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Chloroplasts are the Subcellular Location of Both Soluble and Membrane-associated Homoserine Kinases in Pea (Pisum sativum L.) Leaves
Chloroplasts are the Subcellular Location of Both Soluble and Membrane-associated Homoserine Kinases in Pea (Pisum sativum 1.) Leaves MICHAEL}. MUHITCH*) and KENNETH G. WILSON Botany Department, Miami University, Oxford, OH 45056, USA Received October 5,1982 . Accepted January 11,1983
Summary Pea leaf homoserine kinase was associated with intact chloroplasts, but not with naked thylakoids (broken chloroplasts), on sucrose density gradients, however, when isolated intact chloroplasts were lysed in the presence of a protective media, both soluble and thylakoid-associated activities were found. The thylakoid-associated homoserine kinase activity exhibited apparent Km values of 1.2 and 4.7 mM for homoserine and ATP, respectively. The inhibition of the thylakoid-associated homoserine kinase activity by added amino acids was similar to that of the soluble pea leaf homoserine kinase (Thoen et al., Plant Sci. Let. 13, 103-112, 1978), with the exception of S-adenosylmethionine, which when present at 7.5 mM, inhibits the soluble enzyme by 92 %, but only inhibited the thylakoid-associated enzyme by 57 %. The possible relationship between the soluble and thylakoid forms of the enzyme is discussed.
Key words: Pisum sativum L. var Alaska, chloroplast, homoserine kinase.
Introduction Higher plants, bacteria and fungi synthesize Met and Thr from the common intermediate Hse. In bacteria and fungi, Hse (P) serves as the activated intermediate in Thr synthesis while O-succinylHse or O-acetylHse perform the analogous function in cystathionine, and subsequently Met, synthesis (Umbarger, 1978). In contrast, higher plants appear to use the same activated intermediate for both Thr and Met biosynthesis, namely Hse (P) (Bryan, 1980). Hse kinase (E.C. 2.7.1.39), which catalyzes the formation of Hse (P) from Hse and the terminal phosphate of ATP, has been detected in all plants so far examined (Giovanelli et al., 1974). The distribution of Hse kinase between the supernatant and pellet fractions of crude extracts varies from plant to plant, and in several species, the enzyme is labile, requiring a «protective» isolation media in order to preserve the activity during extraction (Giovanelli et al., 1974). These findings have lead the authors to suggest that plant Hse kinase may exist *) Present address: Botany and Microbiology Department, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73019, USA. Abbreviations: AdoMet, S-adenosylmethionine; Hse, homoserine; Hse (P), homoserine phosphate; Ile, isoleucine; Met, methionine, Om, ornithine; Val, valine.
z. Pjlanzenphysiol. Bd. 110. S.39-46. 1983.
40
MICHAEL J. MUHITCH and KENNETH G. WILSON
in multiple forms with differing degrees of lability and that these various forms may be differentially localized within the cell. Hse kinases have subsequently been partially purified from etiolated barley shoots (Aarnes, 1976, 1978) and from green pea leaves (Thoen et al., 1978 a). In the case of the pea enzyme only 5 % of the activity was associated with the pellet of the crude extract. During subsequent purification, the soluble enzyme eluted as two peaks during gel filtration (MW = 120,000 and 240,000 daltons), however the regulatory properties of the two peaks are similar. In addition, the pea leaf Hse kinase exhibited a relatively low affinity for Hse (Km = 6.7 mM), when compared with the barley or microbial Hse kinases (Km = 1 mM or less, see Thoen et al., 1978 a). In this paper, we report the finding of a membrane-associated Hse kinase in green pea leaves which has a Km for Hse of 1.2 mM. In addition, we present evidence that both the soluble and membrane forms of the enzyme are associated with the chloroplast.
Materials and Methods Plant Material Seeds of Pisum sativum L. var. Alaska were obtained from W. Altee Burpee Co., Warminster, PA, USA. Plants were grown in vermiculite in a controlled environment room at 21°C under 10,000 lux of continuous illumination. Chemicals Adomet (Grade II) was purchased from the Sigma Chemical Co., St. Louis, MO, USA. U-I4C-Hse was obtained from Amersham, Arlington Heights, IL, USA. Crude Enzyme Extracts
Whole leaf extracts were prepared by grinding 5 g of leaf material in 10 ml of «media A» (Giovanelli et al., 1974) at 4°C, using a mortar and pestle. The grindate was filtered through 4 layers of cheesecloth and centrifuged for 15 min at 38,000 xg. The supernatant served as the soluble enzyme source, while the pellet was resuspended in a small volume of the same media and served as the source of the bound (pellet) enzyme.
