Planta (Berl.) 104, 185-194 (1972) 9 by Springer-Verlag 1972

Isolation of Protein Bodies on Sucrose Gradients* C. Schnarrenberger**, A. 0eser, and N. E. To]bert Department of Biochemistry, Michigan State University, East Lansing Received December 1, 1971

Summary. Storage protein bodies from sunflower cotyledons during early stages of seed germination were isolated on sucrose density gradients by isopycnic centrifugation. The density of this organelle on the gradients ranged between 1.26 and 1.36 gcm -3. A proteinase with a pH optimum of 5.2 was associated with this organelle, and is probably responsible for degradation of storage protein. A NADHdependent eytochrome-c reductase, a membrane marker enzyme with a pit optimum of 8.4, was also present in this organelle fraction. Introduction Storage proteins are one of the major reserves which provide substrafes for growth during early seed germination. Storage proteins in a cell are located in bodies surrounded b y a single membrane, as visualized in electron-microscopic studies (Briarty et al., 1970 ; Morton and Raison, 1964; Tronier etal., 1971; Varner and Schidlovsky, 1963). During germination marked decreases occur in total storage protein. The proteins bodies first swell and often fuse to larger vacuoles while their content becomes degraded and less dense as determined by eytoehemical (g~cker, 1967; Briarty et al., 1970) and electron-microscopic examination (Opik, 1966; Smith and Flinn, 1967; Briarty et al., 1970). The degradation of storage protein is generally attributed to proteolytie enzymes (Beevers, 1968; Varner and Schidlovsky, 1963; Wiley and Ashton, 1967; Young and Varner, 1959). As judged b y different p H optima, kinetic studies and responses to inhibitors, there are probably several proteinases in germinating seeds: an acid proteinase with a p t I optimum of 4.8-5:5 (Beevers, 1968; Beevers and Spittstoesser, 1968; Jaeobson ~nd Varner, 1967; Shain and Mayer, 1965), ~ neutral proteinase with a p H optimum of around 7.0 (Beevers, 1968 ; Shain and Mayer, 1965 ; Soedigo and Gruber 1960; Varner and Schidlovsky, 1963; Wiley and Ashton, 1967), and a proteinase with a p H optimum of 6.0 (Shain and Mayer, 1965).

Abbreviations: LPA for L-lysine-p-nitroanilide; LPAase for the peptidase which hydrolyzes this peptidc. * This work was supported in part by the National Science Foundation Grant GB-17543, and published as Journal Article No. 5736 of the Michigan Agricultural Experiment Station. ** Supported by a Deutsche Forschungsgemeinschaft Fellowship. 13 Planta(Berl.),Bd.104

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C. Schnarrenberger, A. Oeser, and N. E. Tolbert:

E a r l i e r isolations of storage p r o t e i n bodies from seeds h a v e been a c h i e v e d b y differential eentrifugation, a n d p a r t i c l e i d e n t i f i c a t i o n b y electron m i c r o s c o p y (Morton a n d Raison, 1964; O r y a n d l t e n n i n g s e n , 1969; Tronier et al., 1971; V a r n e r a n d Schidlovsky, 1963). I n one case (Ory a n d Henningsen, 1969) a n a c i d p r o t e i n a s e a n d a n a c i d p h o s p h a t a s e were r e p o r t e d to be associated with t h e p a r t i c u l a t e fraction. Because of a high p r o t e i n t o f a t r a t i o in storage p r o t e i n bodies, a different a p p r o a c h for t h e i r p u r i f i c a t i o n can be m a d e using t h e i r high isopycnic d e n s i t y , as d e m o n s t r a t e d b y t h e excellent s t u d y of T o m b s (1967). B y this m e t h o d we h a v e s e p a r a t e d storage p r o t e i n bodies f r o m other cell organelles b y isopycnic c e n t r i f u g a t i o n in sucrose d e n s i t y gradients. F o r t h e biochemical c h a r a c t e r i z a t i o n of t h e isolated particles we f o u n d a n a c i d p r o t e i n a s e which would a c c o u n t for s t o r a g e - p r o t e i n d e g r a d a t i o n , a n d a N A D t I e y t o c h r o m e - c reductase, which is a m e m b r a n e m a r k e r e n z y m e for t h e outer membrane.

