Subcellular Coenzyme JOHN Department
Localization of 3-Hydroxy-3-Methylglutaryl A Reductase in Pisum sativum Seedlings’ D. BROOKER2 of Biochemistry,
W. RUSSELL Dunedin,
3-Hydroxy-3-methylglutaryl coenzyme A reductase from seedlings of Pisum satiuum L. is localized in the plastids, mitochondria, and microsomes. Separation of the microsomal fraction into heavy and light subfractions shows that 95% of the microsomal activity is associated with the light subfraction. Definitive localization was achieved by showing that reductase activity comigrated with organelle markers on sucrose density gradients. Differential centrifugation studies showed that the microsomal fraction contained 80% of the total cellular activity, and the mitochondrial and plastid fractions each contained about 10%. The results suggest the existence of three parallel biosynthetic pathways which may be important in regulating the synthesis of isoprenoids characteristic of the individual organelles.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA13 reductase, a regulatory enzyme in the isoprenoid pathway, has been extensively studied in both animals (1, 2) and microorganisms (3). Present evidence indicates that in rat hepatic cells HMG-CoA reductase is associated with the microsomal fraction (4) and that in yeast cells the enzyme is localized in the mitochondria (5). Although HMG-CoA reductase has not been previously studied in higher plants there is good evidence (6, 7) that the isoprenoid pathway does exist. In addition, MVA kinase activity has been detected (8) in both the soluble and the chloroplast fraction, thus providing evidence for more than one possible subcellular localization of the pathway. In the present work extensive cell frac-
tionation studies have been carried out to determine the precise organelle localization of HMG-CoA reductase. Particular attention has been given to submicrosomal fractionation since studies by other groups (9, 10) with animal systems have yielded conflicting results. EXPERIMENTAL
Plant growth. Etiolated and green seedlings of Pisum satiuum (var Alaska) were grown as previously described (11). Materials. HMG-CoA was prepared and purified as previously described (11). Insoluble polyvinyl pyrrolidone (polyclar AT) was obtained from the General Film & Aniline Corporation; 2,6-dichlorophenol-indophenol, succinate (Na salt), and 2-mercaptoethanol from Sigma: 5-methylphenazinium methosulphate from J. Baker Chemical Co.; and cytochrome c from Calbiochem. Enzyme assay. HMG-CoA reductase was assayed as described previously (11). The enzyme activity was expressed as nmol MVA/mg protein/h or enzyme units/g fresh wt of tissue. One enzyme unit is defined as the amount of enzyme required to catalyse the formation of 1 nmol of MVA per min under the assay conditions described. Two control assays were also carried out on each cell fraction. One of these did not contain NADPH and one was ether extracted (12) and the ether extract applied to a thin layer plate. These
’ Supported in part by grants from the University of Otago, and the Department of Scientific and Industrial Research to D. W. Russell. Z Supported by a Post Graduate Scholarship from the University Grants Committee. 3 Abbreviations used are: HMG-CoA, 3-hydroxy-3methylglutaryl coenzyme A; MVA, mevalonic acid. 730
controls ensured that no HMG-CoA cleavage products contaminated the MVA produced. Subcell&r fractionation. In preliminary studies on the subcellular localization of HMG-CoA reductase activity, a (crude cell homogenate was prepared by grinding tissues in a mortar with 10% w/w polyclar AT (131 and buffer containing 0.35 M sucrose: 0.1 M Tris-HCl (pH 7.0); 30 mM EDTA; and 10 mM Z-mercaptoethanol (added fresh); the slurry was squeezed through two layers of muslin and the filtrate, designated crude homogenate, was centrifuged at 500g for 10 min. The pellet was discarded and the supernatant was subjected to a stepwise series of differential centrifugations ‘up to 30,OOOg for 10 min followed by further increments up to 105,OOOg for 15 min. Each resulting pellet was resuspended in 0.1 M potassium phosphate buffer (pH 6.9) containing 10 mM dithiothreitol and assayed for HMG-CoA reductase activity. Mitochondrial fractions were ultrasonicated for 30 s with a MSE ultrasonicator prior to being assayed. hokxtion