Thylakoid Preparations Chloroplasts were obtained from apical pea leaves as described previously (Mills and Wilson, 1978). The resultant chloroplast pellets were washed in a non-ionic buffer (Nakatani and Barber, 1977), repelleted, and resuspended in media A (Giovanelli et al., 1974), then mechanically lysed in a hand-held glass homogenizer. Thylakoids were collected by centrifugation at 2000 xg for 1 min and the resultant pellet was resuspended in media A. Prior to lysing, chloroplast preparations were routinely found to be 85 % or more intact as measured by the ferricyanide method ofMcC Lilley et al. (1975). Solubilization Studies Thylakoids were prepared as described above and collected by centrifugation at 2000 xg for 1 min. The pellets were resuspended in media A which contained the various test components (see text) and the thylakoids were removed by centrifugation at 30,000 xg for 20 min. The resulting supernatants were assayed for Hse kinase activity.
Z. Pjlanzenphysiol. Bd. 110. S. 39-46. 1983.
Homoserine kinase in peas
41
Cell Fractionation Studies Cell fractionation was performed essentially as described by Miflin and Beevers (1974). Thirty grams of apical leaf material were washed in ice cold sterile distilled water and placed in an Omni-mixer vessel along with 30 ml of isolation media, which consisted of 350 mM sorbitol, 50 mM 4-(2-hydroxyethyl)-l-piperazine-ethane sulfonic acid (PH 7.5), 2 mM EDTA, 2 mM MgCh, 0.1 % bovine serum albumin and 3 mM B-mercaptoethanol. The tissue was homogenized by applying three 2 s burst at 75 % line voltage. The homogenate was filtered through 2 layers of cheesecloth and 2 layers of Miracloth (Calbiochem-Behring Corp., LaJolla, CA, USA) and 10 ml of the filtrate was layered on the top of a sucrose gradient. The gradients were composed of a 4ml pad of 60% sucrose (w/w), a 6mllinear segment from 60-42% sucrose, 5 ml of 42 % sucrose, a 10 mllinear segment from 42-30 % sucrose and 3 ml of 30 % sucrose. All of the sucrose solutions contained 50 mM 3-(N-morpholino) propanesulfonic acid (pH 7.5). The tubes were then placed in a Sorvall HB-4 swinging bucket rotor (DuPont Instruments, Newtown, CN, USA) and centrifuged for 5 min at 2880 xg, followed by centrifugation for 10 min at 14,000 xg all in a Sorvall RC-5 high speed centrifuge. Gradients were fractionated from the top using a Densi-Flow lIC fractionator (Buchler Instruments, Fort Lee, NH, USA) in combination with a Gilson Mini-escargot fraction collector (Gilson Medical Electronics, Inc., Middleton, WI, USA). Fraction volumes were 1.67 ml each. The sucrose concentrations of the fractions were monitored by refractometry, while marker enzyme assays were performed as previously described (Miflin and Beevers, 1974). The results which are presented are typical of data obtained on four separate occasions.
Hse Kinase Assay Hse kinase activity was measured by following the procedure of Giovanelli et al. (1974), except that Hse was present in the assay mixture at a final concentration of 4 mM with a specific activity of 9.25 MBq mmol- 1•
Chlorophyll Determination Chlorophyll content was determined by the method of Arnon (1949).
Results
A crude homogenate of ten day old light grown pea shoots extracted in media A (Materials and Methods) exhibited a Hse kinase activity of 403 nmol Hse (P) formed h -I g-I fresh weight. After centrifugation, 19 % of the activity was associated with the pellet fraction (Table 1). In order to determine the cellular locations of the soluble and pellet-associated Hse kinase activities from pea leaves, the various subcellular fractions were separated from one another on sucrose density gradients (Fig. 1). Hse kinase activity was found to cosediment with those fractions containing intact chloroplasts, but not with the fractions containing mitochondria, peroxisomes or broken chloroplasts. Hse kinase activity in the initial homogenate, which was assayed at the same time as the gradient fractions and therefore sat on ice for 30-40 min was only 24% of the total enzyme activity recovered from the gradient (Table 1). The loss of enzyme activity in the homogenate is, at least in part, due to the instability of the enzyme in the gradient isolation media, since whole cell Hse kinase prepared in media A was ca. 3 times more active. However, even when isolated in a «protective» extraction buffer such as media A, not all of the enzyme activity is preserved since, on Z. Pjlanzenphysiol. Bd. 110. S. 39-46. 1983.
42
MICHAEL]. MUHITCH and KENNETH G. WILSON
Table 1: Comparison of Pea Leaf Homoserine Kinase Activities After Extraction by Various Methods. [nmol HS(P) h-1/tg-l] Chlorophyll
% Total
Whole leaf extract Homogenate Supernatant Pellet
2.0 1.62 0.38
100 81 19
Gradient Intact bands Broken bands Total gradient Homogenate
6.04 0.79 2.55 0.69
Source
Intact chloroplasts"d) Total Supernatant Pellet Intact chloroplastsb•d) SupernatantC) Pellet
Activity
Total Activity [nmol HS(P) h- 1]
1051 79 3404 816
4.53 2.35 1.89
100 52 42
2.06 2.30
47 53
.) Intact chloroplasts removed from sucrose density gradient. b) Intact chloroplasts isolated by differential centrifugation. " Corrected for 100% intactness. Chloroplasts were 86 % intact. ) Activities are based on the total chlorophyll content of the chloroplast preparations prior to separation into supernatant (stroma) and pellet (thylakoid) fractions in order that the relative activities exhibited by these two fractions directly reflects the distribution of HS kinase among them.
