Hoppe-Seyler's Z. Physiol. Chem. Bd. 357, S. 177 - 186, Februar 1976

Plant Microbody Proteins, II. Purification and Characterization of the Major Protein Component (SP-63) of Peroxisome Membranes Bernd LUDWIG and Helmut KINDL Biochemie (Fachbereich Chemie), Universität Marburg/Lahn

(Received 31 October 1975)

Summary: The major component of membranes of microbodies from green leaves of Lens culinaris is a protein of a subunit molecular weight of 63000. This protein, referred to as SP-63, seems to be unique to microbodies and could not be detected when plastids or mitochondria were analyzed. It is probably a structural protein and is thus not solubilized by cholate, Triton X-100, chloroform/methanol, or 0.2M KC1. Solubiliza-

tion from purified membranes was achieved with guanidinium chloride or sodium dodecylsulphate. The protein was separated from minor contaminating components by chromatography on Sepharose 4 B or Sephadex G-150 employing 0.1 % sodium dodecylsulphate or 4M urea as eluent. It was shown to be homogeneous upon sodium dodecylsulphate gel electrophoresis and did not give a positive glycoprotein stain.

Proteine pflanzlicher Mikrokörper, H. Reingigung und Charakterisierung eines Strukturproteins (SP-63) aus Membranen von Blatt-Peroxisomen Zusammenfassung: Das Hauptprotein gereinigter lenfraktion festgestellt werden. Das Verhalten bei Mikrokörpermembranen aus Laubblättern von Ablösungsversuchen spricht für ein StrukturLens culinaris besitzt ein minimales Molekularge- protein: es wird durch Guanidiniumchlorid oder wicht von 63000. Dieses Protein (SP-63) konnte Natriumdodecylsulfat, nicht aber durch Cholat, außer in Mikrokörpern in keiner anderen Organel- Triton X-100, Chloroform/Methanol oder 0.2M Enzymes: Alcohol dehydrogenase, alcohol:NAD® oxidoreductase (EC 1.1.1.1); Carbonate dehydratase, carbonate hydro-lyase (EC 4.2.1.1); Catalase, hydrogen-peroxide: hydrogen-peroxide oxidoreductase (EC 1.11.1.6); Citrate (si)-synthase, citrate oxaloacetate-lyase (pro-3S-CH2' COOe->-acetyl-CoA) (EC 4.1.3.7); Exo-l,4-a-glucosidase, 1,4-a-D-glucanglucohydrolase (EC 3.2.1.3) (glucoamylase); Fumarate hydratase, L-malate hydro-lyase (EC 4.2.1.2); (3-Galactosidase, 0-D-galactoside galactohydrolase (EC 3.2.1.23); Glycollate oxidase, glycollate: oxygen oxidoreductase (EC 1.1.3.1); Hydroxypyruvate reductase, D-glycerate:NAD(P)®2-oxidoreductase (EC 1.1.1.81); Isocitrate lyase, i/zra>Ds-isocitrate glyoxylate-lyase (EC 4.1.3.1); Malate synthase, L-malate glyoxylate-lyase (CoA-acetylating) (EC 4.1.3.2); NADH-cytochrome c reductase, NADH: cytochrome c oxidoreductase (EC 1.6.? not listed); Phosphorylase, l,4-a-D-glucan:orthophosphate-a-glucosyltransferase (EC 2.4.1.1); Urate oxidase, urate:oxygen oxidoreductase (EC 1.7.3.3).

Brought to you by | Purdue Universit Authenticated Download Date | 6/5/15 4:05

178

B. Ludwig and H. Kindl

KC1 von der Membran abgelöst. Zur Reinigung eignet sich die Chromatographie über Sephadex G-l 50 oder Sepharose 4B mit 4M Harnstoff bzw. 0.1 proz. Natriumdodecylsulfat als Lösungsmittel.

Bd. 357 (1976)

So erhält man ein Protein, das sich bei Natriumdodecylsulfat-Gelelektrophorese als homogen erweist. Es handelt sich wahrscheinlich nicht um ein Glykoprotein.

Key words: Plant microbodies, peroxisomes, membrane protein, Lens culinaris.

