During senescence ofoat ... olysis characteristic of senescence, since this process involves ..... and negligible cytosolic contamination) were assayed against.
Received for publication October 13, 1988 and in revised form July 14, 1989
Plant Physiol. (1989) 91, 1414-1418 0032-0889/89/91/141 4/05/$01 .00/0
Proteolytic Activity at Alkaline pH in Oat Leaves, Isolation of an Aminopeptidase' Leonardo M. Casano, Marcelo Desimone, and Victorio S. Trippi* Laboratorio de Fisiologia Vegetal, Facultad de Ciencias Exactas, Fisicas y Naturales, Universidad Nacional de C6rdoba, P. 0. Box 395-(5000) C6rdoba, Republica Argentina ABSTRACT
correlation between the activity of the 'acid' protease and the increase of protein degradation. The activity of the 'neutral' protease and possibly of others should be taken into account in assessing the role of the proteolytic enzymes in the proteolysis characteristic of senescence, since this process involves endo- and exoproteases spacially and temporally coordinated. Therefore, it is necessary to know if other proteases exist and their characteristics, such as, biochemical and regulatory aspects as well as subcellular localization. In this paper, we present evidence of an important proteolytic activity at alkaline pH, due to an aminopeptidase which was partially purified. This enzyme seems to be located within the chloroplasts since a high aminopeptidase activity was found in the intact organelles.
Proteolytic activity in oat leaf extracts was measured with both azocasein and ribulose bisphosphate carboxylase (Rubisco) as substrates over a wide range of pH (3.0-9.2). With either azocasein or Rubisco activity peaks appeared at pH 4.8, 6.6, and 8.4. An aminopeptidase (AP) which hydrolyzes leucine-nitroanilide was partially purified. Purification consisted of a series of six steps which included ammonium sulfate precipitation, gel filtration, and two ionic exchange chromatographies. The enzyme was purified more than 100-fold. The apparent Km for leucine-nitroanilide is 0.08 millimolar at its pH optimum of 8.4. AP may be a cystein protease since it is inhibited by heavy metals and activated by 2-mercaptoethanol. Isolated chloroplasts were also able to hydrolyze leucine-nitroanilide at a pH optimum of 8.4, indicating that AP could be localized inside the photosynthetic organelles.
MATERIALS AND METHODS Plant Material
Proteolysis is one of the most conspicuous processes in the life of a plant. It operates during protein turnover and in reserve-protein mobilization. The net loss of proteins in senescence is one of the most extensively studied subjects in plant senescence (3, 9, 11, 12, 13, 17). There is consensus on the key role of proteolytic enzymes in regulating protein breakdown, and efforts have been made in isolation and characterization of proteinases in several plant species. Endoand exopeptidases of leaves of both dicotyledonous and monocotyledonous plants have been purified to different degrees of homogeneity (10, 16, 20). Within each group of enzymes, most of plant proteases found in different species have a number of common properties. For example, aminopeptidases have a mol wt range between 50 to 100 kD, contain essential sulfhydryl groups, are inhibited by metal ions, and have neutral or slight alkaline pH optima (20). In oat leaves, Drivdahl and Thimann (4, 5) have isolated two proteases. One seems to be an endoprotease with an acid pH optimum, and the other, an aminopeptidase, is active at neutral pH. These authors also suggested the presence of other proteolytic
Seeds of Avena sativa cv suregrain were sown in vermiculite and grown for 7 to 8 d under greenhouse conditions (20-23°C and 70-80% RH). Crude Extracts
Subapical 3 cm segments ofthe first leaf were homogenized with chilled mortar and pestle in 100 mm Pi buffer (pH 7.0), containing 1% PVP (1:2, gram of fresh weight:mL of grinding buffer). The resulting slurry was centrifuged at l0,000g for 10 min, and proteolytic activity was determined in the supernatant fraction. Chloroplast Isolation Chloroplasts were obtained from the first leaf of oat plants according to Mills and Joy (12a). They were rinsed three times and then resuspended in a medium containing 0.33 M sorbitol, 2 mm EDTA, 1 mI MgCl2, and 50 mm Hepes buffer (pH 7.6). Integrity was measured by ferricyanide photoreduction assay, as described by Yamagishi et al. (21). Cytosol contamination was tested by measuring a marker enzyme, glucose 6P dehydrogenase according to Kuby and Noltmann (8). Activity of glucose 6-P dehydrogenase ranged from 4 to 5.2 nmol NADPH min-' mg protein-' among chloroplasts preparations, corresponding to 5% of that measured in purified protoplasts (1).