a chlorophyll basis, whole leaf preparations contained only 33 % of the activity found in the gradient isolated intact chloroplasts. As noted above, Hse kinase was associated with intact, but not with broken chloroplasts on sucrose density gradients. This suggests that Hse kinase is a soluble enzyme. However, when intact chloroplast containing bands were removed from the gradients and separated into thylakoid and stromal fractions, the enzyme activity was found to be ca. equally distributed between the two fractions (Table 1). Isolation of chloroplasts by the differential centrifugation method of Nakatani and Barber (1977), which results in a high percentage of intactness and yet yields a sufficient amount of chlorophyll to quantify the chloroplast intactness by the ferricyanide method (McC Lilley et aI., 1975), gave similar results. By comparing the supernatant and the pellet Hse kinase activities from whole cells and isolated chloroplasts on a chlorophyll basis, it appears that, if Hse kinase is totally chloroplast associated, then the majority of the activity which is lost is from the pellet (thylakoids). This would be consistent with the fact that while thylakoid fractions from intact chloroplasts exhibited Hse kinase activity, the gradient isolated broken chloroplasts did not. Z. Pjlanzenphysiol. Bd. 110. S. 39-46. 1983.
Homoserine Kinase in Peas
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Fig. 1: Distribution of chlorophyll, marker enzymes and homoserine kinase activit! in a sucrose density gradient. The activity of homoserine kinase is expressed as nmol h-lml- ; cyto· chrome oxidase, as nmol min-Iml- I; catalase and triose phosphate isomerase as /Lmol min -lml- I. Chlorophyll is expressed in /Lg ml- I.
Since the above results suggest that the thylakoid-associated Hse kinase is easily inactivated during extraction, we therefore chose to isolate the thylakoid-associated Hse kinase activity by first isolating intact chloroplasts, followed by mechanically lysing the chloroplasts into media A. The solubilization, kinetic and regulatory characteristics of this activity were then examined. The thylakoid-associated Hse kinase activity was found to be dependent on ATP and Mg, but not on K, and the radiolabeled product, which co migrated with authenic Hse (P) during paper electrophoresis, was destroyed in the presence of alkaline phosphatase. The pH optima for the thylakoid-associated activity was not sharp, with 80 % or more of the maximal activity observed at pH values between 7.2 and 8.8. Various methods for solubilizing the thylakoid-bound activity were tested (Table 2). Of Z. Pjlanzenphysiol. Bd. 110. S. 39-46.1983.
44
MICHAEL}. MUHITCH and KENNETH G. WILSON
Table 2: Solubilization of Homoserine Kinase Activity from the Thylakoids of Pea Chloroplasts. Treatment
% Control
Centrifugation omitted (Control) None Second centrifugation 5mMMgCb 200mMMgCb 10mMEDTA 40% Sucrose lMKCI 0.5 % Triton-X-l00 5 mM MgCb, 5 mM A TP, 1 mM homoserine 1 mM S-adenosylmethionine 10 mM Dithiothreitol Darkb) Lysing media pH 6.8
100a) 13 17 21 25 20 13 61 91
16 10 12 14 10
") Control activity = 1.12 nmol HS(P) h-'"g-' chlorophyll. b) Thylakoids were prepared in the dark from peas which had been in the dark for 16 hr. Table 3: Effects of Amino Acids on Thylakoid-Associated Homoserine Kinase. Amino Acidb)
% Control")
Isoleucine 13 Valine 18 Ornithine 38 S-adenosylmethionine (7.5 mM) 43 Methionine 76 Threonine 82 .) Control activity = 1.20 nmol homoserine phosphate formed h -I"g-I chlorophyll. b) All amino acids were 10 mM, unless otherwise indicated.
these, only KCI and Triton-X-I00 were effective in releasing significant portions of the Hse kinase activity from the thylakoid membranes. Thylakoid-associated Hse kinase appeared to follow simple Michelis-Menton kinetics, with apparent Km values for ATP : Mg and Hse of 4.7 and 1.2 mM, respectively. The effects of several added amino acids on the thylakoid-associated Hse kinase activity are shown in Table 3. Of the amino acids tested, Ile, Val, Om and AdoMet strongly inhibited the enzyme activity. The effects of a series of AdoMet concentrations on the thylakoid-associated Hse kinase activity are shown in Fig. 2. The AdoMet concentration resulting in one-half maximum inhibition was 0.6 mM. Discussion
A chloroplast location of pea leaf Hse kinase, as determined in the present study, is consistent with the demonstrated ability of isolated pea chloroplasts to synthesize Z. Pjlanzenphysiol. Bd. 110. S. 39--46.1983.