trifugation. Organelle fractions were isolated by the following differential centrifugation procedures for routine assay of HMG-CoA reductase activity associated with each fraction. Microsomes. The membrane fraction containing the microsomal enzyme was routinely prepared as follows. Mitochondria were removed from the crude homogenate (see above) by centrifuging at 10,OOOg for 15 min, then recentrifuging the supernatant at 15,OOOg for 10 min; the postmitochondrial supernatant was centrifuged at 50,OOOg for 1 h. The pellet was suspended in 0.1 ml potassium phosphate buffer (pH 6.9) containing 10 mM dithiothreitol, and the supernatant was recentrifuged at 105,OOOg for a further 1 h. Each resuspended pellet was assayed for HMG-CoA reductase activity. The results showed that 95% of the microsomal reductase activity was associated with the 50,OOOg pellet which was a clear yellow color. This procedure was used as a routine method for the preparation of the microsomal enzyme. Mitochondria and Plastids. Mitochondrial and plastid fractions were prepared from the crude homogenate by the respective differential centrifugation procedures described below for each organelle (see Density gradient isolation). Density gradient isolation ojo~ganelles. For a more definitive localization of HMG-CoA reductase activity, cell organelles prepared by differential centrifugation were purified on sucrose density gradients and the distribution of both HMG-CoA reductase and specific marker enzymes was determined. Microsomes. Apical buds from etiolated seedlings were homogenized in a mortar and pestle with 10% w/w polyclar AT in a medium containing 0.35 M sucrose; 0.1 M Tris-HCl (pH 7.1); 50 mM KCl; 5.0 mM MgCl,; and 10 mM 2mercaptoethanol (added fresh). The homogenate was squeezed through two layers of
muslin and the filtrate centrifuged at 10,OOOg for 15 min. The supernatant was recentrifuged at 15,OOOg for a further 10 min and then 7 ml of supernatant were layered on top of a discontinuous sucrose density gradient. The gradient consisted of various concentrations Of sucrose as fCllOWS (14); 2 ml Of 2.0 M, 5 Id Of 1.3 M, and 7 ml of 0.35 M containing the postmitochondrial supernatant (all solutions contained the above buffer), The gradients were centrifuged in a Beckman SW-40. Ti rotor at 40,000 rpm for 3 h. Fractions (1 ml) were then collected from the interface between 2.0 and 1.3 M sucrose, and 1.3 and 0.35 M sucrose. Each fraction was diluted with homogenizing medium, centrifuged in a Beckman Type 40 rotor at 40,000 rpm for 1 h and each pellet suspended in 0.2 ml of 0.1 M potassium phosphate buffer (pH 6.9). A small aliquot from each fraction was assayed for HMG-CoA reductase, NADPH-cytochrome c reductase (15). and succinate dehydrogenase activity (16). Mitochondria. Apical buds from etiolated seedlings were homogenized in a buffer containing 0.4 M sucrose: 0.1 M Tris-HCl (pH 7.5); 10 mM KCl; 0.1% bovine serum albumin; 1.0 mM EDTA; 0.1 mM MgCl,; and 10 mM 2-mercaptoethanol (added fresh) (17). The homogenate was squeezed through two layers of muslin and then centrifuged at l,OOOg for 10 min. The pellet was discarded and the supernatant recentrifuged at 2,OOOg for 10 min. The resulting supernatant was centrifuged at ll,OOOg for 15 min and the pellet was washed by resuspending in homogenizing medium and centrifuging at 10,OOOg for 15 min. The pellet was resuspended in a small volume of 30% sucrose containing 0.1 M Tris-HCl (pH 7.51. This suspension (2 ml) was layered on top of a linear gradient (17) consisting of 27 ml of 30-60% sucrose over 5 ml of 60% sucrose. The gradient was centrifuged in a Beckman SW-25.1 rotor at 22,500 rpm for 4 h. Fractions were then collected and assayed for succinate dehydrogenase activity. The localized mitochondrial fraction was