Glyoxysomes house enzymes of glyoxylate cycle and fatty acid degradation, thus taking over part of the function ascribed to mitochondria in many other cellsl1'2'. Some of the more than twenty enzyme activities attributed to glyoxysomes are found to be associated with the organelle's membrane!3>4J. During the last few years the role of the microbody membrane in metabolic processes has become more and more interesting and will certainly be the subject of a great deal of study and discussion in the near future. It was, therefore, obvious to extend these studies to leaf peroxisomes, another specialized form of microbodies which is, together with chloroplasts, responsible for photorespiration in green leaves'2!. Investigations on the biogenesis or the differentiation of plant microbodies are hampered, at least in most cases, by the fact that the individual protein components are still not obtainable as pure entities. That applies also to typical membrane components. A knowledge of as many as possible of the components is necessary to study the assembling of the organelles, e.g. the time course of biosynthesis of matrix proteins compared to the timing of membrane formation. The current problems may be summarized in two questions.

Materials and Methods Plant material Lentil seeds (Lens culinaris L.) were soaked in tap water for 12 h and then germinated in vermiculite at 20 °C in the dark. After 5 days, the seedlings were grown under continous light using daylight fluorescence lamps. Cucumber seeds (Cucumis sativus L.) were germinated similarly at 20 °C for 5 days in the dark. 8 week-old leaves of sunflowers (Helianthus annuus L.) were obtained from a local field.

Leaf peroxisomes from Lens culinaris All operations were carried out at 4 °C if not stated otherwise. Sucrose concentrations are given as w/w. Preparations on a small scale were performed as described earlier' 51. Large quantities of microbodies were obtained as follows: 200 g (500 g) leaves (2 - 3 week old) of L. culinaris were homogenized in a chilled grinding medium as described earlier'6!. Bovine serum albumin was strictly omitted as it exhibits a subunit molecular weight close to that of the membrane protein to be characterized. The plant material was twice ground in a blendor with the sucrose medium at low speed for 20 sec, yielding 300 ml (800 m/) homogenate. A crude microbody-enriched fraction was isolated by a 40-min centrifugation at 14000 g of the supernatant resulting from the 10-min centrifugation (2000 g ) of the homogenate. The pellet was carefully suspended in grinding medium by means of a small brush and then made up to Firstly, whether glyoxysomes and leaf peroxisomes a density of 1.192 g/cm 3 by slowly adding 70% sucrose.

are surrounded by a biochemically similar or identical membrane; and secondly, whether comparisons are possible with other intracellular membrane systems. That would enable us to substantiate or to disprove speculations that (a) microbodies might originate from smooth endoplasmic reticulum via dilated cisternae or that (b) leaf peroxisomes derive directly from glyoxysomes during the drastic change from heterotrophic to photoautotrophic metabolism. The present paper deals with a structural protein unique to membranes of peroxisomes.

The sucrose solutions were pumped as follows into a Ti-14 zonal rotor spinning at 3000 rpm. An Isco gradient former (Dialagrade No. 380; Instumentation Specialties Co., Lincoln, Nebr., USA) was used with 30% and 60% sucrose solutions containing 40mM Tris/HCl, pH 7.5. The following settings were used: 0, 15, 30, 45, 50, 53, 56, 60, 65, 70, 100. The How rate was adjusted to 750 ml per h; the programme lasted for 40 min. After pumping 100 ml 30% sucrose, the programme was started and continued until 43% sucrose was reached. At this point the programme was stopped and the rotor inlet was switched to another pump which layered 70 ml organelle suspension (d = 1.192 g/cm3) underneath the

Brought to you by | Purdue Universit Authenticated Download Date | 6/5/15 4:05

Bd. 357 (1976)

Plant Microbody Proteins, II.

already existing gradient. Then the remaining part of the gradient was introduced by the dialagrade pump, continuing the programme. Density gradient centrifugation was carried out for 10 h at 47 000 rpm. After the run, the gradient was pushed out by addition of 60% sucrose at the edge, fractionated and assayed for marker enzymes and sucrose density. When the crude organelle mixture is introduced into the gradient at a density corresponding to 43% sucrose, the bulk of its components, the thylakoids, are forced to float towards the core of the rotor while peroxisomes sediment to their equilibrium densities of 1.235 g/cm3. In some experiments the pellet resulting from centrifugation at 14000 χ g was suspended in grinding medium and then the density of the suspension slowly increased up to 1.245 g/cm 3 by adding 70% sucrose. In this case, all organelles must float in order to reach their equilibrium densities and the microbodies are soon separated from the thylakoids. Preparation of other microbodies Microbodies from cotyledons of C. sativus^\ or leaves of H. annuus^ were prepared by well described methods and characterized by marker enzymes. Glyoxysomes from C. sativus banded at an equilibrium density of 1.25 g/cm 3 (mitochondria at 1.20 g/cm 3 ); peroxisomes from green leaves of H. annuus sedimented to a density of 1.245 g/cm 3 (mitochondria 1.21 g/cm3). Isolation ofmicrobody membranes Microbody membranes were prepared from whole organelles by diluting the fractions highest in catalase activity to a sucrose concentration of 30% and a subsequent centrifugation in a Spinco 50-Ti rotor at 42000 rpm for 40 min. In the case of glyoxysomes, the pellet was resuspended in 50mM MgCl2 and finally sedimented for 30 min at 42000 rpm. Enzyme assays Catalase, fumarate hydratase, citrate synthase, isocitrate lyase, malate synthase, and glycollate oxidase were assayed according to documented methods as summarized elsewhere^6!. NADH-cytochrome c reductase activity was measured according ίο^ 9 '. χηε amount of protein was determined by the method of Lowry et aU101. When necessary, the values obtained were corrected for sucrose concentration^111. Solubilization of membrane proteins Purified membranes (about 100 Mg of protein) were suspended in 50mM phosphate buffer containing one of the various detergents or salts to be tested and were treated for 30 min at 4 °C in a total volume of 500 μ/.