Homoserine Kinase in Peas
45
100
80
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60
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Fig. 2: Effects of a series of S-adenosylmethionine concentrations on pea thylakoid-associated homoserine kinase. Control activity = 1.14 nmol HS(P) h- 1 /-Ig-l chlorophyll.
Thr and He from radiolabeled precursors in vitro (Mills et al., 1980). Although Hse kinase did not appear to be associated with mitochondria or peroxisomes, the possibility of these or other subcellular locations of the enzyme can not be completely rejected due to the labile nature of the enzyme. Hse kinases from barley, parsely and corn are similarly labile (Giovanelli et al., 1974). The inhibition of the thylakoid-associated Hse kinase by added amino acids is similar to that of the soluble pea enzyme (Thoen et al., 1978 a) with the exception of AdoMet, which when present at 7.5 mM inhibited the thylakoid enzyme by only 57 % while inhibiting the soluble enzyme by 92 %. This partial inhibition of the thylakoid-associated Hse kinase by AdoMet suggests that the thylakoid-associated activity may consist of separate AdoMet sensitive and insensitive isozymes. Other interpretations of the results are also possible, however. For instance, thylakoid-associated Hse kinase could be completely insensitive to AdoMet, however, if AdoMet activated a thylakoid-associated Thr synthase (Thoen et al., 1978 b), part of the Hse (P) produced would be further metabolized to Thr under our assay conditions. Whether the thylakoid-associated Hse kinase is the same enzyme as the soluble form reported by Thoen et al. (1978 a), or a separate isozyme is not clear. The similar pH curves, the similar pattern of inhibition by added amino acids (except for AdoMet) and the similar binding characteristics to ion exchange resins (data not shown) of the bound and soluble enzyme activities suggest that they are the same enzyme. On the other hand, the affinity of the thylakoid-associated Hse kinase for Hse is ca. 5 fold higher than that of the soluble enzyme. It is possible that tissue Z. Pjlanzenphysiol. Ed. 110. S.39-46. 1983.
46
MICHAEL J. MUHITCH and KENNETH G. WILSON
homogenation dislodges some of the bound enzyme from the thylakoids, which results in an alteration of the kinetics and regulatory properties of the solubilized enzyme. Anyway, we suggest that this membrane-associated Hse kinase most likely participates in the de novo synthesis of Thr and cystathionine in the intact leaf, since (1) the membrane-associated enzyme is located within the chloroplast, where the aspartate pathway is known to be operative (Mills et aI., 1980) and (2) the Km of the membrane-associated enzyme is sufficiently low so that, in contrast to the soluble pea Hse kinase of Thoen et aI. (1978 a), it would be able to efficiently catalyze the formation of Hse (P) from the 1-5 mM concentrations of Hse which exist in pea chloroplasts (Mills and Joy, 1980). Acknowledgements This study was supported in part by a Grant-in-Aid from Sigma Xi to M. J. M. A portion of this work was included in the Doctoral Dissertation of M. J. M.
References AARNES, H.: Homoserine kinase from barley seedlings. Plant Sci. Let. 7, 187-194 (1976). - Regulation of threonine biosynthesis in barley seedlings (Hordeum vulgare L.). Planta 140, 185-192 (1978). ARNON, D. I.: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant physiol. 24, 1-15 (1949). BRYAN, J. K.: Synthesis of the Aspartate Family and Branched-chain Amino Acids. In: MiFLIN, B. J. (ed.): The biochemistry of plants. A comprehensive treatise 5, 403-452. Academic Press, New York, 1980. GIOVANELLI, J., S. H. MUDD, and A. H. DATKO: Homoserine esterification in green plants. Plant Physiol. 54, 725-736 (1974). McC LILLEY, R., M. P. FITZGERALD, K. G. REINITS, and D. A. WALKER: Criteria of intactness and the photosynthetic activity of spinach chloroplast preparations. New Phytol. 75, 1-10 (1975). MIFLIN, B. J. and H. BEEVERS: Isolation of intact plastids from a range of plant tissues. Plant Physiol. 58, 870-874 (1974). MILLS, W. R. and K. W. JOY: A rapid method for isolation of purified, physiologically active chloroplasts, used to study the intracellular distribution of amino acids in pea leaves. Planta 148,75-83 (1980). MILLS, W. R., P. J. LEA, and B. J. MIFLIN: Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts. Plant Physiol. 65, 1166-1172 (1980). MiLLS, W. R. and K. G. WILSON: Effects of lysine, threonine and methionine on light-driven protein synthesis in isolated pea (Pisum sativum L.) chloroplasts. Plant a 142, 153-160 (1978). NAKATANI, H. Y. and J. BARBER: An improved method for isolating chloroplasts retaining their outer membranes. Biochim. Biophys. Acta 461,510-512 (1977). THOEN, A., S. E. ROGNES, and H. AARNES: Biosynthesis of threonine from homoserine in pea seedlings: I. Homoserine kinase. Plant Sci. Let. 13, 103-112 (1978 a). - - - Biosynthesis of threonine from homoserine in pea seedlings: II. Threonine synthase. Plant Sci. Let. 13, 113-119 (1978 b). UMBARGER, H. E.: Amino acid biosynthesis and its regulation. Ann. Rev. Biochem. 47, 533-606 (1978).