enzymes.
Variations of in vitro assayed proteinases cannot always be correlated with changes in protein levels, specially when only one or few enzymes were analyzed. During senescence of oat leaves, Veierskov and Thimann (1 8) reported a lack of simple 'Supported by Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) and Consejo Investigaciones Cientificas y Tecnol6gicas de la Provincia de C6rboda (CONICOR).
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AMINOPEPTIDASE OF OAT LEAVES
Enzyme Assays
(a) Azocasein digesting activity was assayed in crude extracts by incubating 0.1 mL of crude extracts with 0.3 mL of 0.5% azocasein and 0.6 mL of either 50 mM citrate-phosphate buffer (for pH 3.0-6.8) or 50 mm Tris-Cl buffer (for pH 7.09.2). After 2 h at 37°C, the reaction was stopped by adding 2 mL of ice-cold 12% TCA. Precipitated proteins were removed by centrifugation (l0,OOOg, 10 min), and A340 was measured in the supernatant. One unit of azocaseinolytic activity was defined as causing a 0.01 increase of A340 respective to 0 time values (reactions stopped inmediatly after starting). (b) Rubisco,2 purified according to Hall and Tolbert (6), hydrolyzing activity of crude extracts was assayed as proposed by Saleemudin et al. (14) in a pH range similar to that used for azocasein digesting assays (see paragraph a). (c) Leucine aminopeptidase activity was measured with Leu-NA, according to Cheng and Kao (2). Routinely, 0.02 mL of either enzyme or chloroplast preparation was assayed in the presence of 0.5 mL of 50 mM Tris-Cl (pH 8.4) and 10 mM 2-ME during 30 min at 39°C. The reaction was stopped by adding 0.52 mL of 30% acetic acid, and release of the nitroanilide group was estimated by the increase in A410 respective to 0 time values (reactions stopped immediately after starting). One unit of aminopeptidase activity was considered as causing a 1.0 decrease of A410 per min. Purification of the Aminopeptidase Approximately 700 g of 7 to 8 d old primary leaves of oat plants were homogenized in (1:2, fresh weight:grinding buffer), 50 mM tris-Cl (pH 8.0), 50 mm NaCl, and 1% insoluble PVP, with a Sorvall Omnimixer (40 s, full speed). The filtrate was centrifuged at 27,000g during 30 min. The resulting supernatant fraction was taken as crude extract and purified as described below. All the steps were carried out at 4°C, except the gel filtration on Sephacryl S-300, which was done at room temperature. Fractional Precipitation with Ammonium Sulfate
Solid ammonium sulfate was slowly added to the crude extract to give 35% saturation. After gently stirring during 30 min the inactive precipitate was removed by centrifugation at 27,000g, 30 min. More solid ammonium sulfate was added to give 65% saturation, and after 30 min stirring, the active precipitate was collected by centrifugation. This was resuspended in 20 mL of buffer A, consisting of 50 mm Tris-Cl (pH 8.0), 50 mm NaCl, and 10 mm 2-ME. Filtration through Sephadex G-25 Column The preceding preparation was desalted by passing it through a column of Sephadex G-25 (2 x 40 cm) equilibrated with buffer A. The same buffer was used for the elution and 3.0 mL fractions were collected. The enzyme eluted with the void volume. 2
Abbreviations: Rubisco, ribulose bisphosphate carboxylase; AP, aminopeptidase; Leu-NA, leucine-nitroanilide; 2-ME, 2-mercaptoethanol; TLCK, N,a-p-tosyl-L-lysine chloromethyl ketone.
