A discontinuous sucrose density gradient was used to separate membrane fractions from a homogenate of maize rcottips. Endoplasmic reticulum-, Golgi apparatus-, plasma membrane- and mitochondria-rich fractions were identified by their enzymic characteristics and by their appearance under the electron microscope. Maize roots were incubated in vivo with D-[U-14C]glucose, [Me-14C]choline chloride and diazotized [U-3H]sulphanilic acid. The pattern of incorporation of radioactivity into the various membrane fractions was investigated. Analyses of the polypeptide chains of the membrane fractions by SDS-polyacrylamide gel electrophoresis showed that the mitochondria-rich fraction had a different pattern of polypeptides from that of the other membrane fractions. The results are discussed in relation to the hypotheses of endomembrane flow and differentiation.

The endomembrane system of eukaryotic cells includes the nuclear envelope, rough and smooth endoplasmic reticulum, Golgi apparatus, plasma membrane and various cytoplasmic vesicles (Northcote, 1971, 1974). Some of the components of the system are joined by structural connexions and they all have a functional continuity in the cytoplasm of eukaryotic cells (Morré & Ovtracht, 1977). It is now generally believed that the endomembrane system is present in a dynamic state representing a flow of membranes from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane (Bowles & Northcote, 1974; Morré, Kartenbeck & Franke, 1979).

In the last few years, attention has been directed to the separation of the endomembranes from plant cells. Several fractionation systems have been developed, but most of them aim to separate only one component of the system from a total homogenate of cells (Powell & Brew, 1974; Hodges & Leonard, 1974). The difficulties met in separating and identifying intact endomembranes from plant cells arise from the presence of the cell wall and the lack of reliable markers for the different parts of the endomembrane system (Quail, 1979). The first problem has been overcome for some tissues by the use of protoplasts as starting material (Galbraith & Northcote, 1977).

The purpose of this study was to develop a simple and quick procedure to separate membrane fractions from maize root tips. We avoided pelleting and re-suspension of membranes to decrease the chance of their destruction.

Please address all correspondence to Professor D. H. Northcote at the above address.

Radioactive chemicals

D-[U-14C]glucose (sp. radioact. 10·8 GBq/mmol), UDP-D-[U-14C]glucose (sp. radioact. 10−5 GBq/mmol), [Me-14C]choline chloride (sp. radioact. 2·2 GBq/mmol) and [U-3H]sulph-anilic acid were purchased from the Radiochemical Centre, Amersham, Bucks., U.K. [U-3H]sulphanilic acid was supplied as an impure solution (in GBq) and was purified by thin-layer chromatography in ethyl acetate/propan-2-0l/aq.NH3 (sp.gr. 0 ·880)/water (6:4:2:1, by vol.) on silica gel plates (20 cm × 20 cm, layer 2 mm; Macharey, Nagel und Co., Dilren, FRG). The sp. radioact. of the purified material was 0·1 GBq/mmol.

Plant material

Seedlings of maize (Zea mays-, var. Caldera) were grown under sterile conditions. The seeds (coated with a copper-containing fungicide) were washed in sterile water, soaked in sterile chloramphenicol solution (10 mg I.−1) overnight, and germinated for 3–4 days in the dark at 25 °C as described by Harris & Northcote (1970). Harvesting and all subsequent procedures of fractionation were carried out in the light at 4 °C.

Preparation of membrane fractions

Primary root tips (4–5 mm long) were excised with scissors and washed twice in the homogenization medium. Root tips (5 g fresh weight) were then suspended in 5 ml of the homogenization medium, chopped into small pieces and ground gently in a chilled mortar for 90–120 s, using a squashing action. The homogenization medium consisted of 8 % (w/w) sucrose, 50 mM Tris-HCl buffer at pH 7·4, 1 mM EDTA, and 0 ·1 mM MgCl2.

After filtration through 2 layers of muslin, the homogenate was centrifuged at 800 g for 10 min to remove starch, nuclei, cell wall fragments and unbroken cells. The volume of the supernatant was made up to 12 ml by the addition of homogenization medium and immediately layered on a discontinuous sucrose density gradient. This was prepared in a 38-ml cellulose nitrate tube by layering in succession 4 ml of 45 % (w/w) sucrose and 5’5 ml each of 39, 34, 25 and 14 % (w/w) sucrose solutions, using a peristaltic pump. The sucrose solutions used to make the discontinuous gradient contained all the components of the homogenization medium except that the Tris-HCl buffer, pH 7·4, was at 10 mM. The tube was then centrifuged at 100000 g for 4 h and the particulate material at each sucrose interface was carefully collected with a Pasteur pipette. The membrane fractions collected at the 14–25, 25–34, 34–39 and 39–45 % interfaces are referred to as membrane fractions 1,2,3 and 4 respectively, and all the material left in the tube after the collection of interfaces represents the remainder fraction (Fig. 1).