Z. Pjlanzenphysiol. Ed. 110. S.39--46. 1983.
Summary Pea leaf homoserine kinase was associated with intact chloroplasts, but not with naked thylakoids (broken chloroplasts), on sucrose density gradients, however, when isolated intact chloroplasts were lysed in the presence of a protective media, both soluble and thylakoid-associated activities were found. The thylakoid-associated homoserine kinase activity exhibited apparent Km values of 1.2 and 4.7 mM for homoserine and ATP, respectively. The inhibition of the thylakoid-associated homoserine kinase activity by added amino acids was similar to that of the soluble pea leaf homoserine kinase (Thoen et al., Plant Sci. Let. 13, 103-112, 1978), with the exception of S-adenosylmethionine, which when present at 7.5 mM, inhibits the soluble enzyme by 92 %, but only inhibited the thylakoid-associated enzyme by 57 %. The possible relationship between the soluble and thylakoid forms of the enzyme is discussed.
Key words: Pisum sativum L. var Alaska, chloroplast, homoserine kinase.
Introduction Higher plants, bacteria and fungi synthesize Met and Thr from the common intermediate Hse. In bacteria and fungi, Hse (P) serves as the activated intermediate in Thr synthesis while O-succinylHse or O-acetylHse perform the analogous function in cystathionine, and subsequently Met, synthesis (Umbarger, 1978). In contrast, higher plants appear to use the same activated intermediate for both Thr and Met biosynthesis, namely Hse (P) (Bryan, 1980). Hse kinase (E.C. 2.7.1.39), which catalyzes the formation of Hse (P) from Hse and the terminal phosphate of ATP, has been detected in all plants so far examined (Giovanelli et al., 1974). The distribution of Hse kinase between the supernatant and pellet fractions of crude extracts varies from plant to plant, and in several species, the enzyme is labile, requiring a «protective» isolation media in order to preserve the activity during extraction (Giovanelli et al., 1974). These findings have lead the authors to suggest that plant Hse kinase may exist *) Present address: Botany and Microbiology Department, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73019, USA. Abbreviations: AdoMet, S-adenosylmethionine; Hse, homoserine; Hse (P), homoserine phosphate; Ile, isoleucine; Met, methionine, Om, ornithine; Val, valine.
z. Pjlanzenphysiol. Bd. 110. S.39-46. 1983.
40
MICHAEL J. MUHITCH and KENNETH G. WILSON
in multiple forms with differing degrees of lability and that these various forms may be differentially localized within the cell. Hse kinases have subsequently been partially purified from etiolated barley shoots (Aarnes, 1976, 1978) and from green pea leaves (Thoen et al., 1978 a). In the case of the pea enzyme only 5 % of the activity was associated with the pellet of the crude extract. During subsequent purification, the soluble enzyme eluted as two peaks during gel filtration (MW = 120,000 and 240,000 daltons), however the regulatory properties of the two peaks are similar. In addition, the pea leaf Hse kinase exhibited a relatively low affinity for Hse (Km = 6.7 mM), when compared with the barley or microbial Hse kinases (Km = 1 mM or less, see Thoen et al., 1978 a). In this paper, we report the finding of a membrane-associated Hse kinase in green pea leaves which has a Km for Hse of 1.2 mM. In addition, we present evidence that both the soluble and membrane forms of the enzyme are associated with the chloroplast.
Materials and Methods Plant Material Seeds of Pisum sativum L. var. Alaska were obtained from W. Altee Burpee Co., Warminster, PA, USA. Plants were grown in vermiculite in a controlled environment room at 21°C under 10,000 lux of continuous illumination. Chemicals Adomet (Grade II) was purchased from the Sigma Chemical Co., St. Louis, MO, USA. U-I4C-Hse was obtained from Amersham, Arlington Heights, IL, USA. Crude Enzyme Extracts
Whole leaf extracts were prepared by grinding 5 g of leaf material in 10 ml of «media A» (Giovanelli et al., 1974) at 4°C, using a mortar and pestle. The grindate was filtered through 4 layers of cheesecloth and centrifuged for 15 min at 38,000 xg. The supernatant served as the soluble enzyme source, while the pellet was resuspended in a small volume of the same media and served as the source of the bound (pellet) enzyme.
Thylakoid Preparations Chloroplasts were obtained from apical pea leaves as described previously (Mills and Wilson, 1978). The resultant chloroplast pellets were washed in a non-ionic buffer (Nakatani and Barber, 1977), repelleted, and resuspended in media A (Giovanelli et al., 1974), then mechanically lysed in a hand-held glass homogenizer. Thylakoids were collected by centrifugation at 2000 xg for 1 min and the resultant pellet was resuspended in media A. Prior to lysing, chloroplast preparations were routinely found to be 85 % or more intact as measured by the ferricyanide method ofMcC Lilley et al. (1975). Solubilization Studies Thylakoids were prepared as described above and collected by centrifugation at 2000 xg for 1 min. The pellets were resuspended in media A which contained the various test components (see text) and the thylakoids were removed by centrifugation at 30,000 xg for 20 min. The resulting supernatants were assayed for Hse kinase activity.