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First Ionic Exchange Chromatography on DEAESephacell Column Active fractions from gel filtration were loaded on to a DEAE-Sephacell column (2.5 x 25 cm) equilibrated with buffer A. After loading, the column was washed with one volume of equilibration buffer. Elution was done with buffer A containing a NaCl gradient of 50 to 250 mm at a flow rate of about 2 mL/min. Fractions of 5 mL were collected and the enzyme eluted at around 110 mM NaCl. Active fractions were polled and concentrated in a dialysis bag against polyethylene glycol 8000 to reach a volume of 2 mL.
Gel Filtration using a Sephacryll S-300 Column Following ionic exchange chromatography concentrated active fractions were loaded on to a column (2 x 70 cm) of Sephacryll S-300 equilibrated with 50 mm Tris-Cl (pH 8.0), 150 mm NaCl, and 10 mM 2-ME. Elution was performed with the same buffer at a flow rate of 1 mL/min, and 3 mL fractions were collected. The mol wt was estimated by precalibrating the same column with the following standards: apoferritin (443,000), j3-amylase (200,000), alcohol dehydrogenase (150,000), bovin serum albumin (66,000), carbonic anhydrase (29,000), and Cyt c (12,400).
Second Ionic Exchange Chromatography on DEAESephacell Column The NaCl concentration of peak fractions from the previous step was decreased to 50 mM by adding 50 mM Tris-Cl (pH 8.0) and then adsorbed to a column (1.5 x 17 cm) of DEAESephacell equilibrated with buffer A. After washing, the enzyme was eluted with the same buffer containing a NaCl gradient of 50 to 150 mm, and 2 mL fractions were collected. Estimation of Proteins
During the purification of AP the protein content was followed by measuring A280. In the other experiments proteins were quantified by the method of Sedmark and Grossberg (15).
Aminopeptidase Characterization Enzyme Kinetics Aminopeptidase activity was determined at substrate (LeuNA) concentrations up to 0.7 mm, employing a procedure similar to that described above. Effect of pH In both purified enzyme and chloroplasts, AP activity was assayed as described above except that the pH of the reaction media ranged from 6.0 to 7.0 with 50 mM Pi buffer and 7.2 to 9.2, with 50 mm Tris-ClH buffer. Effect of 2-ME and Inhibitors
The influence of 2-ME concentration in the assay medium ranged up to 10 mM. All the inhibitors were prepared in 50
CASANO ET AL.
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mM Tris-Cl (pH 8.4), previously disolved in dimethyl sulfoxide (1 %, v:v, final concentration), except for the metal salts where dimethyl sulfoxide was omitted. Samples of purified enzyme were preincubated during 40 min for heavy metals and 90 min for the other inhibitors at 4°C. Aliquots of 40 ,uL of preincubated enzyme were assayed as described above.