Fig. 1.

Scheme summarizing the procedure for the preparation of membrane fractions from maize root tips.

Fig. 1.

Scheme summarizing the procedure for the preparation of membrane fractions from maize root tips.

Marker enzyme assays

NADH-, and NADPH-cytochrome c reductases were assayed by following the reduction of cytochrome c (A550 18 · 5 mM−1 cm−1) (Shore & Maclachlan, 1975) using a Beckman Model 25 recording spectrophotometer. The effect of antimycin A (14 μg ml−1) on NADH-cytochrome c reductase was also investigated. Succinate dehydrogenase activity was determined by following spectrophotometrically the reduction of 2,6-dichlorophenol-indophenol (E600 21 mM−1 cm−1) (Veeger, Der Vartanian & Zeylemaker, 1969). Latent IDPase was measured after storing the membrane fractions at 4 °C for 4 days (Ray, Shininger & Ray, 1969). Mg2+-ATPase was assayed at pH 6 · 5 (Leonard & Van Der Woude, 1976; Taussky & Short, 1953). UDPG:sterol glucosyltransferase was assayed by a modification of the method of Letcher & Wojciechowski (1976). The incubation was carried out at 30 °C for 30 min in a solution (0·4 ml) containing membrane fraction (50 μg protein), 50 mM Tris-HCl, pH 7·4, 0·8 mM β-mercaptoethanol, iomM MgCl,, 0·8 μm UDP-D-[U-14C]glucose and 0·2% Triton X-100 with and without 0·5 mM β-sitosterol. The reaction was started by the addition of the membrane fraction and stopped by the addition of 6 ml of chloroform/methanol (3:2, v/v). After centrifugation, the supernatant was washed according to the method of Folch, Lees & Sloane Stanley (1957), roto-evaporated and assayed for radioactivity. UDP-galactose: N-acetylghicosamine galactosyl transferase was assayed as described by Palmiter (1969). Protein was estimated by the method of Lowry, Rosebrough, Farr & Randall (1951) after precipitation of the membranes with 18 % cold trichloroacetic acid. Bovine serum albumin was used as the standard.

Incubation of roots in vivo with diazotized [U-3H]sulphanilic acid

[U-3H]sulphanilic acid was converted to the diazonium salt (Berg & Hirsh, 1975). The diazotization mixture contained [U-’H]sulphanilic acid (925 kBq), 2 μmol HC1 and 50 μI (300 μmol) isoamyl nitrite. Maize seedlings (40·50) were arranged in a perforated plate so that their root tips dipped into a Petri dish containing the diazotized [U-3H]sulphanilic acid (925 kBq) dissolved in 5 ml of sterile water and were then incubated in the dark at 25 °C with reciprocal shaking. After 60 min the roots were washed repeatedly with water to remove root slime and non-covalently bound radioactivity, surface dried, excised, mixed with non-radioactive root tips, homogenized and fractionated.

The various membrane fractions were suspended in the homogenization medium and pelleted at 100000 g and 4 °C for 30 min. The 800 g pellet and membrane pellets were suspended in 18% (w/v) trichloroacetic acid. The remainder fraction was precipitated with 18% (w/v) trichloroacetic acid. The insoluble material was washed 4 times with 18 % (w/v) trichloracetic acid followed by acetone which was removed by heating in an oven at 60 °C for 5 min. It was then dissolved in 50 βl of 1 M NaOH, neutralized with 1 M HC1, re-suspended in 1·9 ml of the scintillation fluid and assayed for radioactivity.

Incubation of roots in vivo with r>-[U-14C] glucose

Maize seedlings (30) were placed radially in circular groups (10 seedlings per group) in a sterile Petri dish so that the tips of the roots were in contact. They were then incubated with sterile D-[U-14C]glucose solution (10 μl, 37 kBq per root) by placing the solution at the point of root contact. The roots were incubated in the dark at 25 °C for 45 min (Bowles & Northcote, 1974). At the end of the incubation, the slime and the incubation medium were removed and the roots were washed repeatedly with sterile water and then surface dried. The root tips were excised, mixed with non-radioactive root tips, homogenized and fractionated.

Analysis of membrane fractions prepared from roots incubated with D-[U-]4C] glucose in vivo

Membrane fractions that had been prepared from roots incubated with D-[U-14C]glucose were divided into 3 equal aliquots, pelleted, washed with trichloroacetic acid and acetone, and dried. The first aliquot was assayed for radioactivity. The second was dissolved in 72 % (w/w) H2SO4 at 20 °C. After 4 h the acid was diluted to 3 % (w/w) H,SO4 and the preparation was hydrolysed in an autoclave at 120 °C and 103 kN m−2 for 1 h. The hydrolysates were neutralized with Amberlite IR-4B resin (CO3−2 form), and roto-evaporated to dryness. The residues were dissolved in water and applied to Whatman no. 1 paper and then run electrophoretically in acetic acid/formic acid/water (4:1:45, by vol.) at pH 2 and 5 kV for 20 min (Harris & Northcote, 1970). At the end of the run, neutral sugars and uronic acids remained near the origin, while peptides and amino acids moved towards the cathode. These were cut from the electrophoretogram and assayed for their radioactivity. The third aliquot was dissolved in chloroform/methanol (3:2, v/v) to extract the lipids. The chloroform/methanol extract was washed (Folch et al. 1957), roto-evaporated to dryness and assayed for radioactivity.

