Planta
Planta 142, 9 1 - 9 5 (1978)
9 by Springer-Verlag 1978
Isolation and Partial Characterization of Pyrenoids from the Brown Alga Pilayella littoralis (L.) Kjellm. N.W. Kerby and L.V. Evans Department of Plant Sciences, University of Leeds, Leeds LS2 9JT, U.K.
Abstract. In order to isolate high yields of pyrenoids
from the brown alga Pilayella littoralis it is necessary to pretreat them with 0.1% HgC12 in sea water for 3 h. Without this pretreatment there is a substantial loss of pyrenoid ground substance and yields are low. Pyrenoid fractions of high purity have been obtained using silica sol gradients. A partial characterization has shown the pyrenoid to be proteinaceous and lacking chlorophyll. SDS polyacrylamide gel electrophoresis has shown that the majority of protein present is accounted for by two polypeptides which resemble the large and small subunits of ribulose-l,5bisphosphate carboxylase (EC 4.1.1.39). Key words: Pilayella -
Pyrenoids bisphosphate carboxylase.
Ribulose-l,5-
having a high specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase). The presence of ribose-5-phosphate isomerase and ribulose-5-phosphate kinase in the pyrenoid extracts was also noted. In the present communication a method is reported for isolating pyrenoids from the brown alga Pilayella littoralis, and a preliminary characterization of the extracted bodies is given.
Materials and Methods Pilayella littoralis ( L.) Kjellm. Material was collected from Filey Brig, North Yorkshire and around Holy Island, Anglesey. Prior to storage at 4 ~ in filtered sea water the material was thoroughly washed and cleaned.
Pyrenoid Isolation
Introduction
Pyrenoids are associated with the chloroplasts of many algae and some species of liverworts, (see reviews Griffiths, 1970; Dodge, 1973). Although they have attracted a lot of attention from microscopists little physiological work has been carried out, so that the nature and function of pyrenoids is still unclear, despite some speculation. It has been shown by histochemical stains that the bulk of the pyrenoid ground substance consists of protein (Simon, 1954; Brown and Arnott, 1970) and the work of Holdsworth (1971) on isolated pyrenoids from the green alga Eremosphaera viridis further showed that about 90% of the protein present was similar to Fraction I protein in Abbreviations." D T T = d i t h i o t h r e i t o I ; H E P E S - N - 2 - h y d r o x y e t h y l piperazine-N1-2-ethanesulfonic acid; P E G - p o l y e t h y l e n e glycol; PVPP = polyvinylpolypyrrolidone; RuBP = ribulose- 1,5-blsphosphate; R u B P C a s e = r i b u l o s e - l , 5 - b i s p h o s p h a t e carboxylase; SDS = s o d i u m dodecyl sulphate.
The alga was incubated in 0.1% HgC12 in filtered sea water at 4 ~ for 3 h, washed repeatedly in sea water and gently homogenized in a prechilled pestle and mortar in buffered grinding medium (GM) containing 25 m M H E P E S - N a O H pH 6.8 ; 0.15 M sorbitol; 0.I5 M sucrose; 1 m M MgCI2; 1 m M MnC12; 1 m M D T T and 3% PVPP. The homogenate was then passed through four layers of muslin, centrifuged at 100 x g for 3 min and the resulting supernatant centrifuged at 1500 x g for 15 min. The pellet was resuspended in 5 cm 3 of G M and layered onto a discontinuous gradient as described below. All steps were carried out at 4 ~ C unless otherwise stated. Ludox A M (E.I. du Pont de N e m o u r s and Co.), a silica sol, was purified as described by Morgenthaler et al. (1974) and 10% (w/v) P E G (6000) then added. The density of Ludox/PEG/ G M was determined gravimetrically and a calibration curve of density vs. refractive index was constructed after Walbot (1977).
Density Gradient Centrifugation The discontinuous L n d o x / P E G / G M gradient which consisted of two steps, 30% and 70% (v/v) Ludox: PEG, was centrifuged at 1500 x g for 30 min. The interface was then diluted with GM, layered onto a 28 cm 3 linear gradient of 30 70% (v/v) Lndox: P E G (with the same concentration and composition of G M throughout) and
0032-0935/78/0142/0091/$01.00
92 centrifuged at 25000xg for 1 h using an 8• 3 rotor in a MSE-'Superspeed'65. Gradients were fractionated by pumping 70% (w/v) sucrose into the bottom of the tubes and approximately 0.7 cm 3 fractions were collected.
N.W. Kerby and L.V. Evans: Brown Algal Pyrenoids Sigma, London or the British Drug Houses, Poole, Dorset at the purest grades commercially available.