diluted with 0.1 M potassium phosphate buffer (pH 6.9) containing 10 mM 2-mercaptoethanol and centrifuged at 12,000g for 15 min. The mitochondria were resuspended in a small volume of potassium phosphate buffer and ruptured by ultrasonication for 3 x 15 s. The solution was then diluted with the same buffer and centrifuged in a Beckman Type 50 rotor at 25,000 rpm for 60 min. The resulting mitochondrial membrane pellet was resuspended in 0.2 ml of 0.1 M potassium phosphate buffer (pH 6.9) containing 10 mM dithiothreitol, and assayed for HMG-CoA reductase using the radiochemical assay (111, succinate dehydrogenase, and NADPHcytochrome c reductase activity. Fractions on either side of the main mitochondrial band were similarly treated and assayed for HMG-CoA reductase activity. Chloroplasts. Bud and leaf tissue (approx 10 g) from light-grown pea seedlings were homogenized in buffer (approx 100 ml) using a Waring Blendor at full
speed for 10 s. The buffer contained 0.4 M sucrose; 0.1 Tris-HCl (pH 7.2); 10 mM KCl; 0.1% bovine serum albumin; 1.0 mM EDTA; 0.1 mM MgCl,; and 10 mM 2-mercaptoethanol (added fresh). The homogenate was filtered through two layers of muslin, and then centrifuged at 400g for 10 min. The supernatant was recentrifuged at 3,000g for 10 min and the pellet was resuspended in homogenizing medium to give a chloroplast suspension which was analyzed on a discontinuous sucrose gradient. The gradient consisted of 5 ml of 66% sucrose, 10 ml of 45% sucrose, 10 ml of 30% sucrose, and 6 ml of chloroplast suspension. Gradient solutions all contained 0.1 M Tris-HCI (pH 7.2) and 10 mM 2-mercaptoethanol. The gradients were centrifuged in a Beckman SW-25.1 rotor at 12,000 rpm for 2 h. Fractions were collected and aliquots were analyzed for chlorophyll content by measuring the absorbance at 620 nm in 80% aqueous acetone. Chloroplast-containing fractions were diluted with homogenizing medium and centrifuged at 10,OOOg for 10 min. The chloroplasts were osmotically ruptured by resuspending in 40 ml of 0.1 M Tris-HCl (pH 7.0) containing 10 mM 2-mercaptoethanol. The ruptured chloroplasts were centrifuged at 15,000g for 15 min to sediment the membranes which were then resuspended in 0.2 ml of 0.1 M potassium phosphate buffer (pH 6.9) containing 10 mM dithiothreitol and assayed for HMG-CoA reductase activity. Fractions on each side of the chloroplast bands were similarly treated and assayed. Chloroplast fractions were also examined under a phase contrast microscope.
Organelle localization of HMG-CoA reductase has been studied using several methods. Subcellular fractions obtained by stepwise centrifugation show a profile of reductase activity (Fig. 1) which indicates that HMG-CoA reductase is present in more than one fraction. Preliminary investigation of individual organelle fractions isolated by differential centrifugation suggested that activity was present in the microsomes, mitochondria, and plastids. These results were substantiated by more rigorous methods using sucrose density gradients and marker enzymes. Gradient isolations. The total microsomal fraction present in the 15,000g supernatant was fractionated into heavy and light microsomes by centrifugation in a discontinuous sucrose density gradient. Heavy microsomes migrated to the interface of 2.0;1.3 M sucrose as a white band, and light microsomes were seen as a dis-
40 50 EC 70 80 103 x centrifugal force
FIG. 1. Profile of HMG-CoA reductase activity in subcellular fractions prepared by stepwise centrifugation. A crude homogenate from the apical buds of etiolated seedlings was subjected to a series of centrifugations of increasing force. The pellets formed were collected separately, resuspended in a small volume of 0.1 M potassium phosphate buffer (pH 6.9), and assayed for HMG-CoA reductase activity. Mitochondrial fractions were disrupted by sonication for 30 s prior to being assayed. Fumarate hydratase (24) (fumarase) activity was used as a marker for ruptured mitochondria. HMG-CoA reductase activity is expressed as nmoles of MVA/mg protein/h.
