly specific for this enzyme, finally made it possible to purify the protein in three steps with a high yield to apparent homogeneity (Table II). The enzyme substrate histidinol was coupled to Sepharose 4B and the gel packed into a column. Before using the affinity gel efficiently, the protein extract was purified on a DEAEToyopearl ion-exchanger. The protein fraction with activity was eluted from the column before the main protein peak, at a concentration of 150 mM sodium chloride in the buffer. The pooled active fractions were directly loaded on the histidinol-Sepharose 4B affinity column. All activity was bound to the gel, probably very tightly, since only 40% of the activity could be eluted specifically with a one-step addition of a high concentration of imidazole

TABLE

step

I / I I .: 6 -.-.

Purification

precipitated and desalted on Sephadex G-25) from various monocotyledon and dicotyledon species (Table I). Cultured rose cells and spring cabbage showed the highest specific enzyme activities. Wheat germ had an extremely high extractable activity probably due to its low content of water; however, its specific activity was modest. Also, shoots of cabbage and cucumber and spring cabbage heads contained a high extractable activity. No activity could be detected in 2-week-old pimento shoots and cultured tobacco cells.

Purification

.

Fraction

3.3 0

= E \ 3 5 h C .? 2

.

!

o

the calculation of the extractable activity, the wet weight material was used. The weight of cells from submersed was determined after collection by filtration with a cloth. activity per milligram of protein. activity per gram of plant material.

Purification

;

0

0.4 0.3 0

i

200

0.5-

L : ::

1.6 0.3

0.4

/

z 2

3.3

0.9

,,.---.-..,.....,.. ...(_ ,-,.....,_... -‘-\ . I (1II i

Extractable activity bU/d *

1.6

129

CABBAGE

1.01

of Histidinol Dehydrogenase in Different Plant Species and Preparations

Cabbage shoots (Brassica oleracea L. var capitata L.) Asparagus shoots (Asparagus oficinalis) Lettuce shoots (Lactuca satiua) Eggplant shoots (Solarium melongena) Cucumber shoots (Cuxumis satiuus) Pimento shoots (Capsicun annum) Spring cabbage (Bras&a oleracea L. var capitata L.) Wheat germ Wheat cell culture (Triticum aestiuum var Chinese Spring) Rose cell culture (Rosa “Paul’s Scarlet”) Tobacco cell culture (Nicotiana tabacum L. cv Samsun NN) Note. For of the plant tissue cultures a Units of * Units of

DEHYDROGENASE

Dehydrogenase

activity NJ)

Recovery (%)

Specific activity W/W

Purity (-fold)

n.d.

n.d.

n.d.

23

100

0.0048

1

0.029

6

20.1

87

7.6

33

Note. In this typical purification, 10 kg of cabbage heads were processed. All activities 25 or dialysis. The activity in the crude extract could not be detected (n.d.).

were measured

nd.

10.16

after

desalting

2116

with

either

Sephadex

G-

130

NAGAI

AND

(700 mM) in the elution buffer (Fig. 1). By this step alone, the specific enzyme activity was increased by a factor of 350 to 10.16 U/mg of protein, showing this chromatography to be very specific for histidinol dehydrogenase. The overall 2116-fold purification does not include the first step of fractionated ammonium sulfate precipitation, since no activity could be detected in crude extract. A heavily loaded SDS-polyacrylamide gel showed one major band (Fig. 2). Storage of the enzyme for more than 4 months at -80°C did not change significantly the activity. At 4°C the enzyme lost 10% of its activity during 1 week. By the addition of glycerol to a final concentration of 20%, full activity could be maintained at 4°C for more than 1 week. Molecular Weight and Structure The native molecular weight of the enzyme was determined by Superose 12 gel filtration to be 103,000. Under denaturing and reducing conditions, the enzyme migrated as a single band if exposed to SDS-polyacrylamide gel electrophoresis, suggesting a dimeric quarternary structure of the enzyme. A molecular mass of 52 kDa was calculated for the monomer. The exposure of purified enzyme to analytical isoelectric focusing lead to its separation into six protein bands within the pH range 5.1-5.4 (Fig. 3). Five bands showed histidinol dehydrogenase activity by employing activity staining. The sixth band had only very slight or no activity. In control experiments lacking either histidinol or