Incubation of roots in vivo with [Me-14C]choline chloride

Maize seeds (100) were germinated under sterile conditions. After 2 days they were incubated with 1110 kBq [Me-14C]choline chloride in 10 ml sterile water by addition of the radioactive solution to the dish. After a further 48 h, the seedlings were removed and the roots were thoroughly washed with water and then surface dried. The radioactive root tips were excised, mixed with non-radioactive root tips, homogenized and fractionated. After pelleting the membrane fractions, lipids were extracted in chloroform/methanol (3:2, v/v). The extract was washed (Folch et al. 1957), roto-evaporated to dryness and assayed for radioactivity.

To investigate the incorporation of [Me-14C]choline into intact maize roots, radioactive root tips (10) were homogenized at 4 °C, with a pestle and mortar in chloroform/methanol (3:2, v/v; 5 ml) and the homogenate was stored at 4 °C overnight to extract the lipids and centrifuged at 10000 g for 30 min at 4 °C. The lipid extract was decanted and the tissue residue was further extracted with 3 ml of chloroform/methanol (3:2, v/v). The pooled lipid extracts were then washed (Folch et al. 1957), roto-evaporated to a small volume and analysed by thin-layer chromatography. Thin-layer chromatography was carried out in chloroform/methanol/water (65:2514, by vol.) on silica-gel G plates (20 × 20 cm, layer 0-25 mm, without gypsum; Camlab, Cambridge, U.K.). The tissue residue was washed several times with 80% (v/v) methanol (Sharma, Babczinski, Lehle & Tanner, 1974), dried and assayed for radioactivity.

Radioactivity determination

Radioactivity was determined using a Searle Mark III scintillation counter. Samples were assayed for radioactivity for 10 min in a mixture of scintillation fluid made up of 2,5-di-phenyloxazole (PPO), 4 g, 1,2-6w-(5-phenyloxazol-2-yl)benzene (POPOP), 0·075 g, Triton X-100, 750 ml, in 1·5 1. of sulphur-free toluene. The efficiency for 14C was 90% and that for 3H was 50 %.

SDS-polycrylamide gel electrophoresis

Membrane pellets were analysed on slab gels (Studier, 1973) using the system of Laemmli (1970). Gels (15 %) were run at 200 V for approx. 5 h. After electrophoresis the proteins were fixed in the gel with a mixture of methanol/acetic acid/water (9:2:9, by vol.), stained for 30 min with 0 · 125 % Coomassie brilliant blue made up in the fixer and destained by repeated washing in a mixture of methanol/acetic acid/water (1:1:8, by vol). A mixture of marker proteins (each 5 μg) was run on each gel. The mixture consisted of lysozyme (mol. wt 14300), creatine kinase (mol. wt 40000) and bovine serum albumin (mol. wt 68000).

Amino acid analysis

A sample of the 800 g pellet was dried in vacuo and was then hydrolysed with 6 M HC1 in an evacuated sealed tube at 105 °C for 24 h. The hydrolysed sample was dried under vacuum and then dissolved in 1 ml of 0 · 2 M citrate buffer, pH 2 · 2 (10·5 g citric acid, 11 · 7 g sodium chloride, 3 · 5 ml Brj-35-(detergent) and 2 · 5 ml thiodiglycol 1.−1). Quantitative amino acid analysis was performed using Rank Hilger Chromaspex J 180 Mark I amino acid analyser.

Electron microscopy

Membrane fractions were examined by negative staining with 2 % phosphotungstic acid (adjusted with NaOH to pH 6·8) and they were prepared for thin sectioning, stained with uranyl acetate and alkaline lead citrate and then examined in an AEI EM6B electron microscope at 60 kV (Brett & Northcote, 1975).

Distribution of enzyme activities

NADH-cytochrome c reductase, NADPH-cytochrome c reductase, succinate dehydrogenase, Mg2+-ATPase at pH 6·5, UDPG: sterol glucosyltransferase and UDP-galactose: N-acetylglucosamine galactosyltransferase were assayed immediately after the preparation of fractions. Latent IDPase activity was assayed after storage of the membranes at 4 °C for 4 days. The distribution of these enzyme activities is shown in Table 1.

Table 1.