tinct yellow band at the interface of 1.3-0.35 M sucrose. Above this was a creamy opalescent band which probably comprised Golgi vesicles and plasma membranes (10). The visible microsomal bands were collected with a syringe and fractions also taken on either side of the bands. Assays of heavy and light fractions for NADPH-cytochrome c reductase showed equivalent activity in both fractions and assays for HMG-CoA reductase showed that 95% of the total microsomal activity was associated with the light fraction which was yellow in color (Fig. 2). No succinate dehydrogenase activity was present in the microsomal bands, demonstrating that they contained no mitochondrial membrane contaminants. The specific activity of HMG-CoA reductase in the light fraction was more than lo-fold higher than in the heavy fraction although total protein per fraction was similar. When heavy and light microsomal fractions were prepared in the presence of 10 mM MgCl, instead of 5.0 mM as used above, 60% of the total microsomal activity was associated with the heavy fraction, although the specific activity of the light fraction was highest (Table I). Mitochondria were isolated in a linear 30-60s sucrose density gradient. Succinate dehydrogenase assays of the fractions
showed that the mitochondria banded at an apparent density of about 1.2 g cme3 (18). Two difficulties were encountered in the assay of HMG-CoA reductase activity in mitochondria. Firstly, intact mitochondria showed no reductase activity and secondly, only relatively low activity could be demonstrated in ruptured unfractionated mitochondrial preparations. Assays with ruptured unfractionated mitochondria yielded a product with an Rf similar to acetate on tic. However, when mitochondria were ruptured by sonication and the membranes separated from the soluble constituents by sedimentation, reductase activity could be readily demonstrated in the resuspended mitochondrial membrane preparation. This procedure was therefore followed in measuring HMG-CoA reductase activity in mitochondria. Gradient profiles (Fig. 3) of both HMG-CoA reductase and succinate dehydrogenase activity show that the two enzymes comigrate. The absence of both HMG-CoA reductase and NADPH-cytochrome c reductase activity in intact mitochondrial preparations demonstrated that
FIG. 2. Submicrosomal localization of HMG-CoA reductase. Total microsomes from a 15,OOOg supernatant were fractionated into heavy and light subfractions by centrifuging through a discontinuous sucrose density gradient. Fractions (1 ml) were collected from the interfaces between 2.0 and 1.3 M sucrose, and 1.3 and 0.35 M sucrose, diluted with homogenizing medium, and centrifuged at 105,OOOg for 1 h. Each pellet was suspended in 0.2 ml 0.1 M potassium phosphate buffer (pH 6.9) and a small aliquot assayed for HMGCoA reductase, NADPH-cytochrome c reductase (---), and succinate dehydrogenase activity.
EFFECT OF Mg 2+ CONCENTRATIONON THE FRACTIONATIONOF MICROSOMAL HMG-CoA REDUCTASP Microsomal subf;;-
Hew Light Heavy Light
Mg*+ cone (mM)
5 o ’ 10.0
Specific activity (nmo!/mg protein/h)
dpm MVY fraction
3.5 39.5 22.5 36.0
2928 51920 41256 28560
% Total dpm
5 95 60 40
OA 15,000g supernatant containing total microsomes was prepared in the presence of either 5.0 mM or 10.0 mM MgCl, and fractionated in a discontinuous sucrose density gradient containing the same MgCl, concentrations as the supernatant. Heavy and light microsomal bands were collected, diluted with homogenizing medium, and centrifuged in a Beckman type 40 rotor at 40,000 rpm for 1 h. Each pellet was resuspended in 0.2 ml of 0.1 M potassium phosphate buffer (pH 6.9) containing 10 mM dithiothreitol, and an aliquot was assayed for HMG-CoA reductase activity.
the mitochondria were not contaminated by microsomal membranes. Intact chloroplasts banded at the interface between 45% and 60% sucrose on a discontinuous sucrose density gradient. Some plastids which had been stripped of their outer membrane (19) formed a band at the interface between 45 and 30% sucrose. The integrity of the plastids was verified by examination under a phase contrast microscope. These two bands plus fractions on either side were assayed for HMG-CoA reductase activity and for chlorophyll content. The results (Fig. 4) show that HMG-CoA reductase activity corresponds with the chlorophyll peaks associated with both intact and naked chloroplasts. The highest HMG-CoA reductase activity corresponds with the major chlorophyll peak which marks the position of the main (intact) chloroplast band. Some enzyme activity is also located in the same position as the minor chlorophyll peak which marks the location of the naked chloroplasts. Quantitative
The quantitative distribution of reductase activity in both green and etiolated seed-
L VIZ& 015 % E 0 -2 *a o,02z $2 005: ii
FIG. 3. Localization of HMG-CoA reductase activity in mitochondria. Mitochondria prepared by differential centrifugation were suspended in a small volume of 30% sucrose containing 0.1 M Tris-HCl (pH 7.5). and layered on top of a linear 30-6070 sucrose density gradient. The gradient was centrifuged in a Beckman SW-25.1 rotor at 22,500 rpm for 4 h, and fractions were then collected and assayed for succinate dehydrogenase activity. The localized mitochondrial fraction was diluted with 0.1 M potassium phosphate buffer (pH 6.9) containing 10 mM 2-mercaptoethanol, and centrifuged at 12,000g for 15 min. The mitochondrial pellet was resuspended in a small volume of potassium phosphate buffer and ruptured by ultrasonication for 3 x 15 s. The solution was then diluted and the mitochondrial membranes sedimented by ultracentrifugation. The pellet was resuspended in 0.2 ml 0.1 M potassium phosphate buffer (pH 6.91, containing 10 mM dithiothreitol and assayed for HMG-CoA reductase, succinate dehydrogenase f---l, and NADPHcytochrome c reductase activity. Fractions on either side of the main mitochondrial band were similarly treated
FIG. 4. Localization of HMG-CoA reductase activity in chloroplasts. Intact chloroplasts from green seedlings were prepared by differential centrifugation, and 6 ml of suspension were layered on top of a discontinuous sucrose gradient. The gradients were centrifuged in a Beckman SW-25.1 rotor at 12,000 rpm for 2 h. Fractions were collected and aliquots were analysed for chlorophyll content f---l. Chloroplast containing fractions were then diluted with homogenising medium and centrifuged at 10,OOOg for 10 min. The chloroplasts were osmotically ruptured and then centrifuged at 15,000g for 15 min. The final pellets were resuspended in 0.2 ml 0.1 M potassium phosphate buffer (pH 6.9) containing 10 mM dithiothreitol and assayed for HMG-CoA reductase activity.
