THE ISOLATION AND PARTIAL CHARACTERIZATION OF THE PYRENOID PROTEIN OF EREMOSPHAERA VIRIDIS ROBERT H . HOLDSWORTH From the Department of Biology, Princeton University, Princeton, New Jersey 08540. Dr. Holdsworth's present address is the Department of Biology, George Mason College of the Uni- versity of Virginia, Fairfax, Virginia 22030 . ABSTRACT The pyrenoids of Eremosphaera viridis, a green alga, were isolated by density gradient cen- trifugation and their physical and enzymatic properties were studied . The ultraviolet absorption spectrum of sodium dodecyl sulfate (SDS) extracts of pyrenoids showed a single peak at a wavelength of 277 nm, indicating the presence of protein and the probable absence of nucleic acid . Upon electrophoresis on polyacrylamide gels containing SDS, 16 bands were resolved of which two, together, accounted for 90% of the total protein on the gels . The molecular weights of these two proteins were estimated to be 59,000 and 12,300 and the ratio by weight of the larger to the smaller protein was found to be 2 :1 . The physical and enzymatic properties of these two proteins were found to closely resemble the properties reported in the literature for the subunits of fraction I protein . Both pyrenoids and fraction I protein are localized in the chloroplast, and both have two principal protein components . The molecular weights and relative ratio of the two pyrenoid components are very similar to those of the two components of fraction I protein . The pyrenoid was found to contain a high specific activity of ribulose-1 , 5-diphosphate carboxylase which is the same enzymatic activity exhibited by fraction I protein. The presence of ribose-5-phosphate isomerase and ribulose-5-phosphate kinase activities was also noted in pyrenoid preparations . It is sug- gested that the pyrenoid contains fraction I protein and possibly other enzymes of the Calvin-Bassham carbon dioxide fixing pathway. INTRODUCTION In the chloroplasts of many species of algae and of 11, 12, 28, 39, 44, 54) . It should be noted, however, some species of liverworts are found round, that pyrenoids of certain algae, in particular some angular, or irregularly shaped bodies called of the Phaeophytes (54), Rhodophyes (20), and pyrenoids (20, 55) . Most studies of the pyrenoid Chrysophytes (41), do not show a strong affinity have concentrated on its structural aspects (see for these stains. Brown and Arnott (9) found that 22, 23, and 26 for reviews) ; however, some at- the pyrenoids of Tetracystis excentrica stained deeply tempts have been made to determine the chemical with mercuric bromphenol blue, a stain shown by composition of pyrenoids by using histochemical Mazia et al. (43) to be specific for proteins . Several techniques . That protein constitutes the bulk of studies have shown that proteolytic enzymes used the pyrenoid has been deduced from its staining as histochemical reagents are capable of digesting properties. Among the most frequently cited stains the pyrenoid core (9, 17, 19) . used successfully on pyrenoids are safranin of Studies to determine the enzyme content of Fleming's triple stain and hematoxylin (1, 3, 4, 6, pyrenoids have been limited to those enzymes THE JOURNAL OF CELL BIOLOGY . VOLUME 51, 1971 • pages 499-513 499 which can be detected histochemically . Simon mately 130 ft-c intensity and at a temperature of (54) found alkaline phosphatase in and around the approximately 20 °C. pyrenoids of Enteromorpha, Rhodochorton, and Pylaiella by using the Gomori reaction . Cook, also Pyrenoid Isolation using the Gomori reaction, reported positive Cultures 5 or 6 wk old were harvested by centrif- results for acid phosphatase in and around pyre- ugation, yielding about 15 ml of packed cells . noids of Closterium (13) . Empty cell walls which formed a white layer above Perhaps the most direct approach to solving the the packed cells were discarded. The cells were problems of pyrenoid composition and function rinsed twice in ice-cold 0 .02 M sodium phosphate would be to isolate this organelle in bulk and buffer at pH 7 .0 and were broken in the cold by 25 study its properties in vitro . A preliminary account strokes of a teflon pestle in a hand homogenizer . The of the isolation of pyrenoids of Zygnema has been homogenate was filtered three times through 200- mesh bolting cloth (Carolina Biological Supply Co., reported by Rosowski and Hoshaw (51) . They have Elon College, N. C.) to remove cell walls and un- found isolated pyrenoids to be "firm, spherical, broken cells . The resulting suspension was centrifuged colorless refractive bodies" when studied with the in the cold for 25 min at 175 g . The pellet was resus- light microscope . The inability of the isolated pended in buffer and centrifuged in the cold at 175 g pyrenoids to bind propiocarmine stain, as could for 3 min. The supernatant liquid, denoted S, con- the in situ pyrenoids, led the authors to suggest