Distribution of protein, and enzyme activities in subcellular fractions of maize root tips obtained by discontinuous sucrose density gradient centrifugation.

Distribution of protein, and enzyme activities in subcellular fractions of maize root tips obtained by discontinuous sucrose density gradient centrifugation.
Distribution of protein, and enzyme activities in subcellular fractions of maize root tips obtained by discontinuous sucrose density gradient centrifugation.

The presence of NADH-cytochrome c reductase and NADPH-cytochrome c reductase is often used to identify the endoplasmic reticulum (Nagahashi & Beevers, 1978).

Fraction 1 contained 45·9 and 51·9% of the total activity of NADH- and NADPH-cytochrome c reductases respectively. The activity of NADH-cytochrome c reductase was found to be about 26 times higher than that of NADPH-cytochrome c reductase (Table 1). When assayed in the presence of antimycin A, the activity of NADH-cytochrome c reductase decreased markedly in some membrane fractions. It was inhibited by about 4·9, 9·5, 21·5 and 21·5% in membrane fractions 1, 2, 3, and 4, respectively.

IDPase has been shown to be a marker for the Golgi apparatus in plant cells, both histochemically (Dauwalder, Whaley & Kephart, 1969) and biochemically (Ray et al. 1969; Coughlan & Evans, 1978). The highest latent IDPase specific activity was recovered in fraction 2. This fraction as well as the other isolated membrane fractions did not show any activity when assayed for UDP-galactose: 7V-acetylglucosamine galactosyltransferase.

Mg2+-ATPase, assayed at pH 6·5, has been demonstrated to be a plasma membrane marker in plants (Galbraith & Northcote, 1977; Boss & Ruesink, 1979). The highest specific activity of this enzyme was found in fraction 3 and the remainder fraction contained high levels as well as IDPase. These activities could be due to non-specific phosphatases contained in vacuoles which were broken during homogenization. The high recovery of Mg2+-ATPase (155 %) is probably due to the dilution of an inhibitor such as phosphate ions and is in agreement with results found in soybean suspension cultures (Galbraith & Northcote, 1977). Hartmann, Fonteneau & Benveniste (1977) showed that UDPG:sterol glucosyltransferase was located in the plasma membrane of maize. When membrane fractions were assayed for this enzyme, the bulk of radioactivity incorporated from UDP-D-[U-14C]glucose was found in fractions 3 and 4, but the highest specific activity occurred in fraction 3 and corresponded to that of Mg2+-ATPase at pH 6·5. The enzymic activity was investigated with endogenous sterols as well as with added β-sitosterol. The addition of β-sitosterol enhanced the activity by 2-4-fold in the various membrane fractions. The high recovery (135 %) could be due to dilution of an inhibitor or activation of the enzyme upon homogenization and fractionation.

Endogenous sterols were roughly estimated by comparing the incorporation of radioactivity with and without added β-sitosterol (Table 2). The results showed that fraction 3 contained more sterols mg−1 protein when compared with other isolated fractions.

Table 2.

Rough estimate of sterol in membrane fractions

Rough estimate of sterol in membrane fractions
Rough estimate of sterol in membrane fractions

Succinate dehydrogenase, which is localized in the inner mitochondrial membrane (Janiszowska, Sobocinska & Kasprzyk, 1979), was found mainly in fraction 4 (73 · 5 % of total activity). No activity was found in fractions 1 and 2.

These data suggest that fraction 1 was enriched in endoplasmic reticulum, fraction 2 in Golgi apparatus, fraction 3 in plasma membrane and fraction 4 in mitochondria.

Electron-microscopic investigations of the membrane fractions

800 g pellet

It was not possible to obtain electron micrographs of sufficient quality from this pellet because of the large amounts of non-membranous material. Thin sections showed that it was enriched in cell wall fragments. However, broken nuclei, a few intact nuclei, plastids with starch grains, mitochondria and trapped membranes were also present.

Fraction 1

Negative staining and thin sections of this fraction showed smooth and some rough surface vesicular membranes of different sizes (Figs. 2, 3).

Fig. 2.

Electron micrograph of fraction 1 (endoplasmic reticulum-rich) negatively stained with 1 % sodium phosphotungstate, pH 6·8. × 10000.

Fig. 2.

Electron micrograph of fraction 1 (endoplasmic reticulum-rich) negatively stained with 1 % sodium phosphotungstate, pH 6·8. × 10000.

Fig. 3.

Electron micrograph of sectioned material from fraction 1 (endoplasmic reticulum-rich) stained with uranyl acetate and alkaline lead citrate, × 10000.

Fig. 3.

Electron micrograph of sectioned material from fraction 1 (endoplasmic reticulum-rich) stained with uranyl acetate and alkaline lead citrate, × 10000.