lings was studied using differential centrifugation techniques. The results (Table II) show that the microsomal fraction from both green and etiolated seedlings contains about 80% of the total cellular activity, and mitochondria and plastids each contain approx 10%. However the specific activity of the microsomal enzyme from etiolated seedlings is more than 2-fold higher than from green seedlings. This contrasts with similar specific activities of the reductase in mitochondria from green and etiolated seedlings and in plastids from green and etiolated seedlings. DISCUSSION
Subcellular fractionation studies have shown that HMG-CoA reductase from pea seedlings is localized in three distinctive components: microsomes, mitochondria, and plastids. The microsomal fraction contains about 80% of the total cellular activity and submicrosomal localization studies have shown that the activity is associated with the light microsomes. This result supports
LOCALIZATION TABLE II OF HMG-CoA
Mitochondria Microsomes Plastids Total cellular-activity
IN PEA SEEDLINGS
% of total cellular activity (fresh wt)
(units/g fresh wt)
4.0 45.0 3.0 52.0
3.0 20.0 4.0 27.0
10 83 7
8 76 16
1.50 0.12 1.80
“Microsomal, mitochondrial, and plastid fractions were isolated from a crude cell homogenate by differential centrifugation. Mitochondria were ultrasonically disrupted and the membranes sedimented. The pelleted membranes were resuspended in 0.2 ml of 0.1 M potassium phosphate buffer (pH 6.9) containing 10 mM dithiothreitol and assayed for HMG-CoA reductase activity. Chloroplasts were osmotically ruptured by suspending in 0.01 M Tris-HCl (pH 7.0) containing 10 mM 2-mercaptoethanol and the membranes sedimented by centrifugation. The chlomplast membranes were suspended in 0.2 ml of 0.1 M potassium phosphate buffer (pH 6.9) containing 10 mM dithiothreitol, and assayed for HMG-CoA reductase activity. * Etiolated.
Goldfarb’s localization studies (10) which showed that the reductase in rat liver also is associated with the light microsomal fraction. In contrast Guder et al. (9) have reported that the rat liver enzyme is localized in the heavy microsomal fraction. The conditions employed by these two groups differ significantly in that the isolation medium used by Goldfarb contained 1 mM Mg2+ whereas that of Guder et al. contained 10 mM Mg2+. It is recognised (20) that some Mg2+ is essential to maintain the integrity of the heavy microsomes but Dallner (14) has shown that relatively high Mg2+ concentrations cause irreversible aggregation of the smooth microsomal membranes, thus affecting their sedimentation behavior. This evidence, together with our .data, suggests an explanation for the above conflicting results. We have shown that when the microsomal fraction from pea seedlings is prepared and fractionated in the presence of 10 mM Mgz+, 60% of the total reductase activity is associated with a heavy microsomal fraction which is pale yellow in color. However, when the fractionation procedure is carried out in the presence of 5 mM Mgz+, 95% of the reductase activity is associated with the light microsomal fraction which forms a distinct yellow band. at the interface between 1.3 and 0.35 M sucrose. The heavy fraction
isolated under these conditions (5 I’nM Mg2+) forms a translucent white band at the 1.3-2.0 M sucrose interface. Thus our evidence strongly suggests that high Mg*+ concentrations cause significant quantities of light microsomal membranes to sediment with the heavy membrane fraction, possibly due to aggregation. This conclusion is supported by the absence of any significant change in specific activity of the reductase in the light microsomal fraction isolated under different Mg2+ concentrations. However, there is a large change in the specific activity of the enzyme in the heavy fraction isolated under similar conditions. It is thus concluded that the microsomal reductase is associated with the light microsomes. Differential centrifugation studies strongly suggested that HMG-CoA reductase activity also occurred in the mitochondrial fraction. This was confirmed by showing that reductase activity in this fraction comigrated with succinate dehydrogenase on a linear sucrose density gradient. Mitochondrial localization of HMG-CoA reductase has also been reported for yeast cells (5) and for the intestinal crypt cells of rats (21), but not for rat liver mitochondria. However, it is known (21) that rat liver mitochondria contain a lyase which rapidly converts HMG-CoA to acetyl-CoA and ace-