a tained fragments of chloroplasts and was saved . The difference in the chemical state between in situ and pellet containing mostly pyrenoids and stroma-starch isolated pyrenoids. was resuspended, centrifuged, and decanted as before . This paper reports the isolation and characteri- The resulting pellet was resuspended in 0 .5 ml of a CsC1-sucrose solution having a density of 1 .55 g/ml zation of the pyrenoids of Eremosphaera viridis, a (42 .6% w/w CsCl, 12.4% w/w sucrose, 55.0% w/w large (250 µ in diameter), spherical, fresh-water homogenization buffer) . The suspension was layered Chlorophyte . In the present context the term on a 5 ml continuous CsCl-sucrose gradient running "pyrenoid" will refer to both the core substance from a density of 1 .61 g/ml at the bottom (45 .5% w/w and its surrounding starch sheath (designated as CsCl, 13 .3% w/w sucrose, 41 .2% w/w buffer) to a pyrenoid starch) . "Stroma-starch" will be used to density of 1 .55 g/ml at the top . The preparation was designate the starch of the cell which is not asso- centrifuged for 5 hr at 32,600 g in a Beckman Model ciated with a pyrenoid core . L centrifuge by which time equilibrium of the particles in the gradient was reached. The stroma- MATERIALS AND METHODS starch formed a band at a density of approximately 1 .60 g/ml while the pyrenoids formed a somewhat Algal Culture broader zone just above the starch . Stroma-starch and pyrenoid fractions were removed from the Axenic cultures of Eremosphaera viridis strain E-4 1 gradient by hypodermic needles inserted through the were grown in a medium slightly modified from the side of the cellulose nitrate centrifuge tubes . The BEV medium devised by Smith and Bold (56) . The material in each fraction was rinsed three times with MoO3 employed in Smith's medium was replaced by the buffer used for homogenization . the more soluble Na2Mo04, and the concentrations The chloroplast fragments in the supernatant of the two vitamins used, cyanocobalamin and thia- liquid, S, were subjected to rehomogenization and mine hydrochloride, were increased to 0 .1 mg/liter centrifugation to remove the pyrenoids and starch each. 5% CO2 in compressed air was continuously grains remaining in the chloroplast material . The bubbled through a cotton filter into the medium chloroplast fragments were then pelleted by centrif- during culture growth giving a final pH of approxi- ugation at 18,400 g for 20 min . mately 5 .5. A flow diagram of the over-all isolation procedure Every 4-7 days during growth the cells were is shown in Fig . 1 . allowed to settle in the culture flask and the depleted medium was siphoned off and replaced with fresh Light and Electron Microscopy medium from a 6 liter reservoir bottle . Cultures were grown under conditions of constant light of approxi- Cells of Eremosphaera viridis were fixed for electron microscopy for 18 hr in the cold in a solution of 3% 1 The author is greatly indebted to Dr . Richard L . glutaraldehyde buffered at pH 7 .2 with 0 .01 M Smith (Department of Botany, Eastern Illinois sodium cacodylate followed by a 3 hr postfixation in University, Charleston, Illinois) for supplying the 1 % osmium tetroxide in the same buffer . After stock cultures used in this study. several rinses with buffer, the cells were dehydrated 500 THE JOURNAL OF CELL BIOLOGY . VOLUME 51, 1971 HOMOGENATE Filter (200-mesh bolting cloth) I Cell walls and unbroken cells Filtrate Rehomogenize and Filter (2X) Cell walls Filter (2X) and large debris (bolting cloth) (discard) Centrifuge (25 min at 175 g) I Pellet Supernatant liquid Resuspend Centrifuge (15 min at Centrifuge (3X) 4000 g) (3 min at 175 g) I Pellet Supernatant (pyrenoids liquid and stroma starch) (S) I I Resuspend Rehomogenize (in CsCl-sucrose) I Density gradient Centrifuge (3X) centrifugation (3 min at 175 g) (32,600 g) Pellet Supernatant (discard) liquid Centrifuge (20 min at 8400 g) Stroma Chloroplast Homogenization starch Pyrenoids fragments supernatant identified by light and identified by electron microscopy light microscopy FIGURE 1 Flow diagram of procedure for isolating pyrenoids, stroma-starch, and chloroplast frag- ments from Eremosphaera viridis. through an ethanol series and embedded in Spurr's were prepared for electron microscopy by using the plastic (57) . same procedures as were used for whole cells . Samples of isolated pyrenoids either untreated or Sections cut with a duPont diamond knife (E . I . extracted with a 1 % solution of sodium dodecyl du Pont de Nemours & Co., Inc ., Wilmington, Del .) sulfate (SDS) in pH 7 .0 sodium phosphate buffer on a Porter-Blum model MT-2 ultramictotome were ROBERT H. HonnswoRTH The Pyrenoid Protein of Eremosphaera viridis 5 0 1 picked up on 200-mesh copper grids coated with microdensitometer and the relative amount of Formvar and carbon, and stained with uranyl protein-stain complex present in the bands of a given acetate and lead citrate .