Fraction 2

On examination of negatively stained samples, these membranes showed high proportions of individual Golgi cisternae (Fig. 4). They had characteristic structures identical to those described by Brett & Northcote (1975). Thin sections revealed the presence of structures similar to individual cisternae (Fig. 5). Vesicles of various sizes were also present.

Fig. 4.

Electron micrograph of fraction 2 (Golgi apparatus-rich) negatively stained with 1 % sodium phosphotungstate, pH 6·8. Arrows show structures which may represent individual cisternae. × 10000.

Fig. 4.

Electron micrograph of fraction 2 (Golgi apparatus-rich) negatively stained with 1 % sodium phosphotungstate, pH 6·8. Arrows show structures which may represent individual cisternae. × 10000.

Fig. 5.

Electron micrograph of sectioned material from fraction 2 (Golgi apparatusrich) stained with uranyl acetate and alkaline lead citrate. Arrows show structures which may represent individual cisternae. × 16000.

Fig. 5.

Electron micrograph of sectioned material from fraction 2 (Golgi apparatusrich) stained with uranyl acetate and alkaline lead citrate. Arrows show structures which may represent individual cisternae. × 16000.

Fraction 3

Examination of negatively stained and thin-sectioned material of this fraction showed that it was rich in vesicles with smooth membranes. A few mitochondria were also present (Figs. 6, 7).

Fig. 6.

Electron micrograph of fraction 3 (plasma membrane-rich) negatively stained with i % sodium phosphotungstate, pH 6·8. × 10000.

Fig. 6.

Electron micrograph of fraction 3 (plasma membrane-rich) negatively stained with i % sodium phosphotungstate, pH 6·8. × 10000.

Fig. 7.

Electron micrograph of sectioned material from fraction 3 (plasma membranerich) stained with uranyl acetate and alkaline lead citrate, × 10000.

Fig. 7.

Electron micrograph of sectioned material from fraction 3 (plasma membranerich) stained with uranyl acetate and alkaline lead citrate, × 10000.

Fraction 4

Thin sections of this fraction showed enrichment in intact mitochondria. A few broken mitochondria and vesicular material were also present (Fig. 8).

Fig. 8.

Electron micrograph of sectioned material from fraction 4 (mitochondria-rich) stained with uranyl acetate and alkaline lead citrate, × 10000.

Fig. 8.

Electron micrograph of sectioned material from fraction 4 (mitochondria-rich) stained with uranyl acetate and alkaline lead citrate, × 10000.

Distribution of radioactivity incorporated from diazotized [U-3H]sulphamlic acid

The distribution of radioactivity incorporated from diazotized [U-3H]sulphanilic acid between the various membrane fractions is given in Table 3. Most of the radioactivity was found to be in the 800-g pellet. This could be because of the presence of unbroken cells in this pellet. However, it could also be due to structural proteins and entrapped plasma membrane within the cell wall fragments of the 800-g pellet. Among the other membrane fractions the highest specific activity occurred in membrane fraction 3 which had been shown by enzyme assays to be plasma membrane-rich.

Table 3.

Distribution of radioactivity incorporated from diazotized [U-3H]sulphanilic acid into various membrane fractions of maize roots

Distribution of radioactivity incorporated from diazotized [U-3H]sulphanilic acid into various membrane fractions of maize roots
Distribution of radioactivity incorporated from diazotized [U-3H]sulphanilic acid into various membrane fractions of maize roots

Incorporation of [Me-14C]choline into maize root lipids

Roots were incubated with [Afe-14C]choline chloride for 48 h. Lipid extraction of the roots removed 100% of the radioactivity. After Folch washing, all the radioactivity in the lipid phase co-chromatographed with authentic phosphatidylcholine (Fig. 9). Most of the radioactive phosphatidylcholine was found in the endoplasmic reticulum-rich fraction (Table 4).

Table 4.

Distribution of radioactivity incorporated from [Me-14C]choline chloride into various membrane fractions of maize roots

Distribution of radioactivity incorporated from [Me-14C]choline chloride into various membrane fractions of maize roots
Distribution of radioactivity incorporated from [Me-14C]choline chloride into various membrane fractions of maize roots
Fig. 9.

Thin-layer chromatography of the radioactive lipids extracted from maize roots incubated in vivo with [Me-14C]choline chloride. Roots were incubated with [Me-14C]choline chloride for 48 h in vivo. Lipids were extracted with chloroform/methanol (3: 2, v/v). The lipids were chromatographed on silica gel thin-layer plates, which were then sliced and assayed to determine the distribution of radioactivity.

Fig. 9.

Thin-layer chromatography of the radioactive lipids extracted from maize roots incubated in vivo with [Me-14C]choline chloride. Roots were incubated with [Me-14C]choline chloride for 48 h in vivo. Lipids were extracted with chloroform/methanol (3: 2, v/v). The lipids were chromatographed on silica gel thin-layer plates, which were then sliced and assayed to determine the distribution of radioactivity.