Results Light and Electron Microscopy Fresh material was either examined directly, or, after staining with mercuric bromophenol blue (0.I% HgCI2, 0.05% bromophenol blue in sea-water) for 3 h followed by rinsing in sea water. Gradient fractions were diluted with GM and centrifuged at 3000 x g for 15min. Pellets were fixed in 4% glutaraldehyde in a 0.1 M cacodylate buffer (pH 7.0) containing 0.25 M sucrose and post-osmicated, as described by Evans and Holligan (1972). After embedding in Epon 812 sections cut with a Reichert 'OMU2' were stained with uranyl acetate and lead citrate (Reynolds, 1963) and examined with a Siemens 102 electron microscope at 80 kv.
Purification of RuBPCase Fresh material was homogenized in an ice-cold medium (HM) containing 50mM Tris-HC1 pH 8.0; 2raM EDTA Na2; 3ram DTT and 3% PVPP and then passed through four layers of muslin and centrifuged at 100,000 x g for l h. Proteins present in the supernatant were precipitated with 65% (NH,)zSO 4 saturation, and were dissolved in 5 cm 3 HM without PVP and layered onto a Sephadex G-200 column (Z2 x 45 cm). Fractions were collected and assayed for RuBPCase after Callow (1974), by incorporation of NaH14CO3 into acid stable products. Assays were preincubated at 25 ~ C for 10 min before adding RuBP. After the reaction was stopped by the addition of 1 cm3 1 N HC1 and after incubation overnight at room temperature (to remove the unused NaH14CO3) aliquots were counted for 14C incorporation by standard scintillation methods, using a Nuclear Chicago Unilux 11 scintillation counter.
Polyacrylamide Gel Elctrophoresis Purified RuBPCase was tested electrophoretically by the method of Davies (1964), omitting spacer gels, using 7.5% acrylamide. The quaternary structure of purified RuBPCase and pyrenoid protein was investigated after incubation at 37 ~ C for 3 h, or by boiling for 10 min in 10 mM phosphate buffer (pH 7.0) containing 1% (w/v)SDS and 1% (v/v) ~-mercaptoethanol. Electrophoresis of samples containing 50=-100 lag protein was performed according to Weber and Osborne (1969) using gels polymerized from 10% acrylamide at a constant current of 5 mA/tube. Molecular weights of polypeptides were determined using BDH molecular weight markers (MW range 14,300-71,500). Gels were stained using Coomassie blue G 250 after Fairbanks et al. (1971) and were destained using 10% acetic acid.
Other Measurements Protein concentration was measured according to Lowry et al. (1951), using bovine serum albumin as a standard, and chlorophyll concentration according to Arnon (1949).
Chemicals NaH14CO3 was purchased from the Radiochemical Centre, Amersham, Bucks., and all other chemicals were purchased either from
The pyrenoid of Pilayella is typical of this group of algae, being a stalked body protruding from the chloroplast (Fig. 2) bounded by three separate pairs of membranes. The innermost pair are in continuity with the chloroplast envelope, the next pair are in continuity with the chloroplast endoplasmic reticulum (CER) and the outermost pair are sometimes dilated and form a cap terminating in the region of the pyrenoid neck. No thylakoids enter the ribosome-free pyrenoid core. The pyrenoid of Pilayella appears colourless when seen under the light microscope (Fig. 1), and stains deeply with mercuric bromophenol blue, a stain shown by Mazia et al. (1953) to be specific for proteins. When viewed by fluorescence microscopy the pyrenoid is not observed, indicating that the core does not contain chlorophyll. The structure of brown algal pyrenoids is described by Bouck (1965) and Evans (1966, 1968). Since pyrenoids are easily disrupted during gentle homogenization and the cores are readily solubilized during purification procedures, pretreatement with HgC12 is necessary to stabilize the pyrenoid core. Following this treatment, pyrenoid preparations of a high degree of purity may be obtained when silica sol gradients are employed (Fig. 3), and isolated pyrenoids can be recognised with the electron microscope by the characteristic granular appearance of the core material and by the pairs of membranes surrounding the core (Fig. 4). The