179

The membranes were then pelleted by centrifugation at 100000 χ g for 30 min, suspended in buffer and exhaustively dialysed against 0.1% sodium dodecylsulphate in 25mM sodium phosphate buffer, pH 7.5. The supernatant of the high speed run was subjected to dialysis in the same way. Both the membrane suspension and the solution containing the solubilized proteins were concentrated to a final volume of 20 μ/ or to dryness and then dissolved in 20 μ/ of sodium dodecylsulphate/urea medium (see gel electrophoresis). Gel filtration of membrane proteins Microbody membranes containing about 30 mg of protein were solubilized with sodium dodecylsulphate (see below) and applied to a column (r = 1.4 cm, Λ = 60 cm) with Sepharose 4B which had previously been equilibrated with 25mM sodium phosphate containing 0.1% sodium dodecylsulphate. India ink, as used for drawing, marked the front. A flow rate of 20 ml per h was adjusted. The eluent was identical with the equilibration buffer. Re-concentrating of protein solutions Concentration of solutions in sodium dodecylsulphate was achieved by precipitation with acetone (yielding a final concentration of 80%, v/v), by Ultrafiltration with Minicon B15 cells or by lyophilization. Frequently, diluted solutions were dialysed in Visking bags and then concentrated by surrounding these bags with dry Sephadex powder. The use of polyethylene glycol caused interferences and insufficient migration in the gel; upon addition of polyethylene glycol proteins consistently migrated at altered velocities and the zones became more diffuse. The proteins were also concentrated electrophoretically according to Stephens^12!. Gel electrophoresis Membrane samples or concentrated protein solutions containing about 100 μg of protein were dissolved in 20 μ/ of the following extraction medium: 25mM sodium phosphate (pH 7.2), 2% sodium dodecylsulphate, 8M urea, and 1 % mercaptoethanol. In some cases the membranes were delipidated prior to use, either with chloroform/methanol (2:1, v/v) or with acetone (80%, v/v). The incubation of the samples with the extraction medium was in a tube in a 100 °C bath for 2 min. Then, sucrose (to a final concentration of 15%) and bromophenol blue were added. The gel (3 mm thick) was prepared in a slab gel apparatW13! by mixing the following components according tol14'15!; 17.5 m/ 0.2M sodium phosphate (pH 7.2) containing 0.8% sodium dodecylsulphate, 10.0 m/^,7V,^',^v"-ietramethylethylenediamine (1%), 23.4 ml of a mixture containing acrylamide (28.5%)

Brought to you by | Purdue Universit Authenticated Download Date | 6/5/15 4:05

180

B. Ludwig and H. Kindl

and^,^"-methylenebis(acrylamide) (1.5%), 5.0 ml sodium sulphite (1%) and 78.5 ml H2O. Finally, 5.6 ml ammonium persulphate (1%) was added and the mixture deaerated. Usually, electrophoresis was performed at 100 mA and 150 V at 15 °C and was terminated when the bromophenol blue tracking dye had migrated 15 cm (about 4 h). Immediately after the electrophoresis the front was labelled with India ink and the gels were stained for protein with Coomassie Brilliant Blue R250. Excess dye was removed by diffusion. Carbohydrate-containing proteins were stained according to^ 16 L In this case glucoamylase from Rhizopus niveus was used as test. A series of marker proteins with known subunit molecular weightt14'15! was co-electrophoresed with the membrane samples: 0-galactosidase (Escherichia coli): 130000; phosphorylase (muscle): 96000; serum albumin (bovine): 68000; catalase (liver): 58000; alcohol dehydrogenase (yeast): 37000; carbonate dehydratase (erythrocytes): 29000; cytochrome c (muscle): 12400. The calibration curve of log subunit molecular weight against relative mobility was found to be linear over a range of at least 25 000 -100000.