Incorporation of D-U-14C]glucose into maize roots

The incorporation pattern of radioactivity into neutral sugars and uronic acids, amino acids and peptides and lipids in the various membrane fractions is shown in Table 5. Most of the radioactivity was found in the 800-g pellet, owing to the presence of cell wall fragments. The bulk of the radioactivity in this pellet, after hydrolysis, was recovered in the neutral sugars and uronic acids which arose from cell wall polysaccharides. The ratio of incorporation of radioactivity into carbohydrate: protein increased in the sequence endoplasmic reticulum-, < Golgi apparatus-, < plasma membrane-rich fraction.

Table 5.

Relative amounts of radioactivity incorporated from D-[U-14C]glucose into carbohydrate, protein and lipid components in various membrane fractions of maize roots

Relative amounts of radioactivity incorporated from D-[U-14C]glucose into carbohydrate, protein and lipid components in various membrane fractions of maize roots
Relative amounts of radioactivity incorporated from D-[U-14C]glucose into carbohydrate, protein and lipid components in various membrane fractions of maize roots

Electrophoretic patterns of the polypeptides of membrane fractions

The pattern of polypeptides produced on electrophoresis of the isolated membrane fractions is shown in Fig. 10. A comparison of gel patterns revealed similarities and differences between the various membrane fractions. The polypeptide pattern of the mitochondria-rich fraction was unlike that of the other membrane fractions. The plasma membrane-rich fraction showed some bands which were also present in the mitochondria. This could be due to contamination by mitochondria. Electron microscopy and marker enzyme determinations confirmed that the plasma membranerich fraction was contaminated with mitochondria.

Fig. 10.

SDS-polyacrylamide gel electrophoresis of the isolated membrane fractions. Tracks 1 and 2; mitochondria-rich fraction. Tracks 3 and 4; plasma membrane-rich fraction. Tracks 5 and 6; Golgi apparatus-rich fraction. Tracks 7 and 8; endoplasmic reticulum-rich fraction. Tracks 9 and 10: 800-g pellet. Approx. 150 μg of protein were applied per track. Tracks 11 and 12; protein markers with molecular weights as indicated. Membrane fractions were isolated from maize roots as shown in Fig. 1. After pelleting, membranes were analysed on a 15 % gel at 200 V for approx. 5 h. Staining was with Coomassie brilliant blue.

Fig. 10.

SDS-polyacrylamide gel electrophoresis of the isolated membrane fractions. Tracks 1 and 2; mitochondria-rich fraction. Tracks 3 and 4; plasma membrane-rich fraction. Tracks 5 and 6; Golgi apparatus-rich fraction. Tracks 7 and 8; endoplasmic reticulum-rich fraction. Tracks 9 and 10: 800-g pellet. Approx. 150 μg of protein were applied per track. Tracks 11 and 12; protein markers with molecular weights as indicated. Membrane fractions were isolated from maize roots as shown in Fig. 1. After pelleting, membranes were analysed on a 15 % gel at 200 V for approx. 5 h. Staining was with Coomassie brilliant blue.

Endoplasmic reticulum-, Golgi apparatus-, and plasma membrane-rich fractions were similar. However, the Golgi apparatus-rich fraction showed some intense bands in the mol. wt range of 40000 to 68000. The endoplasmic reticulum-rich fraction contained a higher ratio of low molecular weight to high molecular weight polypeptides than the Golgi apparatus-rich fraction.

The 800-g-pellet (cell wall-rich) showed 2 characteristic bands of mobility close to that of the bovine serum albumin marker and only few other bands which were mainly of low molecular weight.

The cell wall of higher plants is known to contain a hydroxyproline-rich glycoprotein (Lamport & Northcote, 1960). The cell wall-rich pellet was hydrolysed and analysed for amino acid composition. The results (Table 6) showed that hydroxyproline accounted for 7 · 9 % of the total of mole % of amino acids in this pellet.

Table 6.

Amino acid composition of the 800-g pellet

Amino acid composition of the 800-g pellet
Amino acid composition of the 800-g pellet

The discontinuous gradient described in this work provided a partial separation of several enzyme activities and therefore of different membrane fractions. The identification of these membrane fractions was based on marker enzyme enrichment as well as on morphological criteria derived from electron-microscopic investigation.

Fraction 1 (density < 1 ·1g ml1) was identified as endoplasmic reticulum-rich fraction. It showed enrichment in antimycin A-insensitive NADH-cytochrome c reductase and NADPH-cytochrome c reductase. In the absence of EDTA, the endoplasmic reticulum sediments at a higher density than rig ml− 1 since EDTA brings about the release of ribosomal subunits (Green & Northcote, 1979). EDTA was included in the homogenization medium and gradient solutions that we used to prevent membrane aggregation and to ensure better separation of membranes. The inhibition of NADH-cytochrome c reductase with antimycin A was very low in the endoplasmic reticulum-rich fraction as compared with the mitochondria-rich fraction which showed maximum inhibition.