pyrenoid sac is lost during isolation, and in some cases portions of the CER are lost also, but the pyrenoid is usually bounded by at least one pair of membranes. In a preliminary investigation of the pyrenoid core, gradient fractions were prepared for microscopic examination and it was found that the greatest number of pyrenoids were present in fractions 41-47, i.e. they band at a density of 1.135cm -3. Protein and chlorophyll measurements of gradient fractions reveal a protein peak in this region which is characterized by the absence of chlorophyll (Fig. 5). Maximum chlorophyll was recorded in fractions 13-15 and these were found to be composed mainly of chloroplasts. Following treatment with 1% SDS to solubilize the pyrenoid core, the majority of protein present is accounted for by two polypeptides, as revealed by SDS polyacrylamide gel electrophoresis (Fig. 6). The ratio of peak area of the larger (1) to the smaller (2) peak is approximately 2: 1. A third (3) smaller peak was evident just in front of the larger of the
N.W. Kerby and L.V. Evans: Brown Algal Pyrenoids
93
Figs. 14. Vegetative filament of Pilayella showing discoid chloroplasts and pyrenoids (arrows). Light micrograph • 600. 2. Stalked pyrenoid of Pilayella showing continuity of membranes around pyrenoid and chloroplast. Electron micrograph (EM) x 37,500.3. Isolated pyrenoids in fraction 43 after 0.1% HgCl~ pretreatment and fractionation on silica sol gradients. EM x4000. 4. Isolated pyrenoids in fraction 43 showing remains of chloroplast endoplasmic reticulum (two arrows) and presence of chloroplast envelope (single arrow). EM x 30,000
two m a j o r p o l y p e p t i d e s . T h e p e a k r e c o r d e d in front o f the origin p r o b a b l y represents u n d i s s o c i a t e d proteins, as it is n o t present in gels where the s a m p l e was b o i l e d for 10 min in the presence o f SDS. O t h e r b a n d s a p p e a r to be p r e s e n t in the centre p o r t i o n o f
the gel b u t the b a n d s are very faint a n d n o t e v i d e n t in all gels in each o p e r a t i o n . T h e m o l e c u l a r weight estimates are 56,000, 14,000 a n d 50,000 for 1, 2 a n d 3 respectively. F o l l o w i n g p u r i f i c a t i o n o f R u B P C a s e f r o m Pi-
N.W. Kerby and L.V. Evans: Brown Algal Pyrenoids
94 3.0
--
5O
2.0
2O
o~ ~o
--
1.25
_
1.20
--
1.15
--
1.10
1.0 I0
0t,
10
2o
3o
4o . . . . Fr~c tion
To--
-T6""
A
.05
-7o
number
Fig. 5. Distribution of protein and chlorophyll in fractions collected from continuous silica sol gradients (20-70% Ludox/PEG/HM) after centrifugation at 25,000 xg, 1 h. Pilayella pretreated with 0.1% HgCI2 3 h. 9 9 chlorophyll concentration (gg cm-a). A-A protein concentration (lag c m - 3). _.... density Ludox/PEG/HM ( g c m 3)
[5511
IZSZ
7-1125115
1
tfa
c B
2~ Origin
5'0
f5 BPB
Fig. 6. S D S polyacrylamide gel electrophoresis. Gel and corresponding scan at 575 n m of pyrenoid fraction obtained by pretreatment with 0.1% HgC12 and Ludox/PEG/HM density gradient centrifugation. Peaks 1, 2 and 3 represent major polypeptides. BPB (bromophenol blue)
u-x
-g
;4 i
25 5O Distance (m 0
layella on Sephadex G-200, fractions collected from the column were assayed for carboxylase activity and only those showing such activity were used for studies on quaternary structure. The homogeneity of bulked fractions was tested by polyacrylamide gel electro-
Orig~
1
75 I BPB
Fig. 7. SDS polyacrylam~de gel electrophoresis. Gel and corresponding scan at 575 nm of RuBPCase purified on Sephadex G-200. A, B and C represent major polypeptides. BPB (bromophenol blue)
N.W. Kerby and L.V. Evans: Brown Algal Pyrenoids
phoresis and the protein present was found to migrate as a single band. The purified RuBPCase was treated with SDS and run on polyacrylamide gels containing SDS and 3 major peaks were observed (Fig. 7). Peaks A, B and C closely resemble peaks 1, 2 and 3 respectively in Figure 6. The molecular weights are 55,000 (peak A), 15,000 (peak B) and 50,000 (peak C). The molecular weights of peaks A and B correspond closely to the reported values of RuBPCase from other organisms (Jensen and Bahr, 1977).