Results

1) Separation of organelles and purification of membranes Several plants were tested with respect to their microbodies and, especially, to the microbody membranes. Surveying different microbody membranes for characteristic features, e.g. structural proteins, preliminary studies gave a first hint that membranes of leaf peroxisomes provide a better material for isolating certain pure protein components than glyoxysomes do; the membranes of the latter comprise too many proteinaceous components. The membrane of peroxisomes from leaves of L. culinaris appeared to be especially suited for the studies intended.

t 2

I •S

5 °*

/V\

ι /V'

»ί 40

1

Bd. 357 (1976)

t

!§ . · · * * . · . ,f/ l ^ '' .·

3000 —

\ j • f\vj \\ · "'" / """*"··,\ . .

5l 1

:·''''·!

10 Fract. no. ->

1000

s

&

20

Fig. 1. Distribution of catalase on a sucrose gradient centrifuged in a zonal rotor. In this representative example, the crude organelle fraction prepared from 200 g leaves of L. culinaris was entered into the gradient as suspension in 43% sucrose (i). The sucrose gradient formed, as described in Materials and Methods, was centrifuged for 10 h at 47 000 rpm in a Ti-14 zonal rotor. TH: position of thylakoids; M: position of mitochondria, ··· sucrose; ··· catalase; *** protein. Further data not included in the diagramme: Fumarate hydratase activity (U per fraction) 0.2 (Fr. 10), 0.5 (Fr. 12), 0.8 (Fr. 13), 0.3 (Fr. 14), 0.1 (Fr. 15); chlorophyll G4 660 /0.1 m/) 0.4 (Fr. 9), 0.6 (Fr. 10), 0.2 (Fr. 11), 0.2(Fr. 12).

suspension of the 14000 χ g pellet and during the following operations scarcely migrate under these conditions and should therefore be localized around 43% sucrose. With respect to the organelles, this type of gradient centrifugation includes flotation (thylakoids) as well as sedimentation (microbodies). Another example which enables one to separate even higher amounts of microbodies from the other organelles is presented in Fig. 2. In this case the crude microbody Fig. 1 shows the preparation of large quantities of enriched pellet, though originally suspended in microbodies from L. culinaris leaves using a zonal 20% sucrose medium, was brought to 51.5 % sucrose and then layered into the corresponding rotor. The distribution of marker enzymes in the gradient of this large-scale preparation indicates a position during the filling of the zonal rotor. The separation of peroxisomes (at densities of about utilization of a large Ti-15 zonal rotor allows the 1.23 g/cm3) from mitochondria (about 1.20 g/ preparation of more than 100 mg peroxisomal 3 3 cm ) and thylakoids (about 1.17 g/cm ). This is protein. After the flotation the soluble proteins an example of the procedure used for routine originating from broken organelles are ascribed work; here, the crude organelle preparation is to the fractions 60 - 68, corresponding to 51% applied as suspension in 43% sucrose. Any soluble sucrose, while peroxisomes are positioned at 48 - 49% sucrose in this very shallow gradient. proteins released by damage to organelles during

Brought to you by | Purdue Universit Authenticated Download Date | 6/5/15 4:05

Plant Microbody Proteins, II.

Bd. 357 (1976)

Independently, Fig. 3 presents a correlation between another gradient, but comparable to that in Fig. 1, and the electrophoretic patterns of the membrane proteins of the corresponding fractions. While fractions (No. 9, 10) consisting of microbodies are almost free of contaminating organelles, the fractions with slightly lower densities (No. 8, 7, 6, 5) contain mitochondrial markers besides catalase. These findings are paralleled by profiles of membrane-bound proteins obtained from the corresponding membranes. The sodium dodecylsulphate gel electrophoresis clearly demonstrates that membrane preparations originating from organelles banding at 1.215 1.230 g/cm3 contain primarily a protein with a subunit molecular weight of 63000 (SP-63); the lower the equilibrium density of the whole organelles (down to 1.190 g/cm3), the more prominent another protein, molecular weight 56000, and the smaller the proportion of SP-63. Thus, the maximum of membrane protein SP-63 corresponds to the maximum of microbody markers, while protein at 56 000 is attributable to mito2000

Iι·!·i ' .···1 ··' "

t

talase act.