RESULTS AND DISCUSSION
Proteolytic Activity in Crude Extracts
When crude extracts from oat leaves were assayed with both azocasein and purified Rubisco, peaks of proteolytic activity were found at pH 4.8, 6.6, and 8.4 (Fig. 1). In oat leaves Drivdahl and Thimann (4) previously identified and isolated two proteases, one active at pH 4.2 and other at pH 6.6. Proteolytic activity in crude extracts at acid pH had a higher pH optimum than that reported by Drivdahl and Thimann (4) for the isolated protease (4.8 versus 4.2). This difference could be due to varietal differences between the plant material used by these authors and by us, and/or to factors present in crude extracts but not in purified preparations, which could have shifted the pH optimum in our assays. Proteolytic activity at pH 8.4 has not previously been reported in oat leaves. On the other hand, exoproteases, that break peptide bonds from the amino-terminal end (called aminopeptidases), are active at neutral and slight alkaline pH. Hence, we measured aminopeptidase activity at pH 8.4 employing a specific substrate, Leu-NA. Crude extracts hydrolyzed Leu-NA at a rate of 0.50 unit mg protein-', suggesting that oat leaves could contain at least one aminopeptidase active at alkaline pH. Aminopeptidase Isolation
The protocol used for the isolation and partial purification of AP consisted of a series of six fractionation steps, summarized in Table I. Freshly prepared crude extracts were treated with 35% saturation ammonium sulfate, and the supernatant fraction contained all the AP activity (data not shown). More
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Figure 1. pH dependence of proteolysis in crude extracts of oat leaves. Aliquots of extracts and either azocasein or Rubisco were incubated at 37°C during 2 h in the presence of 50 mm citrate-Pi buffer (pH 3.0-6.8) or 50 mM Tris-CIH (pH 7.0-9.2). For details see "Materials and Methods."
Plant Physiol. Vol. 91,1989
ammonium sulfate was then added to give 65% saturation. After filtration on Sephadex G-25 the preparation was run through a DEAE-Sephacell column as previously described. Aminopeptidase activity appeared as a complex peak (Fig. 2A), which was run through a Sephacryl S-300 column. The enzyme now appeared as a single peak with an apparent mol wt of 65,000. The peak fractions from the Sephacryl column were pooled, run through a second DEAE-Sephacell column, and eluted with a NaCl gradient from 50 to 150 mm. The specific activity of AP was increased more than 100-fold by this isolation procedure. However, both SDS and nondenaturing PAGE of the final preparation and staining with Coomassie brilliant blue showed that the enzyme was still not homogeneous, since several protein bands were still present (data not shown).
Aminopeptidase Characterization: Enzyme Kinetics In Figure 3A the AP activity is plotted against the concentration of Leu-NA in the assay medium. A Linewaver-Burk double-reciprocal plot of these data (Fig. 3B) shows the apparent Km value of Leu-NA to be 0.08 mm. This result is in agreement with Km values reported for aminopeptidase from other species (7, 19); even though differences in Km values for the same enzyme against different substrates were previously observed ( 19). Effect of pH on Reaction Rate The activity of AP increased with the pH, reaching a maximum rate of about 8.4, and then decreased (data not shown). This pH optimum is intermediate to that for other aminopeptidases from oat leaves (pH 6.6) (4), pea chloroplasts (pH 7.7) (10), and barley (pH 8.5-10.5) (16). However, the optimum pH of our purified AP coincided with the alkaline peak of proteolytic activity of crude extracts (Fig. 1), suggesting that this aminopeptidase could at least partially account for the in vivo alkaline proteolysis. Effects of Mercaptoethanol and Some Inhibitors The presence of a sulfhydryl reagent such as 2-ME, was essential for the full activation of AP (Table II), with saturation occurring at 2 mM 2-ME. The cation chelator EDTA only slightly inhibited enzyme activity, even at 5 mm concentration (Table II). Neither the serine protease inhibitor PMSF, the vacuolar protease inhibitor leupeptin, nor the aspartic acid protease inhibitor pepstatin had any effect on AP activity. It was, however, slightly inhibited by the histidine modifying reagent, TLCK. Among heavy metals tested, Zn strongly inhibited AP, but its effect was overcome by further incubation with 2-ME (Table II). This observation is consistent with the requirement of 2-ME for the enzyme activation, and it indicates that this AP of oat leaves is a cysteine-protease, agreeing with the nature of other previously studied aminopeptidases (19).