toacetate. Hence failure to detect reductase activity in liver mitochondria could be due to the presence of this enzyme. Our results from assays performed on disrupted pea seedling mitochondria suggest that these mitochondria also contain an active soluble enzyme which attacks the substrate causing low apparent activity of the reductase. However, when the mitochondria were disrupted and the membranes then separated from the soluble fraction by centrifugation, HMG-CoA reductase activity was readily detected in the mitochrondrial membrane fraction. Intact mitochondria showed no HMG-CoA reductase activity and no NADPH-cytochrome c reductase activity indicating negligible contamination of the mitochondria with the microsomal fraction. Hence it is concluded that in pea seedlings, a proportion of the total cellular HMG-CoA reductase activity is located in the mitochondria, and is associated with the mitochondrial membranes. HMG-CoA reductase activity was found in both etioplasts and mature chloroplasts isolated by differential centrifugation. This localization was verified by isolating chloroplasts from green seedlings on a discontinuous sucrose density gradient. Activity was associated with both the main and minor chlorophyll bands, which corresponded, respectively, to intact chloroplasts and those which were devoid of their outer envelope. It is known (22) that the levels of isoprenoid electron carriers, carotenoids, and internal membranes all increase dramatically during chloroplast development but the subcellular origin of the isoprenoid precursors is incompletely understood. Previous studies with intact plant tissues (6) showed that radioactivity was incorporated into carotenoids from “CO, but not from [14C]acetate or [l%]MVA. On the basis of these results it was suggested that all of the enzymes of the isoprenoid pathway were present in the chloroplast and that compartmentation is important in the regulation of the chloroplast and cytoplasmic pathways. In contrast to the above results, studies on fatty acid synthesis (23) have demonstrated that [%]acetate is readily incorporated into fatty acids by intact isolated chloroplasts.
Hence suggestions of compartmentalized synthesis which are based only on lack of acetate incorporation are open to question, and the existence of the complete pathway in chloroplasts must be established by demonstrating the presence of the necessary enzymes. Some more conclusive evidence has been reported (8) showing that MVA is converted to MVA-&phosphate and MVA-5-pyrophosphate in isolated ruptured chloroplast preparations, thus indicating the presence in the chloroplast of enzymes which metabolize MVA. In the present work the demonstration that HMG-CoA reductase activity is present in purified chloroplast membranes provides evidence for the synthesis of MVA from HMG-CoA within the chloroplast. In the absence of evidence for the localization of the preceding enzymes it is uncertain whether HMG-CoA is synthesised in the chloroplast or cytoplasm, although synthesis in the plastid appears most likely. Studies on the quantitative distribution of HMG-CoA reductase in etiolated and green seedlings have shown that approx 80% of the total cellular activity is associated with the microsomal fraction and that the mitochondria and plastids each contain about 8-10s. However the microsomal reductase activity (units/g wt and specific activity) is more than 2-fold higher in etiolated seedlings than in green seedlings. This large difference may be related to the fundamental changes in growth and development which are initiated when etiolated seedlings are exposed to light. In contrast, mitochondria from green or etiolated seedlings contain similar HMG-CoA reductase activity, and the activity present in plastids from green or etiolated seedlings is also similar. Thus definitive organelle isolation studies have shown that HMG-CoA reductase from pea seedlings is localized in the microsomes, mitochondria, and plastids, which suggests that the organelles and cytoplasm contain independent but parallel pathways for isoprenoid biosynthesis from HMG-CoA. This raises questions as to whether the distinct activities represent identical proteins or whether they are isozymes, and where in the ceil they are
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