S

TH' >M/ i' -·A\ iV / 76

52

48

64

Fract. no. ->

-50 t

4 .40-

.20

'80

Fig. 2. Preparation of peroxisomes by flotation gradient centrifugation in a Ti-15 rotor. Prior to centrifugation, the microbody-enriched fraction obtained from 5 00 g of leaves was brought to 51.5% sucrose and then introduced in a gradient (;) at the corresponding position. The centrifugation was performed for 15 h at 32 000 rpm. Fractions of 20 ml each were tested for enzyme activities spectrophotometrically. Symbols as in Fig. 1. Further data not included in the diagramme: Fumarate hydratase activity (U per fraction) 0.1 (Fr. 30), 0.4 (Fr. 34), 1.2 (Fr. 36), 1.3 (Fr. 38), 1.3 (Fr.40), 0.7 (Fr. 42), 0.2 (Fr. 44); chlorophyll C4660/0.1 m/) 0.3 (Fr. 16); 0.7 (Fr. 20), 0.9 (Fr. 24), 0.2 (Fr. 28).

181

chondria. Thylakoids and chloroplasts are well below these equilibrium densities. When the electrophoretic patterns of fractions obtained by common differential centrifugation of crude homogenates are compared, it can be seen that SP-63 represents a considerable percentage of the total protein in the 14000 χ g pellet, which contains a mixture of thylakoids, mitochondria, peroxisomes and various membranes. Purified membrane fractions (e.g. Fig.3/No. 11) do not contain enzyme activities, except a very low antimycin Α-insensitive NADH-cytochrome c reductase (5 mU/mg protein). Neither malate synthase, which is very tightly bound to membranes of glyoxysomes, nor significant amounts of catalase were detectable. 2) Solubilization and solubility of SP-63 A series of treatments was carried out to clarify the question of how SP-63 is bound to the microbody membrane. Fig.4 summarizes the solvent systems which can be used to solubilize SP-63 or other membrane proteins. It becomes evident from these experiments that cholate (0.1%), Triton X-100 (0.1%), n-butanol or acetone (not shown), chloroform/methanol (2-1, v/v), or KC1 at concentrations up to 0.2M do not solubilize SP-63, but in certain cases do separate other proteins from the membrane. Thus, the mitochondrial protein with molecular weight 56 000 can be readily eliminated from the partially purified microbody membranes by treatment with Triton X-100 or 0.2M KC1. Increasing the ionic strength leads to a partial solubilization of SP-63 even with salts: treatment with 0.5M or I.OM KC1 removes approximately 10% of the SP-63 bound to the membrane. In order to test the solubility of SP-63 in various solvents, microbody membranes were at first delipidated with acetone or chloroform/methanol (2-1, v/v). The insoluble protein pellet thus obtained was processed with sodium dodecylsulphate (1 %), KC1 (0.2M), Triton X-100 (0.1 %), guanidinium chloride (OM) or urea (8M). Only sodium dodecylsulphate, guanidinium chloride and urea were suitable solvents; the same systems were capable of directly solubilizing SP-63 from intact membranes.

Brought to you by | Purdue Universit Authenticated Download Date | 6/5/15 4:05

182

B. Ludwig and H. Kindl

Bd. 357 (1976)

sp63

Fig. 3. Correlation between the enzyme distribution on a sucrose density gradient and the electrophoretic patterns of the membrane proteins of the respective fractions. After fractionation of the gradient, 12 samples were selected according to enzyme distribution and processed individually for the electrophoretic separation of membrane proteins on SDS gels as detailed in "Materials and Methods", For the determination of chlorophyll, portions of the gradient fractions were diluted with 2 vol. of water; absorption was measured at 660 nm in a Gilford spectrophotometer. M, marker proteins on the gel, were as follows: a) /3-galactosidase (E. coli; 130000); b) phosphorylase (muscle; 96000); c) serum albumin (bovine; 68000); d) catalase (liver; 58000); e) alcohol dehydrogenase (yeast; 37000); f) carbonate dehydratase (erythrocytes; 29000); g) cytochrome c (muscle; 12400); Sample no.

Catalase

Fumarate hydratase

Membrane protein

lg/cm ]

[U/m/1

[mU/m/]

[mg/m/]

1.198

0 4 6 65 82

6 13 27 30 36

0.3 6.6 11.0 2.4 1.8

120 205 230 250 260 150 48

23 12 6 2 2 2 2

Equilibrium density [% sucrose]

1 2 3 4 5

35.0 39.5 41.5 43.5 44.5

6 7 8 9 10 11 12

46.0 47.0 48.0 49.0 50.0 51.5 53.0

3

1.209 1.220 1.230

< < <