peptide bond. Some other proteins such as BSA are hydro- lyzed by MRP, albeit much less rapidly than NR, whereas others like glutamate dehydrogenase and ...
Plant Physiol. (1993) 101: 415-419
Characterization of a Maize Root Proteinase' Valerie I. Goodfellow, Larry P. Solomonson, and Ann Oaks* Botany Department, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 (V.J.G., A.O.); and Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa, Florida 33612 (L.P.S.)
small subset of a11 the possible amino acid sequences and they do not take into account other factors involved in the determination of specificity, such as the three-dimensional structure of the substrate, it was necessary to determine cleavage patterns with protein substrates. It has been established that this proteinase belongs to the class of Ser endopeptidases based on its sensitivity to a number of inhibitors. The classic inhibitor for proteinases that have a Ser residue at their active center is diisopropyl fluorophosphonate, and it completely inhibits MRP (Batt and Wallace, 1989). Elastinal inhibits elastase-type Ser proteinases, whereas chymostatin inhibits chymotrypsin-like Ser proteinases. Both of these inhibitors inactivate MRP (Batt and Wallace, 1989; Long and Oaks, 1990). MRP and other limited-action proteinases have been used to help define structural domains within the NR molecule (Solomonson et al., 1984, 1986; Kubo et al., 1988). NADH NR activity is destroyed by the proteinase, whereas the methyl viologen NR activity is insensitive (Yamaya et al., 1980a, 1980b; Solomonson et al., 1984; Poulle et al., 1987). A hinge region between two domains of the native NR protein is the probable cleavage site (Solomonson et al., 1986; Kubo et al., 1988). In this paper, we have examined the specificity of this endopeptidase with two protein substrates, BSA and Chlorella NR. Using the techniques of microsequence analysis for Nterminal analysis and carboxypeptidase Y identification of the new C-terminal amino acid, the cleavage site was again identified as an Ala residue on the amino side of the scissile bond. The new peptides generated with this proteinase on a model substrate, BSA, were compared with peptides obtained with the standard Ser proteinases trypsin, chymotrypsin, and elastase, and a distinct array of polypeptides was obtained. This demonstrates that MRP has a specificity toward protein substrates that is quite different from the other Ser proteases. The stringent specificity of this proteinase should render it a valuable diagnostic agent both in the elucidation of the primary structure and in defining functional domains of proteins.
The major proteinase in maize (Zea mays) roots behaves as a serine endopeptidase. A possible physiological role of this enzyme could be i n the turnover of nitrate reductase (NR) and, as such, it could be of great importance in regulating the assimilation of nitrate. The objective of this research was to elucidate the specificity and uniqueness of maize root proteinase. When bovine serum albumin and an NR purified from Chlorella vulgaris were used as substrates, the maize root proteinase exhibited a preference for cleavages such that the amino acid on the amino side of the scissile bond was alanine. This information was established by microsequence analysis of the N termini of proteolytic fragments, and carboxypeptidase Y analysis of the C termini of proteolytic fragments of substrates hydrolyzed by the proteinase. Cleavage occurred at the sequence Ala/Ala-Ala-Ala-Pro-Glu in Chlorella NR, and at the sequence Ala-Asp-Glu-Ser-His-Ala-Gln in bovine serum albumin. When bovine serum albumin was the substrate, the maize root proteinase yielded a peptide map that is unique relative to those created with the other serine endopeptidases elastase, trypsin, or chymotrypsin. Based on our data, the maize root proteinase appears to cleave peptide bonds at the carboxy side of alanine. Because of its specificity, i t should have useful applications i n protein chemistry.