kDa

A

B

94 67

43

SCHEIDEGGER PI

A

B

C

6.55 5.85

5.2

4.55

4.15

FIG. 3. Isoelectric focusing of purified histidinol dehydrogenase. (A) Isoelectric point standards, human carbonic anhydrase B (pZ = 6.55), bovine carbonic anhydrase B (pZ = 5.85), P-lactoglobulin A (pZ = 5.2), soybean trypsin inhibitor (pZ = 4.55), and glucose oxidase (pZ = 4.15). (B) Purified histidinol dehydrogenase; protein was stained with Coomassie brilliant blue R-250. (C) Purified histidinol dehydrogenase; activity staining was performed as described under Materials and Methods after cutting the gel.

NAD+ in the reaction mixture, p-nitroblue tetrazolium was not reduced to formazan. The activity of each of the five bands correlated with their color intensities obtained by protein staining (Fig. 3). To rule out artifacts of the purification procedure, several independent purifications were carried out, starting from cabbage heads in different growth stages. In all cases, six protein bands were obtained. N-Terminal Am,ino Acids N-Terminal amino acids were determined in duplicate by Edman degradation. The first sequence run showed a recovery of 15% of the protein concentration determined by amino acid analysis, indicating that 85% of the Nterminus was blocked for unknown reason. The first Nterminal amino acid of the 15% of unblocked enzyme could not be determined unambiguously in either experiment. Ala and Lys were found most abundantly, but also Ser, Gly, Glu, Val, and Leu were detected, all in similar concentrations. The following two amino acids were unequivocally identified as Met and Lys. Reaction and Kinetics

FIG. 2. SDS-polyacrylamide gel of purified histidinol dehydrogenase. (A) Molecular weight standards, phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (ZO,lOO), and a-lactalbumin (14,400). (B) Purified histidinol dehydrogenase. Protein was stained with Coomassie brilliant blue R-250. The direction of protein migration was from the cathode (top) to the anode (bottom).

Histidinol dehydrogenase in microorganisms catalyzes the two last reactions to form the endproduct His. To verify this catalytic role for the isolated protein from cabbage, its catalyzed reaction was analyzed stoichiometritally with regard to His formation and NAD+ reduction. The amount of enzymatically produced NADH was determined from the absorbance at 340 nm, using the absorbance coefficient of 6220. The other formed product was identified by amino acid analysis as His. Authentic His added to reacted sample was coeluted with enzymat-

HISTIDINOL TABLE Stoichiometry Regard

Reaction time (min)

FROM

131

CABBAGE

III

of Histidinol Dehydrogenase to the Formation of His His concentration ( wml/ml) (a)

DEHYDROGENASE

NADH

Reaction NAD+

and

concentration (wWm1) (b)

0

0

0

2 5 30

17.6

35.6

36.6 143.7

80.0 316.0

with

b/a

2.02 2.18 2.2

Note. The enzyme reaction was stopped after 2,5, and 30 min reaction time and the sample analyzed by automated amino acid analysis. Histidinol does not react with ninhydrin and cannot be analyzed by amino acid analysis (at 50 nmol).

PH

FIG. 4.

Dependency of histidinol dehydrogenase activity on pH. (B) reaction in 50 mM Hepes buffer; (0) reaction in 50 mM Gly/NaOH buffer; (0) reaction in 50 mM Ches buffer. In all buffers, 0.5 mM MnCl, was included. The reaction was performed under standard conditions as described under Materials and Methods. In 50 mM phosphate buffer, pH 7.5, no activity was detected.