Z. Pjlanzenphysiol. Bd. 110. S. 39-46. 1983.
Homoserine kinase in peas
41
Cell Fractionation Studies Cell fractionation was performed essentially as described by Miflin and Beevers (1974). Thirty grams of apical leaf material were washed in ice cold sterile distilled water and placed in an Omni-mixer vessel along with 30 ml of isolation media, which consisted of 350 mM sorbitol, 50 mM 4-(2-hydroxyethyl)-l-piperazine-ethane sulfonic acid (PH 7.5), 2 mM EDTA, 2 mM MgCh, 0.1 % bovine serum albumin and 3 mM B-mercaptoethanol. The tissue was homogenized by applying three 2 s burst at 75 % line voltage. The homogenate was filtered through 2 layers of cheesecloth and 2 layers of Miracloth (Calbiochem-Behring Corp., LaJolla, CA, USA) and 10 ml of the filtrate was layered on the top of a sucrose gradient. The gradients were composed of a 4ml pad of 60% sucrose (w/w), a 6mllinear segment from 60-42% sucrose, 5 ml of 42 % sucrose, a 10 mllinear segment from 42-30 % sucrose and 3 ml of 30 % sucrose. All of the sucrose solutions contained 50 mM 3-(N-morpholino) propanesulfonic acid (pH 7.5). The tubes were then placed in a Sorvall HB-4 swinging bucket rotor (DuPont Instruments, Newtown, CN, USA) and centrifuged for 5 min at 2880 xg, followed by centrifugation for 10 min at 14,000 xg all in a Sorvall RC-5 high speed centrifuge. Gradients were fractionated from the top using a Densi-Flow lIC fractionator (Buchler Instruments, Fort Lee, NH, USA) in combination with a Gilson Mini-escargot fraction collector (Gilson Medical Electronics, Inc., Middleton, WI, USA). Fraction volumes were 1.67 ml each. The sucrose concentrations of the fractions were monitored by refractometry, while marker enzyme assays were performed as previously described (Miflin and Beevers, 1974). The results which are presented are typical of data obtained on four separate occasions.
Hse Kinase Assay Hse kinase activity was measured by following the procedure of Giovanelli et al. (1974), except that Hse was present in the assay mixture at a final concentration of 4 mM with a specific activity of 9.25 MBq mmol- 1•
Chlorophyll Determination Chlorophyll content was determined by the method of Arnon (1949).
Results
A crude homogenate of ten day old light grown pea shoots extracted in media A (Materials and Methods) exhibited a Hse kinase activity of 403 nmol Hse (P) formed h -I g-I fresh weight. After centrifugation, 19 % of the activity was associated with the pellet fraction (Table 1). In order to determine the cellular locations of the soluble and pellet-associated Hse kinase activities from pea leaves, the various subcellular fractions were separated from one another on sucrose density gradients (Fig. 1). Hse kinase activity was found to cosediment with those fractions containing intact chloroplasts, but not with the fractions containing mitochondria, peroxisomes or broken chloroplasts. Hse kinase activity in the initial homogenate, which was assayed at the same time as the gradient fractions and therefore sat on ice for 30-40 min was only 24% of the total enzyme activity recovered from the gradient (Table 1). The loss of enzyme activity in the homogenate is, at least in part, due to the instability of the enzyme in the gradient isolation media, since whole cell Hse kinase prepared in media A was ca. 3 times more active. However, even when isolated in a «protective» extraction buffer such as media A, not all of the enzyme activity is preserved since, on Z. Pjlanzenphysiol. Bd. 110. S. 39-46. 1983.
42
MICHAEL]. MUHITCH and KENNETH G. WILSON
Table 1: Comparison of Pea Leaf Homoserine Kinase Activities After Extraction by Various Methods. [nmol HS(P) h-1/tg-l] Chlorophyll
% Total
Whole leaf extract Homogenate Supernatant Pellet
2.0 1.62 0.38
100 81 19
Gradient Intact bands Broken bands Total gradient Homogenate
6.04 0.79 2.55 0.69
Source
Intact chloroplasts"d) Total Supernatant Pellet Intact chloroplastsb•d) SupernatantC) Pellet
Activity
Total Activity [nmol HS(P) h- 1]
1051 79 3404 816
4.53 2.35 1.89
100 52 42
2.06 2.30
47 53
.) Intact chloroplasts removed from sucrose density gradient. b) Intact chloroplasts isolated by differential centrifugation. " Corrected for 100% intactness. Chloroplasts were 86 % intact. ) Activities are based on the total chlorophyll content of the chloroplast preparations prior to separation into supernatant (stroma) and pellet (thylakoid) fractions in order that the relative activities exhibited by these two fractions directly reflects the distribution of HS kinase among them.