Aminopeptidase Activity in Isolated Oat Chloroplasts When oat chloroplasts carefully isolated (75-80% intact and negligible cytosolic contamination) were assayed against
AMINOPEPTIDASE OF OAT LEAVES
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Table I. Purification of Aminopeptidase from Oat Leaves Details of each purification step are described in "Materials and Methods." Step
Extract 35-65% (NH4)2S04 Sephadex G-25 First DEAE-Sephacell Sephacryl S-300 Second DEAE-Sephacell
Protein mg
Activity units
Specific Activity units mg protein-'
Recovery %
Purification -fold
3149 1750 1315 159 38 4
1632 1453 1502 702 604 229
0.52 0.83 1.14 4.41 15.81 56.30
100 89 92 43 37 14
1.6 2.2 8.5 30.5 108.6
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Figure 3. Substrate concentration dependence of aminopeptidase activity (A) and Km determination (B). Purified AP was assessed against different concentrations of Leu-NA, under standard conditions. For details see "Materials and Methods." Table II. Influence of Effectors on the AP from Oat Leaves After incubation with inhibitors, AP activity was assayed under standard conditions. The effect of 2-ME was assayed in the reaction medium. For details see "Materials and Methods."
15o
Inhibitor
Concentration
Inhibition % of control
EDTA
50
40 30 20 Fraction N Figure 2. Elution patterns of the aminopeptidase of oat leaves. A, First ion exchange chromatography on DEAE-Sephacell column. Relative activity of aminopeptidase (@@, A280 ( ), and (- - -) NaCI concentration in each fraction are shown. B, Gel filtration through Sephacryl S-300 column; (0) represents relative enzymatic activity, and the elution pattern of mol wt standard are indicated as (V); a, apoferritin (443,000); b, ,B-amylase (200,000); c, alcohol dehydrogenase (150,000); d, bovine serum albumin (66,000); e, carbonic anhydrase (29,000); and f, Cyt c (12,400). 10
-
Leu-NA in a pH range from 6.0 to 9.0, two pH optima values were obtained, 7.0 and 8.4 (Fig. 4). The neutral peak of aminopeptidase activity might be due to the presence in plastids of a protease other than isolated in the present work, since the pH curve of the purified enzyme only exhibited less than 50% of the peak activity at pH 7.0 (data not shown). In the slightly alkaline region (pH 8.0-9.0) pH curves of the isolated plastids and the purified enzyme preparations are
PMSF Pepstatin Leupeptin TLCK AgNO3 CuS04 ZnC12 ZnCI2+ 2-ME 2-ME
2.5 mM 5.0 mM 1.0 mM 0.1 mg/mL 0.1 mg/mL 1.0 mM 0.5 mM 0.5 mM 0.5 mM 0.5 ± 5 mM 0 mm 1 mM 2mM
8 12 0 0 0 23 24 34 75 5 82 11 0
nearly coincident. Moreover, chloroplasts showed a higher AP activity than crude extracts, 0.72 versus 0.50 unit mg. protein-', respectively. In agreement with these observations, aminopeptidases have already been demonstrated in wheat chloroplasts (18) and Liu and Jagendorf (10) isolated three aminopeptidases from the stroma of pea chloroplasts. It is possible, therefore, that the alkaline aminopeptidase is located inside the chloroplasts, as might be expected from the physi-
CASANO ET AL.
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Figure 4. Assay-pH optimum of aminopeptidase activity of isolated oat chloroplasts. Freshly isolated oat chloroplasts were assayed for aminopeptidase activity, under standard conditions (see "Materials and Methods") at indicated pH values, except that buffers were 50 mM Pi for pH values 6.0 to 7.0, or 50 mm Tris-CIH for pH values 7.2 to 9.2. Maximal activity was 0.72 unit mg protein-'.