ically formed His. Neither His nor NADH were formed in the absence of NAD’, histidinol, or by using cooked enzyme. Two moles of NADH were formed for 0.99 mol purified protein is histidinol dehydrogenase was obtained of His after a reaction time of 2 min (Table III). The from activity staining of the protein on the gel after isoformation of both products, histidine and NADH, was electric focusing. The enzymatically formed product time dependent and correlated. starting from histidinol was identified as His. His and Optimum pH for the activity was at 9.2 in 50 mM Gly/ NADH were produced stoichiometrically, showing the NaOH buffer (Fig. 4). At pH values below 9, activity desame reduction of 2 mol of NAD+ for 1 mol of formed His creased in both Gly/NaOH buffer and 50 mM Hepes as previously described for this reaction in microorganbuffer. At pH 9.2-10, almost full activity was maintained, isms (23). The present results demonstrate that the last but above 10, a rapid loss of activity occurred. two reaction steps in the His biosynthetic pathway in The apparent K,,, values of the enzyme for L-histidinol cabbage are identical to those in microorganisms and are and NAD+ were determined under standard assay concatalyzed by the same single enzyme. The K,,, of this enditions as 15.5 and 42 PM, respectively. zyme for L-histidinol (15.5 PM at pH 9.2) is very similar The influence of divalent metal ions on the enzyme to those (16 and 8.8 PM) of the pure enzyme from S. tyreaction is shown in Table IV. The reaction was stimuphimurium (24) and the partially purified one from wheat lated 26% by the addition of Mn2+ compared to control germ (6). The K,,, (42 PM) for NAD+ is considerably lower conditions without the addition of any divalent metal ion than that of the enzymes from S. typhimurium (1 mM) to the reaction buffer (150 InM Gly/NaOH, pH 9.2). The (24) and from wheat germ [ 140 PM, using partially purified addition of Ba’+, M$+, Ni2+, Ca”, Zn”, and Cu2+ caused enzyme (6)]. The pH optimum of the reaction between inhibition of the reaction. DISCUSSION TABLE

Histidinol dehydrogenase activity has been detected in 10 different plant species: asparagus, cabbage, cucumber, eggplant, lettuce, radish (6), rose, squash (6), turnip (6), and wheat [ (6) and this work] and in five preparations of distinct cell differentiation: germ [ (6) and this work), root (6), fruit [(6) and this work], shoot, and cultured cell. Even though we could not determine enzyme activity in pimento shoots and cultured tissues of tobacco, probably due to activities below detection level, it can be assumed that this enzyme is common in plants. Histidinol dehydrogenase from cabbage is the first His biosynthesis enzyme that has been purified to homogeneity from higher plants. By using a new affinity chromatography, the purification of this enzyme and its investigation became possible. Direct evidence that the

Effect

Addition None Mn2+ Ba2+ Mg2+ Ni2+ Ca” Zn2+ cl?+

of Divalent Metal Dehydrogenase

IV Ions on Histidinol Activity Relative

activity

(%)

100 126 65 54 46 36 34 24

Note. The concentration of metal ion was 0.5 mM. The activity determined after 3 min reaction time, while the velocity was still stant.

was con-

132

NAGAI

AND

pH 9.2-9.4 is identical to that of histidinol dehydrogenases from other organisms. No other divalent metal ion than Mn2+ showed a stimulation of the enzyme activity. This is in line with the results obtained with the enzyme from S. typhimurium, whose catalyzed reaction is also stimulated in the presence of Mn2+ (25). The molecular weight of 103,000 of native cabbage enzyme is considerably higher than that of the enzyme from S. typhimurium [82,000 (26) and 90,000 (27)]. The enzymes from cabbage and S. typhimurium are each composed of two subunits with a similar molecular weight of 52,000 and 43,000 (26), respectively. However, the subunit molecular weight of the trifunctional enzyme from S. cereuisiae was calculated from the nucleotide sequence to be 87,935 (28) and estimated on SDS gels as about 95,000 (12). According to these data, the enzymes from yeast, S. typhimurium and cabbage differ significantly in their molecular weights. The occurrence of multiple forms of plant enzymes distinguishable by charge, as observed with the cabbage enzyme after isoelectric focusing, is not unknown. The enzyme ferredoxin-NADP oxidoreductase from spinach separated into five to six bands on the isoelectric focusing gel (29). The nature of differently migrating forms of glutathione reductase from leaves of pea on isoelectric focusing was attributed to its cytosolic, chloroplastic, and mitochondrial isoforms (30). Histidinol dehydrogenase from cabbage eluted as a single peak from gel filtration and reverse phase HPLC (data not shown), and appeared as a single band on SDS gel. Even though multiple bands on the isoelectric focusing gel were found after each purification, some of them with an even different procedure, proteolytic modification of the enzyme can not be ruled out. Modifications at the N-terminus of the protein could in part also explain why 85% of the N-terminus was blocked for amino acid sequencing. The occurrence of single amino acid substitutions at the genomic level is certainly a further possibility for the occurrence of multiple isoforms. To elucidate the nature of the isoforms of histidinol dehydrogenase, studies of the subcellular distribution of the enzyme and of the gene(s) coding for the enzyme(s) are necessary. ACKNOWLEDGMENTS We are indebted to Jui-Yoa Chang for his support in the determination of N-terminal amino acids. The authors thank Ryo Sato and Kenji Soda for reviewing the manuscript.