a chlorophyll basis, whole leaf preparations contained only 33 % of the activity found in the gradient isolated intact chloroplasts. As noted above, Hse kinase was associated with intact, but not with broken chloroplasts on sucrose density gradients. This suggests that Hse kinase is a soluble enzyme. However, when intact chloroplast containing bands were removed from the gradients and separated into thylakoid and stromal fractions, the enzyme activity was found to be ca. equally distributed between the two fractions (Table 1). Isolation of chloroplasts by the differential centrifugation method of Nakatani and Barber (1977), which results in a high percentage of intactness and yet yields a sufficient amount of chlorophyll to quantify the chloroplast intactness by the ferricyanide method (McC Lilley et aI., 1975), gave similar results. By comparing the supernatant and the pellet Hse kinase activities from whole cells and isolated chloroplasts on a chlorophyll basis, it appears that, if Hse kinase is totally chloroplast associated, then the majority of the activity which is lost is from the pellet (thylakoids). This would be consistent with the fact that while thylakoid fractions from intact chloroplasts exhibited Hse kinase activity, the gradient isolated broken chloroplasts did not. Z. Pjlanzenphysiol. Bd. 110. S. 39-46. 1983.
Homoserine Kinase in Peas
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Fig. 1: Distribution of chlorophyll, marker enzymes and homoserine kinase activit! in a sucrose density gradient. The activity of homoserine kinase is expressed as nmol h-lml- ; cyto· chrome oxidase, as nmol min-Iml- I; catalase and triose phosphate isomerase as /Lmol min -lml- I. Chlorophyll is expressed in /Lg ml- I.
Since the above results suggest that the thylakoid-associated Hse kinase is easily inactivated during extraction, we therefore chose to isolate the thylakoid-associated Hse kinase activity by first isolating intact chloroplasts, followed by mechanically lysing the chloroplasts into media A. The solubilization, kinetic and regulatory characteristics of this activity were then examined. The thylakoid-associated Hse kinase activity was found to be dependent on ATP and Mg, but not on K, and the radiolabeled product, which co migrated with authenic Hse (P) during paper electrophoresis, was destroyed in the presence of alkaline phosphatase. The pH optima for the thylakoid-associated activity was not sharp, with 80 % or more of the maximal activity observed at pH values between 7.2 and 8.8. Various methods for solubilizing the thylakoid-bound activity were tested (Table 2). Of Z. Pjlanzenphysiol. Bd. 110. S. 39-46.1983.
44
MICHAEL}. MUHITCH and KENNETH G. WILSON
Table 2: Solubilization of Homoserine Kinase Activity from the Thylakoids of Pea Chloroplasts. Treatment
% Control
Centrifugation omitted (Control) None Second centrifugation 5mMMgCb 200mMMgCb 10mMEDTA 40% Sucrose lMKCI 0.5 % Triton-X-l00 5 mM MgCb, 5 mM A TP, 1 mM homoserine 1 mM S-adenosylmethionine 10 mM Dithiothreitol Darkb) Lysing media pH 6.8
100a) 13 17 21 25 20 13 61 91
16 10 12 14 10
") Control activity = 1.12 nmol HS(P) h-'"g-' chlorophyll. b) Thylakoids were prepared in the dark from peas which had been in the dark for 16 hr. Table 3: Effects of Amino Acids on Thylakoid-Associated Homoserine Kinase. Amino Acidb)
% Control")
Isoleucine 13 Valine 18 Ornithine 38 S-adenosylmethionine (7.5 mM) 43 Methionine 76 Threonine 82 .) Control activity = 1.20 nmol homoserine phosphate formed h -I"g-I chlorophyll. b) All amino acids were 10 mM, unless otherwise indicated.
these, only KCI and Triton-X-I00 were effective in releasing significant portions of the Hse kinase activity from the thylakoid membranes. Thylakoid-associated Hse kinase appeared to follow simple Michelis-Menton kinetics, with apparent Km values for ATP : Mg and Hse of 4.7 and 1.2 mM, respectively. The effects of several added amino acids on the thylakoid-associated Hse kinase activity are shown in Table 3. Of the amino acids tested, Ile, Val, Om and AdoMet strongly inhibited the enzyme activity. The effects of a series of AdoMet concentrations on the thylakoid-associated Hse kinase activity are shown in Fig. 2. The AdoMet concentration resulting in one-half maximum inhibition was 0.6 mM. Discussion
A chloroplast location of pea leaf Hse kinase, as determined in the present study, is consistent with the demonstrated ability of isolated pea chloroplasts to synthesize Z. Pjlanzenphysiol. Bd. 110. S. 39--46.1983.
Homoserine Kinase in Peas
45
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Fig. 2: Effects of a series of S-adenosylmethionine concentrations on pea thylakoid-associated homoserine kinase. Control activity = 1.14 nmol HS(P) h- 1 /-Ig-l chlorophyll.