Material and Methods Plant Material. Sunflower (Helianthus annuus L., cv. Hale) seeds were obtained from Vaughn's Seed Company, Ovid, Michigan. Dehusked seeds were treated with about 5 mg/ml of the fungicide Captan (Chevron Chemical Company, San Francisco, California) for 5 min and then rinsed with distilled water. Seedlings were grown in vermiculite soaked with half strength ttoagland's solution in a growth chamber at 25 ~ in continuous darkness or in 800-1000 f.c. of light from white fluorescent lamps between the 4th and 6th day of germination. Particle Preparation and Sucrose Density Gradients. ttomogenates of 10 g of cotyledons were prepared by a gentle, incomplete grinding procedure in a Sorvall Omni-Mixer. Sucrose gradients in tubes for the SW 25.2 rotor of the Beckman L2 ultracentrifuge were prepared and used as described previously (Schnarrenberger et al., 1971). The sucrose density gradients contained 2.5 ml layers of 70, 70, 66, 63, 60, 56.5, 51.5, 50, 47.5, 45, 42.5, 37.5, 35, 32.5, 30, 27.5, and 25% (w/w) sucrose in 10-~ M EDTA at pH 7.5. The gradients were centrifuged for 4 h at 25000 rpm, and then drained from the bottom into fractions of 2.5 ml. A pellet on the bottom of the centrifuge tubes was discarded. Enzyme Assays. Catalase (EC 1.11.1.6) was assayed by the initial disappearance of 12.5 mM I-I202 as measured by a decrease in absorbance at 240 nm (Lfick, 1965). Cytochrome-c oxidase (EC 1.11.1.5) was measured spectrophotemetrieally at 550 nm by the oxidation of reduced cytochrome c (Tolbert et al., 1968). The initial ratio of ODbs0 to ODbes was about 9-10. Activities were calculated as described earlier (Sehnarrenberger et al., 1971). Triose-P isomerase (EC 5.3.1.1) activity was coupled to a glycerol-P dehydrogenase reaction and followed by the oxidation of NADH at 340 nm during the reaction at pH 7.6. NADH-cytechrome-c-reductase (EC 1.6.2.1) was assayed spectrophotometrically at two pH values, as the NADtt dependent reduction of oxidized eytochrome c at 550 nm (Martin and Morton, 1956). The reaction mixture contained in a total volume of 0.27 ml: 20 ~tmoles of sodium phosphate at pH 8.4 or 6.0, 38 nmoles of KCN, 25 nmoles of cytochrome e from horse heart (type II, Sigma), 148 nmoles of NADH, and enzyme. The reaction was started by the addition of NADH and corrected for endogenous rates that had been allowed r level off as far as possible. Enzyme concentrations were used which did not reduce in the endogenous reaction more than 1/3 of the oxidized cytoehrome c.

Isolation of Protein Bodies

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Proteinase was measured by its caseolytic activity (Beevers, 1968). After protein precipitation by trichloroacetic acid (TCA), the amino nitrogen in the soluble hydrolyzate was determined by a modified ninhydrin method (u and Cocking, 1955). The proteinase assay was run for 100 rain at 40 ~ C in a volume of 0.85 ml containing 100 tzmoles citrate at pI{ 5.2, enzyme, and 0.18 % casein. The reaction was stopped by 0.15 ml of 50% TCA and centrifuged. A 0.5 ml aliquot of the supernatant liquid was mixed with 1.5 ml of ninhydrin solution. This solution had been prepared by combining 400 mg of SnCI~.2H20 dissolved in 250 ml of 0.6 M citrate at pH 4.7 with 10 g ninhydrin dissolved in 250 ml methylcellosolve. After heating for 20 rain in a boiling water-bath and cooling, 6.0 ml of 50% aqueous propanol were added, and the absorbance at 550 nm determined. For controls the enzyme was incubated as above, but casein was added after the addition of TCA. Peptidase activity was measured spectrophotometrically by the formation of p-nitroaniline from L-lysine-p-nitroanilide (LPA) at 390 nm (Erlanger et al., 1961), and referred to as LPAase activity. The assay mixture in a total volume of 1.0 ml contained 0.16 M phosphate at pH 7.6, enzyme, and 3 • 10-4 M LPA. A molar extiction coefficient of 1.3 • 10-4 mole-4 cm -1 was used at 390 nm for p-nitroaniline. Protein was determined by the Lowry method (Lowry et al., 1951), chlorophyll by its absorption at 652 nm (Arnon, 1949), and sucrose densities (d 10 ~ C) by the refractive index. Results Particulate homogenates of cotyledons from germinating sunflower seedlings were layered on sucrose gradients and centrifuged for 4 h to separate the subcellular organelles (Figs. 1, 2). The markers for organelles in representative gradients were catalasc for microbodies, cytochrome-c oxidase for mitochondria, triose-P isomerase for whole plastids, and chlorophyll for broken chloroplasts (Schnarrenberger etal., 1971; Schnarrenberger et al., 1972). Soluble activity at the top of the gradient in the volume above 40 ml represented solubilized particulate enzymes or a soluble form of an enzyme. Peaks of total protein in the gradient fractions corresponded to the mitochondrial and plastid fractions, and a small shoulder of protein coincided with the m i c r o b o d y fraction. W h e n homogenates of seedlings germinated for less t h a n 4 days were fractionated on sucrose density gradients, considerable protein was present at densities higher t h a n 1.26 g c m -3. This protein appeared to overlap into the microbody fraction, and it did not coincide with other k n o w n particles. The occurrence of protein in these fractions, the first 12.5 m] (upper p a r t of Fig. 1), suggested t h a t a cell organelle with a high isopycnie density was present in the cotyledons during early stages of germination. This fraction has been designated as protein bodies. B y the time seedlings h a d germinated for 6 days, as shown in the b o t t o m p a r t of Fig. 1, these particles had disappeared, for no protein was present in these dense sucrose fractions below the microbodies. During the first 2 days of germination a nearly constant a m o u n t of protein was recovered from the sucrose gradients in the gradient area attributed to protein bodies (Fig. 3). Lower recovery of protein bodies 13"

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