Discussion
The data presented here show that pyrenoids can be isolated successfully from vegetative cells of Pilayella littoralis, after pretreatment with 0.1% HgC12. Without this pretreatment loss of core material occurs to a greater or lesser extent and hence yields are very low. The results from the preliminary characterization indicate that the pyrenoid core is proteinaceous and does not contain chlorophyll. This is consistent with the findings of previous authors (Brown and Arnott, 1970; Holdsworth, 1971). SDS polyacrylamide gel electrophoresis has revealed that two polypeptides account for the majority of the protein present in the pyrenoid core and the relative mobilities of these resemble those of the large and small subunits of RuBPCase. When RuBPCase was purified and the quaternary structure investigated, three major peaks were recorded. If the pyrenoid core contains RuBPCase, a purified carboxylase fraction would be expected to contain the enzyme from both the chloroplast stroma and the pyrenoid core. One possibility to account for the third peak is that the large subunit in the pyrenoid may be different from the large subunit in the chloroplast stroma. Peak 3 (Fig. 6) which corresponds closely to peak C (Fig. 7) and which is much reduced in the pyrenoid extract, could represent contamination by stromal carboxylase derived from chloroplast fragments. The other possibility is that peak C may be due to the action of a protease on the RuBPCase (Ellis, 1973). This would not occur in the pyrenoid preparation due to the pretreatment with HgC12. Further work is in progress and it is hoped that a more complete picture of the subunits of RuBPCase in this alga will emerge. As HgClz is used, direct assays for carboxylase activity are not possible at present. However confirmation that the two major polypeptides found in pure pyrenoid extracts are the subunits of RuBPCase should be possible by the use of immunological techniques.
95 The authors wish to thank Dr. Maureen E. Callow for valuable advice on many aspects of this project. The work was made possible through research support to N.W.K. from the Science Research Council.
References Arnon, D.I. : Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1 15, (1949) Bouck, G.B.: Fine structure and organelle associations in brown algae. J. Cell Biol. 26, 523 537 (1965) Brown, R.M., Arnott, H. J.: Structure and function of the algal pyrenoid. 1. Ultrastructure and cytochemistry during zoo-sporogenesis of Tetracystis excentrica. J. Phycol. 6, 14 22 (1970) Callow, J.A.: Ribosomal RNA, Fraction I protein synthesis and ribulose diphosphate carboxylase activity in developing and senescing leaves of cucumber. New Phytol. 73, I3-20 (1974) Davies, B.J.: Disc electrophoresis. II. Method and application to human proteins. Ann. N.Y. Acad. Sci. 121, 404-427 (i964) Dodge, J.D.: The fine structure of algal cells. London: Academic Press 1973 Ellis, R.J. : Fraction I protein. Comment. Plant Sci. 4, 29 36 (1973) Evans, L.V. : Distribution of pyrenoids among some brown algae. J. Cell Sci. 1,449-454 (1966) Evans, L.V. : Chloroplast morphology and fine structure in some British fucoids. New Phytol. 67, 173-178 (1968) Evans, L.V., Holligan, M.S.: Correlated light and electron microscope studies on brown algae. 1. Localization of alginic acid and sulphated polysaccharides in Dictyota. New Phytol. 71, 1161 I172 (1972) Fairbanks, G., Stech, T.L., Wallach, D.F.H. : Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-2617 (1971) Griffiths~ D.J. : The pyrenoid. Bot. Rev. 36, 29 58 (1970) Holdsworth, R.H.: The isolation and partial characterization of the pyrenoid protein of Eremosphaera viridis. J. Cell Biol. 15, 499-513 (1971) Jensen, R.G., Bahr, J.T. : Ribulose 1,5-bisphosphate carboxylaseoxygenase. Ann. Rev. Plant Physiol. 28, 379400 (1977) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265 275 (1951) Mazia, D., Brewer, P.A., Alfert, M.: The cytochemicaI staining and measurement of protein with mercuric bromophenol blue. Biol. Bull. mar. biol. Lab. (Woods Hole) 104, 57 67 (1953) Morgenthaler, J.-J., Price, C.A., Robinson, J.M., Gibbs, M. : Photosynthetic activity of spinach chloroplasts after isopycnic centrifugation in gradients of silica. Plant Physiol. 54, 532 534 (1974) Reynolds, E.S. : The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208 213 (1963) Simon, M.F. : Recherches sur les pyrenoids des ph+ophyc6es. Rev. Cytol. Biol. V~g. 15, 73 105 (1954) Walbot, V.: Use of silica sol step gradients to prepare bundle sheath and mesophyll chloroplasts from Parnicium maximum. Plant Physiol. 60, 102 108 (1977) Weber, K., Osborne, M.: The reliability of molecular weight determinations by dodecyl-sulphate polyacrylamide gel electrophoresis. J. biol. Chem. 244, 44064412 (1969)
Received 3 April; accepted 3 May 1978
Planta 142, 9 1 - 9 5 (1978)
9 by Springer-Verlag 1978
Isolation and Partial Characterization of Pyrenoids from the Brown Alga Pilayella littoralis (L.) Kjellm. N.W. Kerby and L.V. Evans Department of Plant Sciences, University of Leeds, Leeds LS2 9JT, U.K.