Thr and He from radiolabeled precursors in vitro (Mills et al., 1980). Although Hse kinase did not appear to be associated with mitochondria or peroxisomes, the possibility of these or other subcellular locations of the enzyme can not be completely rejected due to the labile nature of the enzyme. Hse kinases from barley, parsely and corn are similarly labile (Giovanelli et al., 1974). The inhibition of the thylakoid-associated Hse kinase by added amino acids is similar to that of the soluble pea enzyme (Thoen et al., 1978 a) with the exception of AdoMet, which when present at 7.5 mM inhibited the thylakoid enzyme by only 57 % while inhibiting the soluble enzyme by 92 %. This partial inhibition of the thylakoid-associated Hse kinase by AdoMet suggests that the thylakoid-associated activity may consist of separate AdoMet sensitive and insensitive isozymes. Other interpretations of the results are also possible, however. For instance, thylakoid-associated Hse kinase could be completely insensitive to AdoMet, however, if AdoMet activated a thylakoid-associated Thr synthase (Thoen et al., 1978 b), part of the Hse (P) produced would be further metabolized to Thr under our assay conditions. Whether the thylakoid-associated Hse kinase is the same enzyme as the soluble form reported by Thoen et al. (1978 a), or a separate isozyme is not clear. The similar pH curves, the similar pattern of inhibition by added amino acids (except for AdoMet) and the similar binding characteristics to ion exchange resins (data not shown) of the bound and soluble enzyme activities suggest that they are the same enzyme. On the other hand, the affinity of the thylakoid-associated Hse kinase for Hse is ca. 5 fold higher than that of the soluble enzyme. It is possible that tissue Z. Pjlanzenphysiol. Ed. 110. S.39-46. 1983.
46
MICHAEL J. MUHITCH and KENNETH G. WILSON
homogenation dislodges some of the bound enzyme from the thylakoids, which results in an alteration of the kinetics and regulatory properties of the solubilized enzyme. Anyway, we suggest that this membrane-associated Hse kinase most likely participates in the de novo synthesis of Thr and cystathionine in the intact leaf, since (1) the membrane-associated enzyme is located within the chloroplast, where the aspartate pathway is known to be operative (Mills et aI., 1980) and (2) the Km of the membrane-associated enzyme is sufficiently low so that, in contrast to the soluble pea Hse kinase of Thoen et aI. (1978 a), it would be able to efficiently catalyze the formation of Hse (P) from the 1-5 mM concentrations of Hse which exist in pea chloroplasts (Mills and Joy, 1980). Acknowledgements This study was supported in part by a Grant-in-Aid from Sigma Xi to M. J. M. A portion of this work was included in the Doctoral Dissertation of M. J. M.
References AARNES, H.: Homoserine kinase from barley seedlings. Plant Sci. Let. 7, 187-194 (1976). - Regulation of threonine biosynthesis in barley seedlings (Hordeum vulgare L.). Planta 140, 185-192 (1978). ARNON, D. I.: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant physiol. 24, 1-15 (1949). BRYAN, J. K.: Synthesis of the Aspartate Family and Branched-chain Amino Acids. In: MiFLIN, B. J. (ed.): The biochemistry of plants. A comprehensive treatise 5, 403-452. Academic Press, New York, 1980. GIOVANELLI, J., S. H. MUDD, and A. H. DATKO: Homoserine esterification in green plants. Plant Physiol. 54, 725-736 (1974). McC LILLEY, R., M. P. FITZGERALD, K. G. REINITS, and D. A. WALKER: Criteria of intactness and the photosynthetic activity of spinach chloroplast preparations. New Phytol. 75, 1-10 (1975). MIFLIN, B. J. and H. BEEVERS: Isolation of intact plastids from a range of plant tissues. Plant Physiol. 58, 870-874 (1974). MILLS, W. R. and K. W. JOY: A rapid method for isolation of purified, physiologically active chloroplasts, used to study the intracellular distribution of amino acids in pea leaves. Planta 148,75-83 (1980). MILLS, W. R., P. J. LEA, and B. J. MIFLIN: Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts. Plant Physiol. 65, 1166-1172 (1980). MiLLS, W. R. and K. G. WILSON: Effects of lysine, threonine and methionine on light-driven protein synthesis in isolated pea (Pisum sativum L.) chloroplasts. Plant a 142, 153-160 (1978). NAKATANI, H. Y. and J. BARBER: An improved method for isolating chloroplasts retaining their outer membranes. Biochim. Biophys. Acta 461,510-512 (1977). THOEN, A., S. E. ROGNES, and H. AARNES: Biosynthesis of threonine from homoserine in pea seedlings: I. Homoserine kinase. Plant Sci. Let. 13, 103-112 (1978 a). - - - Biosynthesis of threonine from homoserine in pea seedlings: II. Threonine synthase. Plant Sci. Let. 13, 113-119 (1978 b). UMBARGER, H. E.: Amino acid biosynthesis and its regulation. Ann. Rev. Biochem. 47, 533-606 (1978).
Z. Pjlanzenphysiol. Ed. 110. S.39--46. 1983.