ologycal pH ofthese organelles. These topics are priority areas for current investigations. Many proteins are degraded during the life of the oat leaves. However, they are not located in the same cell compartment nor are they hydrolyzed at equal rates. Thus, a diversity of proteolytic enzymes would be an appropriate feature to ensure protein breakdown. The results presented here show that in oat leaves proteolysis can occur at alkaline pH as well as the known ranges of acid and neutral activity. At least one of the enzymes responsible is an aminopeptidase with similar properties to those isolated from other plant species. LITERATURE CITED 1. Boller T, Kende H (1979) Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol 63: 1123-1132 2. Cheng SH, Kao CH (1984) The role of proteolytic enzymes in protein degradation during senescence of rice leaves. Physiol Plant 63: 231-237 3. De Luca D'Oro GM, Trippi VS (1982) Regulaci6n de clorofilas y proteinas solubles por cinetina y cicloheximida, en condiciones de luz y oscuridad, durante la senescencia foliar de Phaseolus vulgaris L. 0yton 42: 73-82
Plant Physiol. Vol. 91,1989
4. Drivdahl RH, Thimann KV (1977) Proteases of senescent oat leaves. I Purification and general properties. Plant Physiol 59: 1059-1063 5. Drivdahl RH, Thimann KV (1978) Proteases of senescent oat leaves. II. Reaction to substrates and inhibitors. Plant Physiol 61: 501-505 6. Hall NP, Tolbert NE (1978) A rapid procedure for the isolation of ribulose bisphosphate carboxylase/oxygenase from spinacia leaves. FEBS Lett 96: 167-169 7. Kolehmainen L, Mikola J (1971) Partial purification and enzymatic properties of an aminopeptidase from barley. Arch Biochem Biophys 145: 633-642 8. Kuby SA, Noltmann EA (1966) Glucose 6-phosphate dehydrogenase (crystalline) from brewer's yeast. Methods Enzymol 9: 116-125 9. Lamattina L, Archoveri B, Conde DR, Pont Lezica R (1987) Quantification of the kinetin effect on protein synthesis and degradation in senescing wheat leaves. Plant Physiol 83: 497499 10. Liu X-Q, Jagendorf A (1986) Neutral peptidases in the stroma of pea chloroplasts. Plant Physiol 81: 603-608 11. Mae T, Makino A, Ohira K (1984) Relation between ribulose bisphosphate carboxylase content and chloroplast number in naturally senescent primary leaves of wheat. Plant Cell Physiol 25: 333-336 12. Martin C, Thimann KV (1972) The role of protein synthesis in the senescence of oat leaves. I The formation of protease. Plant Physiol 49: 64-71 12a. Mills WR, Joy KW (1980) A rapid method for isolation of purified, physiologically active chloroplasts used to study the intracellular distribution of amino acids in pea leaves. Planta 148: 75-83 13. Naito K, Hda A, Suzuki H, Tsuji H (1979) The effect of benzyladenine on protein changes and protease activity in intact bean leaves during ageing. Physiol Plant 46: 50-53 14. Saleemudin M, Ahmad H, Hussain A (1980) A simple, rapid and sensitive procedure for the assay of endoprotease using Coomasie Brilliant Blue G-250. Anal Biochem 105: 202-206 15. Sedmark JJ, Grossberg SE (1977) A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G-250. Anal Biochem 79: 544-552 16. Sopanen T, Mikola J (1975) Purification and partial characterization of barley leucine aminopeptidase. Plant Physiol 55: 809-814 17. Veierskov B, Thimann KV (1988) The control of protein breakdown and synthesis in the senescence of oat leaves. Physiol Plant 72: 257-264 18. Waters SP, Noble ER, Dalling MJ (1982) Intracellular localization of peptide hydrolase in wheat (Triticum aestivum L.) leaves. Plant Physiol 69: 575-579 19. Waters SP, Dalling MJ (1984) Isolation and some properties of an aminopeptidase from primary leaves of wheat (Triticum aestivum L.). Plant Physiol 75: 118-124 20. Yamagishi A, Satoh K, Katoh S (1981) The concentration and thermodynamic activities of cations in intact Bryopsis chloroplasts. Biochim Biophys Acta 637: 252-263