Abstract. In order to isolate high yields of pyrenoids
from the brown alga Pilayella littoralis it is necessary to pretreat them with 0.1% HgC12 in sea water for 3 h. Without this pretreatment there is a substantial loss of pyrenoid ground substance and yields are low. Pyrenoid fractions of high purity have been obtained using silica sol gradients. A partial characterization has shown the pyrenoid to be proteinaceous and lacking chlorophyll. SDS polyacrylamide gel electrophoresis has shown that the majority of protein present is accounted for by two polypeptides which resemble the large and small subunits of ribulose-l,5bisphosphate carboxylase (EC 4.1.1.39). Key words: Pilayella -
Pyrenoids bisphosphate carboxylase.
Ribulose-l,5-
having a high specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase). The presence of ribose-5-phosphate isomerase and ribulose-5-phosphate kinase in the pyrenoid extracts was also noted. In the present communication a method is reported for isolating pyrenoids from the brown alga Pilayella littoralis, and a preliminary characterization of the extracted bodies is given.
Materials and Methods Pilayella littoralis ( L.) Kjellm. Material was collected from Filey Brig, North Yorkshire and around Holy Island, Anglesey. Prior to storage at 4 ~ in filtered sea water the material was thoroughly washed and cleaned.
Pyrenoid Isolation
Introduction
Pyrenoids are associated with the chloroplasts of many algae and some species of liverworts, (see reviews Griffiths, 1970; Dodge, 1973). Although they have attracted a lot of attention from microscopists little physiological work has been carried out, so that the nature and function of pyrenoids is still unclear, despite some speculation. It has been shown by histochemical stains that the bulk of the pyrenoid ground substance consists of protein (Simon, 1954; Brown and Arnott, 1970) and the work of Holdsworth (1971) on isolated pyrenoids from the green alga Eremosphaera viridis further showed that about 90% of the protein present was similar to Fraction I protein in Abbreviations." D T T = d i t h i o t h r e i t o I ; H E P E S - N - 2 - h y d r o x y e t h y l piperazine-N1-2-ethanesulfonic acid; P E G - p o l y e t h y l e n e glycol; PVPP = polyvinylpolypyrrolidone; RuBP = ribulose- 1,5-blsphosphate; R u B P C a s e = r i b u l o s e - l , 5 - b i s p h o s p h a t e carboxylase; SDS = s o d i u m dodecyl sulphate.
The alga was incubated in 0.1% HgC12 in filtered sea water at 4 ~ for 3 h, washed repeatedly in sea water and gently homogenized in a prechilled pestle and mortar in buffered grinding medium (GM) containing 25 m M H E P E S - N a O H pH 6.8 ; 0.15 M sorbitol; 0.I5 M sucrose; 1 m M MgCI2; 1 m M MnC12; 1 m M D T T and 3% PVPP. The homogenate was then passed through four layers of muslin, centrifuged at 100 x g for 3 min and the resulting supernatant centrifuged at 1500 x g for 15 min. The pellet was resuspended in 5 cm 3 of G M and layered onto a discontinuous gradient as described below. All steps were carried out at 4 ~ C unless otherwise stated. Ludox A M (E.I. du Pont de N e m o u r s and Co.), a silica sol, was purified as described by Morgenthaler et al. (1974) and 10% (w/v) P E G (6000) then added. The density of Ludox/PEG/ G M was determined gravimetrically and a calibration curve of density vs. refractive index was constructed after Walbot (1977).
Density Gradient Centrifugation The discontinuous L n d o x / P E G / G M gradient which consisted of two steps, 30% and 70% (v/v) Ludox: PEG, was centrifuged at 1500 x g for 30 min. The interface was then diluted with GM, layered onto a 28 cm 3 linear gradient of 30 70% (v/v) Lndox: P E G (with the same concentration and composition of G M throughout) and
0032-0935/78/0142/0091/$01.00
92 centrifuged at 25000xg for 1 h using an 8• 3 rotor in a MSE-'Superspeed'65. Gradients were fractionated by pumping 70% (w/v) sucrose into the bottom of the tubes and approximately 0.7 cm 3 fractions were collected.
N.W. Kerby and L.V. Evans: Brown Algal Pyrenoids Sigma, London or the British Drug Houses, Poole, Dorset at the purest grades commercially available.