Mitochondria are known to contain 2 activities of NADH-cytochrome c reductase. The first, which is located in the outer membrane, is antimycin A insensitive (Douce, Mannella & Bonner, 1973) and similar to that of the activity found in the endoplasmic reticulum. The second is antimycin A-sensitive and is located in the inner mitochondrial membrane (Lord, Kagawa, Moore & Beevers, 1973; Sparace & Moore, 1979). The significant inhibition of NADH-cytochrome c reductase by antimycin A in fractions 3 and 4 could be due to broken mitochondria. No detergents were used in the enzyme assay.

Fraction 2 was identified as the Golgi apparatus-rich fraction because of its association with IDPase. This fraction also showed high activities of antimycin A-insensitive NADH-cytochrome c reductase and NADPH-cytochrome c reductase which could result from contamination with membrane vesicles derived from endoplasmic reticulum, or the presence of the enzymes as components of the Golgi apparatus. Recent studies (Hino, Asano, Sato & Shimizu, 1978; Hino, Asano & Sato, 1978) have shown that NADH- and NADPH-cytochrome c reductases are localized in the Golgi fraction and their presence could arise from membrane flow between the membranes of the endoplasmic reticulum and the Golgi apparatus. Howell, Ito & Palade (1978) reported evidence for the occurrence of NADPH-cytochrome c reductase in isolated Golgi fractions from rat liver. This was further supported by immunological studies (Ito & Palade, 1978).

Identification of isolated Golgi apparatus from plants is chiefly based on their morphology, which is so characteristic that it serves as a reliable marker (Northcote, 1971, 1974). The secretory activity of the Golgi apparatus changes during the cell cycle in maize root tips (Mollenhauer & Mollenhauer, 1978). The great difficulty in isolating intact dictyosomes from plant tissue as distinct from animal tissue lies in the method of homogenization. Plant dictyosomes become unstacked to produce individual cisternae after the comparatively rigorous homogenization required to break open the cell walls. The method most commonly used for overcoming the problem has been to include glutaraldehyde in the homogenization medium as a membrane fixative (Harris & Northcote, 1971; Bowles & Northcote, 1972). Although this treatment inactivates many enzymes (Ray, Eisinger & Robinson, 1976), the preservation of subcellular organelles is good. The treatment of pea roots with cellulase and the inclusion of polyethylene glycol in the homogenization medium prior to homogenization (Brett & Northcote, 1975) enabled intact dictyosomes to be isolated. Powell & Brew (1974) isolated intact dictyosomes from onion stems by chopping the stems with razor blades, followed by homogenization in an all-glass Potter-Elvehjem homogenizer. The homogenization medium contained 10 mM MgCl2, which may result in membrane aggregation.

In the present work, characteristic individual cisternae but not dictyosomes were found. This could be due to the homogenization method (pestle and mortar) and also to the presence of EDTA in the homogenization medium which will cause membrane disaggregation because of the removal of divalent cations.

UDP-galactose: N-acetylglucosamine galactosyltransferase has been used as a reliable biochemical marker for the Golgi apparatus especially in animal tissues (Morré Yunghans, Vigil & Keenan, 1974). It has been reported to be present in the Golgi membrane of onion stems (Powell & Brew 1974) and of the vegetative tissue of the brown alga Fucus serratus (Coughlan & Evans, 1978). We found no activity in any of the isolated membrane fractions.

The highest specific activity of Mg2+-ATPase at pH 6 · 5 was found in fraction 3. This fraction was identified as plasma membrane-rich, in agreement with results obtained for sugar cane cell suspension (Thom, Laetsch & Maretzki, 1975), soybean protoplasts (Galbraith & Northcote, 1977) and maize roots (Leonard & Van Der W ou de, 1976). However, the activity of the Mg2+-ATPase, present in all the membrane fractions we examined, was not enhanced by the addition of 50 mM K+, which contrasts with some other results (Leonard, Hansen & Hodges, 1973; Lurie & Hendrix, 1979), but agrees with those of Boss & Ruesink (1979). It was also not inhibited by the addition of 0·1 mM diethylstilbestrol. Balke & Hodges (1979) reported diethylstilbestrol as a specific inhibitor for the plasma membrane-associated ATPase.

The plasma membrane-rich fraction showed enrichment in the activity of UDPG: sterol glucosyltransferase. Our results are different from those of Lercher & Wojciechowski (1976) and Bowles, Lehle & Kauss (1977), who suggested that the enzyme was a convenient marker for the Golgi apparatus-rich fraction. Other data obtained for eukaryotic cells showed that glucosyltransferase activity could be found in the mitochondria (Ongun & Mudd, 1970) and in the microsomal fraction (Martin & Thorne, 1974). Thus, the role of UDPG:sterol glucosyltransferase as a specific marker enzyme is questionable.