SCHEIDEGGER

REFERENCES 1. Dougall, 945. 2. Negrutiu, Gen.Genet.

D. K., and F&on, I., De Brouwer,

M. M. (1967) D., Dirks,

Plant

Physiol.

R., and Jacobs,

42,

941-

M. (1985)

Mol.

199,330-337.

3. Heim, D. R., and Larrinua, I. M. (1989) Plant Physiol. 1231. 4. Yadava, P. K. (1979) Mol. Gen. Genet. 170,109-111. K., and Klopotowski, 5. Wiater, A., Krajewska-Grynkiewicz, Actu Biochim. Polonica 18, 299-307.

91, 1226-

T. (1971)

6. Wong, Y.-S., and Mazelis, M. (1981) Phytochem. 20, 1831-1834. 7. Yourno, J., and Ino, I. (1968) J. Biol. Chem. 243, 327333276. 8. Gorisch, H., and Holke, W. (1985) Eur. J. &o&em. 150,305-308. 9. Grubmeyer, C. T., Insinga, S., Bhatia, M., and Moazami, N. (1989) Biochemistry 28, 8174-8180. 10. Bitar, K. G., Firca, J. R., and Loper, J. C. (1977) Biochim. Biophys. Acta 493,429-440. 11. Shaffer, B., Edelstein, S., and Fink, G. R. (1972) Brookhaven Symp. Biol. 23, 250-270. 12. Keesey, Jr., J. K., Bigelis, R., and Fink, G. R. (1979) J. Biol. Chem. 254, 7427-7433. 13. Catcheside, 651.

D. G. (1965)

Biochem.

Biophys.

Res. Commun.

l&648-

14. Strauss, A., Fankhauser, H., and King, P. J. (1985) Plantu 163, 554-562. 15. Kumpaisal, R., Hashimoto, T., and Yamada, Y. (1987) Plant Physiol. 85,145-151. 16. Yamada, Y., and Sato, F. (1978) Plant 6 Cell Physiol. 19,691-699. 17. Laemmli, 18. Matsumoto, 1091-1098.

U. K. (1970) I., Mizuno,

19. Kanamori, A., Seno, 363, 231-242. 20. Skyring, Biochem. 21. Bradford,

Nature (London) Y., and Seno,

227, 680-685. N. (1979) J. Biochem.

N., and Matsumoto,

G. W., Miller, 36,511-520. M. M. (1976)

I. (1986)

J. Chromutogr.

R. W., and Purkayastha, Anal.

Biochem.

85,

V. (1970)

Anal.

72,248-254.

22. Strickler, J. E., Hunkapiller, M. W., and Wilson, K. J. (1984) Anal. Biochem. 140,553-566. 23. Adams, E. (1955) J. Biol. Chem. 217, 325-344. H. (1981) Eur. J. Biochem. 116, 1377142. 24. Burger, E., and Giirisch, 25. Grubmeyer, C., Skiadopoulos, M., and Senior, A. E. (1989) Arch. Biochem. Biophys. 272,311-317. 26. Burger, E., Gorisch, H., and Lingens, F. (1979) Biochem. J. 181, 771-774. 27. Eccleston, E. D., Thayer, Chem. 254, 11,399-11,404. T. F., Farabaugh, 28. Donahue,

M. L., and Kirkwood, P. cJ., and Fink,

S. (1979)

G. R. (1982)

J. Biol. Gene

18,

47-59. 29. Ellefson, W. L., and Krogmann, D. W. (1979) phys. 194,593-599. 30. Edwards, E. A., Rawsthorne, S., and Mullineaux,

180,278-284.

Arch.

Biochem.

P. M. (1990)

BioPlanta