Results Light and Electron Microscopy Fresh material was either examined directly, or, after staining with mercuric bromophenol blue (0.I% HgCI2, 0.05% bromophenol blue in sea-water) for 3 h followed by rinsing in sea water. Gradient fractions were diluted with GM and centrifuged at 3000 x g for 15min. Pellets were fixed in 4% glutaraldehyde in a 0.1 M cacodylate buffer (pH 7.0) containing 0.25 M sucrose and post-osmicated, as described by Evans and Holligan (1972). After embedding in Epon 812 sections cut with a Reichert 'OMU2' were stained with uranyl acetate and lead citrate (Reynolds, 1963) and examined with a Siemens 102 electron microscope at 80 kv.
Purification of RuBPCase Fresh material was homogenized in an ice-cold medium (HM) containing 50mM Tris-HC1 pH 8.0; 2raM EDTA Na2; 3ram DTT and 3% PVPP and then passed through four layers of muslin and centrifuged at 100,000 x g for l h. Proteins present in the supernatant were precipitated with 65% (NH,)zSO 4 saturation, and were dissolved in 5 cm 3 HM without PVP and layered onto a Sephadex G-200 column (Z2 x 45 cm). Fractions were collected and assayed for RuBPCase after Callow (1974), by incorporation of NaH14CO3 into acid stable products. Assays were preincubated at 25 ~ C for 10 min before adding RuBP. After the reaction was stopped by the addition of 1 cm3 1 N HC1 and after incubation overnight at room temperature (to remove the unused NaH14CO3) aliquots were counted for 14C incorporation by standard scintillation methods, using a Nuclear Chicago Unilux 11 scintillation counter.
Polyacrylamide Gel Elctrophoresis Purified RuBPCase was tested electrophoretically by the method of Davies (1964), omitting spacer gels, using 7.5% acrylamide. The quaternary structure of purified RuBPCase and pyrenoid protein was investigated after incubation at 37 ~ C for 3 h, or by boiling for 10 min in 10 mM phosphate buffer (pH 7.0) containing 1% (w/v)SDS and 1% (v/v) ~-mercaptoethanol. Electrophoresis of samples containing 50=-100 lag protein was performed according to Weber and Osborne (1969) using gels polymerized from 10% acrylamide at a constant current of 5 mA/tube. Molecular weights of polypeptides were determined using BDH molecular weight markers (MW range 14,300-71,500). Gels were stained using Coomassie blue G 250 after Fairbanks et al. (1971) and were destained using 10% acetic acid.
Other Measurements Protein concentration was measured according to Lowry et al. (1951), using bovine serum albumin as a standard, and chlorophyll concentration according to Arnon (1949).
Chemicals NaH14CO3 was purchased from the Radiochemical Centre, Amersham, Bucks., and all other chemicals were purchased either from
The pyrenoid of Pilayella is typical of this group of algae, being a stalked body protruding from the chloroplast (Fig. 2) bounded by three separate pairs of membranes. The innermost pair are in continuity with the chloroplast envelope, the next pair are in continuity with the chloroplast endoplasmic reticulum (CER) and the outermost pair are sometimes dilated and form a cap terminating in the region of the pyrenoid neck. No thylakoids enter the ribosome-free pyrenoid core. The pyrenoid of Pilayella appears colourless when seen under the light microscope (Fig. 1), and stains deeply with mercuric bromophenol blue, a stain shown by Mazia et al. (1953) to be specific for proteins. When viewed by fluorescence microscopy the pyrenoid is not observed, indicating that the core does not contain chlorophyll. The structure of brown algal pyrenoids is described by Bouck (1965) and Evans (1966, 1968). Since pyrenoids are easily disrupted during gentle homogenization and the cores are readily solubilized during purification procedures, pretreatement with HgC12 is necessary to stabilize the pyrenoid core. Following this treatment, pyrenoid preparations of a high degree of purity may be obtained when silica sol gradients are employed (Fig. 3), and isolated pyrenoids can be recognised with the electron microscope by the characteristic granular appearance of the core material and by the pairs of membranes surrounding the core (Fig. 4). The