In comparison with other membrane fractions, the plasma membranes are rich in sterols (Keenan, Leonard & Hodges, 1973). From our results, a rough estimation of sterol in the various isolated membrane fractions showed that the plasma membranerich fraction had the highest content. A high sterol concentration has been used as a chemical marker to identify the plasma membrane in plants (Hodges & Leonard, 1974; Hartmann & Benveniste, 1978). However, chemical constituents are not sufficiently restricted to any single type of membrane to be used as unambiguous markers.

Fraction 4 identified as the mitochondria-rich fraction showed enrichment in succinate dehydrogenase. This is in good agreement with other observations on plant tissues (Williamson, Morré & Jaffe, 1975). Succinate dehydrogenase activity was absent from the endoplasmic reticulum- and Golgi apparatus-rich fractions, but was found in both the plasma membrane- and mitochondria-rich fractions. This was probably due to contamination between these 2 fractions, as indicated by electron microscopy. Contamination was possible because of the similarity of mitochondrial and plasma membrane densities. In a preparation of the plasma membrane from maize roots, Leonard & Van Der Woude (1976) removed mitochondria from the homogenate by a low-speed differential centrifugation step (13000 g). When this method was attempted in our work, it resulted in major losses of plasma membrane vesicles. Plasma membrane vesicles may also be lost by selective entrapment in the cell wall residue during homogenization because of the close association between the plasma membrane and the cell wall (Bailey & Northcote, 1976).

In our work, although the various isolated membrane fractions showed enrichment in their specific marker enzymes, marker enzyme activity was also observed in other membrane fractions. If all endomembranes are functionally interconnected, then the presence of an enzyme on several components of the endomembrane system is to be expected. However, the results may not only be explained by differentiation of intracellular membranes, but also by contamination between the different membrane fractions during their isolation.

Diazotized sulphanilic acid has been used as a probe for identifying the plasma membrane in animal (Berg & Hirsh, 1975) and plant cells (Galbraith & Northcote, 1977). With maize roots, the radioactivity associated with the remainder fraction was very small, indicating that the compound did not penetrate the cells to label the internal proteins and membranes. Among the particulate fractions, although the plasma membrane-rich showed the highest specific radioactivity, there were significant amounts of radioactivity found in the mitochondria-rich fraction. This could be due to contamination since plasma membrane and mitochondria were difficult to separate. Although the result of this work is in good agreement with that of Galbraith & Northcote (1977), the cell wall remains a serious obstacle in using diazotized sulphanilic acid as a probe for labelling the plasma membrane of intact maize root cells in vivo as distinct from protoplasts.

Phosphatidylcholine has been found to be the major structural phospholipid in the eukaryotic cellular membranes (Phillips & Butcher, 1979). Incorporation of radioactive choline into the various isolated membrane fractions confirmed the results of Dykes, Kay & Harwood (1976) that radioactive choline was incorporated only into phosphatidylcholine during the first 48 h of germination in soybean. The endoplasmic reticulum has been identified as the major site for phospholipid synthesis in various plant and animal tissues (Lord, 1975; Jelsema & Morré, 1978; Quinn & Williams, 1978). However, the Golgi system has also been suggested to be involved in phospholipid synthesis (Montague & Ray, 1977). In our work, although the total activity found in the endoplasmic reticulum was higher than that of the Golgi apparatus-rich fraction, their specific activities were similar. The distribution of radioactivity also showed a decrease in the order endoplasmic reticulum-> Golgi apparatus-> plasma membrane-rich fraction among the various isolated components of the endomembrane system. This could be due to contamination among the isolated fractions or it could result from the transport of synthesized phosphatidylcholine from the endoplasmic reticulum-rich fraction to other membrane fractions. It has been proposed that the Golgi apparatus is involved in the transformation of membranes of the endoplasmic reticulum to the plasma membrane (Northcote, 1974; Morré, 1975).

Radioactive glucose was incorporated into the various chemical substances contributing to membrane composition and into the material within the lumen of the membranes. The Golgi apparatus-rich fraction showed an intermediary position in the distribution of radioactivity as compared to the endoplasmic reticulum- and plasma membrane-rich fractions.

The characteristic patterns of the polypeptide chains in the various isolated fractions were compared using SDS-polyacrylamide gel electrophoresis. Although the mitochondria-rich fraction was distinct from the other membrane fractions, the endoplasmic reticulum-rich fraction, the plasma membrane-rich fraction and the Golgi apparatus-rich fraction resembled one another. These results are consistent with the data published by other workers (Fleischer & Fleischer, 1970; Hodson & Brenchley, 1976). This similarity between the polypeptide chains of various membrane fractions may again be explained by contamination between the membrane fractions or, it may represent intra-cellular membrane turnover and differentiation (Northcote, 1979).

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