pyrenoid sac is lost during isolation, and in some cases portions of the CER are lost also, but the pyrenoid is usually bounded by at least one pair of membranes. In a preliminary investigation of the pyrenoid core, gradient fractions were prepared for microscopic examination and it was found that the greatest number of pyrenoids were present in fractions 41-47, i.e. they band at a density of 1.135cm -3. Protein and chlorophyll measurements of gradient fractions reveal a protein peak in this region which is characterized by the absence of chlorophyll (Fig. 5). Maximum chlorophyll was recorded in fractions 13-15 and these were found to be composed mainly of chloroplasts. Following treatment with 1% SDS to solubilize the pyrenoid core, the majority of protein present is accounted for by two polypeptides, as revealed by SDS polyacrylamide gel electrophoresis (Fig. 6). The ratio of peak area of the larger (1) to the smaller (2) peak is approximately 2: 1. A third (3) smaller peak was evident just in front of the larger of the
N.W. Kerby and L.V. Evans: Brown Algal Pyrenoids
93
Figs. 14. Vegetative filament of Pilayella showing discoid chloroplasts and pyrenoids (arrows). Light micrograph • 600. 2. Stalked pyrenoid of Pilayella showing continuity of membranes around pyrenoid and chloroplast. Electron micrograph (EM) x 37,500.3. Isolated pyrenoids in fraction 43 after 0.1% HgCl~ pretreatment and fractionation on silica sol gradients. EM x4000. 4. Isolated pyrenoids in fraction 43 showing remains of chloroplast endoplasmic reticulum (two arrows) and presence of chloroplast envelope (single arrow). EM x 30,000
two m a j o r p o l y p e p t i d e s . T h e p e a k r e c o r d e d in front o f the origin p r o b a b l y represents u n d i s s o c i a t e d proteins, as it is n o t present in gels where the s a m p l e was b o i l e d for 10 min in the presence o f SDS. O t h e r b a n d s a p p e a r to be p r e s e n t in the centre p o r t i o n o f
the gel b u t the b a n d s are very faint a n d n o t e v i d e n t in all gels in each o p e r a t i o n . T h e m o l e c u l a r weight estimates are 56,000, 14,000 a n d 50,000 for 1, 2 a n d 3 respectively. F o l l o w i n g p u r i f i c a t i o n o f R u B P C a s e f r o m Pi-
N.W. Kerby and L.V. Evans: Brown Algal Pyrenoids
94 3.0
--
5O
2.0
2O
o~ ~o
--
1.25
_
1.20
--
1.15
--
1.10
1.0 I0
0t,
10
2o
3o
4o . . . . Fr~c tion
To--
-T6""
A
.05
-7o
number
Fig. 5. Distribution of protein and chlorophyll in fractions collected from continuous silica sol gradients (20-70% Ludox/PEG/HM) after centrifugation at 25,000 xg, 1 h. Pilayella pretreated with 0.1% HgCI2 3 h. 9 9 chlorophyll concentration (gg cm-a). A-A protein concentration (lag c m - 3). _.... density Ludox/PEG/HM ( g c m 3)
[5511
IZSZ
7-1125115
1
tfa
c B
2~ Origin
5'0
f5 BPB
Fig. 6. S D S polyacrylamide gel electrophoresis. Gel and corresponding scan at 575 n m of pyrenoid fraction obtained by pretreatment with 0.1% HgC12 and Ludox/PEG/HM density gradient centrifugation. Peaks 1, 2 and 3 represent major polypeptides. BPB (bromophenol blue)
u-x
-g
;4 i
25 5O Distance (m 0
layella on Sephadex G-200, fractions collected from the column were assayed for carboxylase activity and only those showing such activity were used for studies on quaternary structure. The homogeneity of bulked fractions was tested by polyacrylamide gel electro-
Orig~
1
75 I BPB
Fig. 7. SDS polyacrylam~de gel electrophoresis. Gel and corresponding scan at 575 nm of RuBPCase purified on Sephadex G-200. A, B and C represent major polypeptides. BPB (bromophenol blue)
N.W. Kerby and L.V. Evans: Brown Algal Pyrenoids
phoresis and the protein present was found to migrate as a single band. The purified RuBPCase was treated with SDS and run on polyacrylamide gels containing SDS and 3 major peaks were observed (Fig. 7). Peaks A, B and C closely resemble peaks 1, 2 and 3 respectively in Figure 6. The molecular weights are 55,000 (peak A), 15,000 (peak B) and 50,000 (peak C). The molecular weights of peaks A and B correspond closely to the reported values of RuBPCase from other organisms (Jensen and Bahr, 1977).
Discussion
The data presented here show that pyrenoids can be isolated successfully from vegetative cells of Pilayella littoralis, after pretreatment with 0.1% HgC12. Without this pretreatment loss of core material occurs to a greater or lesser extent and hence yields are very low. The results from the preliminary characterization indicate that the pyrenoid core is proteinaceous and does not contain chlorophyll. This is consistent with the findings of previous authors (Brown and Arnott, 1970; Holdsworth, 1971). SDS polyacrylamide gel electrophoresis has revealed that two polypeptides account for the majority of the protein present in the pyrenoid core and the relative mobilities of these resemble those of the large and small subunits of RuBPCase. When RuBPCase was purified and the quaternary structure investigated, three major peaks were recorded. If the pyrenoid core contains RuBPCase, a purified carboxylase fraction would be expected to contain the enzyme from both the chloroplast stroma and the pyrenoid core. One possibility to account for the third peak is that the large subunit in the pyrenoid may be different from the large subunit in the chloroplast stroma. Peak 3 (Fig. 6) which corresponds closely to peak C (Fig. 7) and which is much reduced in the pyrenoid extract, could represent contamination by stromal carboxylase derived from chloroplast fragments. The other possibility is that peak C may be due to the action of a protease on the RuBPCase (Ellis, 1973). This would not occur in the pyrenoid preparation due to the pretreatment with HgC12. Further work is in progress and it is hoped that a more complete picture of the subunits of RuBPCase in this alga will emerge. As HgClz is used, direct assays for carboxylase activity are not possible at present. However confirmation that the two major polypeptides found in pure pyrenoid extracts are the subunits of RuBPCase should be possible by the use of immunological techniques.
95 The authors wish to thank Dr. Maureen E. Callow for valuable advice on many aspects of this project. The work was made possible through research support to N.W.K. from the Science Research Council.
References Arnon, D.I. : Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1 15, (1949) Bouck, G.B.: Fine structure and organelle associations in brown algae. J. Cell Biol. 26, 523 537 (1965) Brown, R.M., Arnott, H. J.: Structure and function of the algal pyrenoid. 1. Ultrastructure and cytochemistry during zoo-sporogenesis of Tetracystis excentrica. J. Phycol. 6, 14 22 (1970) Callow, J.A.: Ribosomal RNA, Fraction I protein synthesis and ribulose diphosphate carboxylase activity in developing and senescing leaves of cucumber. New Phytol. 73, I3-20 (1974) Davies, B.J.: Disc electrophoresis. II. Method and application to human proteins. Ann. N.Y. Acad. Sci. 121, 404-427 (i964) Dodge, J.D.: The fine structure of algal cells. London: Academic Press 1973 Ellis, R.J. : Fraction I protein. Comment. Plant Sci. 4, 29 36 (1973) Evans, L.V. : Distribution of pyrenoids among some brown algae. J. Cell Sci. 1,449-454 (1966) Evans, L.V. : Chloroplast morphology and fine structure in some British fucoids. New Phytol. 67, 173-178 (1968) Evans, L.V., Holligan, M.S.: Correlated light and electron microscope studies on brown algae. 1. Localization of alginic acid and sulphated polysaccharides in Dictyota. New Phytol. 71, 1161 I172 (1972) Fairbanks, G., Stech, T.L., Wallach, D.F.H. : Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-2617 (1971) Griffiths~ D.J. : The pyrenoid. Bot. Rev. 36, 29 58 (1970) Holdsworth, R.H.: The isolation and partial characterization of the pyrenoid protein of Eremosphaera viridis. J. Cell Biol. 15, 499-513 (1971) Jensen, R.G., Bahr, J.T. : Ribulose 1,5-bisphosphate carboxylaseoxygenase. Ann. Rev. Plant Physiol. 28, 379400 (1977) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265 275 (1951) Mazia, D., Brewer, P.A., Alfert, M.: The cytochemicaI staining and measurement of protein with mercuric bromophenol blue. Biol. Bull. mar. biol. Lab. (Woods Hole) 104, 57 67 (1953) Morgenthaler, J.-J., Price, C.A., Robinson, J.M., Gibbs, M. : Photosynthetic activity of spinach chloroplasts after isopycnic centrifugation in gradients of silica. Plant Physiol. 54, 532 534 (1974) Reynolds, E.S. : The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208 213 (1963) Simon, M.F. : Recherches sur les pyrenoids des ph+ophyc6es. Rev. Cytol. Biol. V~g. 15, 73 105 (1954) Walbot, V.: Use of silica sol step gradients to prepare bundle sheath and mesophyll chloroplasts from Parnicium maximum. Plant Physiol. 60, 102 108 (1977) Weber, K., Osborne, M.: The reliability of molecular weight determinations by dodecyl-sulphate polyacrylamide gel electrophoresis. J. biol. Chem. 244, 44064412 (1969)
Received 3 April; accepted 3 May 1978