A typical Ser endopeptidase isolated from maize roots (MRP) (Shannon et al., 1979) may be responsible for the relatively short half-life of NR (Wallace and Oaks, 1986). It inactivates NR from severa1 species of plants (Wallace, 1975). The action of MRP on Chlorella NR results in the formation of two major polypeptide products (Yamaya et al., 1980b; Solomonson et al., 1986), indicative of cleavage of a single peptide bond. Some other proteins such as BSA are hydrolyzed by MRP, albeit much less rapidly than NR, whereas others like glutamate dehydrogenase and nitrite reductase are insensitive to its action (Wallace, 1973). Thus, although MRP does not selectively attack NR, NR seems to be a preferred target. A complete list of proteins that have been tested for hydrolysis by MRP is given in Table I. Batt and Wallace (1989) cleaved the oxidized p chain of insulin with MRP, separated the resulting fragments, and analyzed the amino acids in each fragment. They found that the endopeptidase cleaved at the Ala'4-Lys'5 peptide bond of insulin (Batt and Wallace, 1989). The proteinase also exhibited the ability to cleave Ala esters. Because these substrates contain only a
MATERIALS A N D METHODS Materials
BSA, trypsin, chymotrypsin, and elastase were purchased from Sigma (St. Louis, MO). Carboxypeptidase Y and OPA
Supported by Natural Sciences and Engineering Research Council (Canada) grant A2818, and National Science Foundation grant DCB90 12390. * Corresponding author; fax 1-519-767-1991.
Abbreviations: CAPS, 3-(cyclohexylamino)-l-propanesulfonic acid; MRP, maize root proteinase; NR, nitrate reductase; OPA, ophthaldialdehyde; PVDF, polyvinylidene fluoride.
415
Goodfellow et al.
41 6
Table 1. Effect of MRP on various substrates CBZ, N-Carbobenzoxy; ONp, p-nitrophenyl ester; Boc, N-t-bu-
toxv carbonvl. Hydrolyzed
NADH-NR (Zea mays)” ( Pisum)b (Ch/ore//a)b Cyt c Reductase (NO, in-
duced) (Zea mays)” NADPH-NR (Neurospora)b lnsuline Casein‘ Haemoglobin‘ BSA~ P-GalactosidaseB CBZ-Ala-ONp‘ Boc-Ala-ONp‘ a
Not Hydrolyzed
NADH-NR (Pseudomonas)b
(Nitrobacter)b Methyl viologen-NR (Zea mays)’ Flavin adenine dinucleotide-NR (Zea maysPd
Nitrite reductase’ Glutamate dehydrogenase” Xant h i n e oxidase” lsocitrate lyase” Lysozyme8 Ribonuclease A8 CBZ-Vai-ONp‘ C BZ-G ly-ON p‘
1973. Wallace, 1975. ‘Yamaya et ai., Yamaya et ai., 1980b. e Batt and Wallace, Shannon and Wallace, 1979. 8 Unpublished results.
Wallace,
1980a.
1989.
This investieation.
were purchased from Pierce Chemical Co. (Rockford, IL). Westran was purchased from Schleicher & Schuell (Keene, NH). Maize kernels (Zea mays W64A X 182E) were purchased from Wisconsin Seed Foundation.
Plant Physiol. Vol. 101, 1993
Preparation of Peptides for Microsequence Analysis
Peptides were separated by SDS-PAGE (Laemmli, 1970) and transferred to PVDF membrane (Westran), according to the methods previously described (Matsudaira, 1987). The transfer buffer contained 10 mM Caps, pH 11.0, with 10% methanol. The peptides were then made visible by staining with Coomassie brilliant blue R-250. The desired bands were excised and sequenced. Sequencing was performed by the Biotechnology Service Centre, the Hospital for Sick Children, Toronto, Ontario. C-Terminal Analysis
Carboxypeptidase Y was used to determine the C-terminal amino acid sequence of a peptide, with a modification of the method of Martin et al. (1971). It is an enzyme that sequentially cleaves the C-terminal amino acid from a peptide substrate, and it exhibits broad specificity toward the amino acids it cleaves. The large peptide fragment from the reaction of MRP with Cklorella NR was incubated with carboxypeptidase Y, and 0.25% SDS (w/v) at 28OC for various times. The reaction was terminated by the addition of an equal volume of methanol, and the precipitated protein was removed by centrifugation. Small peptides and free amino acids derived from the reaction were contained in the supernatant fraction. The amino acids were derivatized with OPA and separated and quantitated by reverse-phase HPLC (Winspear and Oaks, 1983). The determination of the C-terminal sequence was based on the relative rates of release of amino acids.
MRP Purification
The proteinase from maize roots was purified by the procedures previously described (Shannon and Wallace, 1979) with the following modifications. The Sephadex G-100 column was replaced by a hydroxyl apatite/brushite 1:3 (v/v) column equilibrated in 10 mM potassium phosphate buffer, pH 7.0. Elution of the protein from this matrix was accomplished by a linear gradient of 10 to 500 mM potassium phosphate buffer, pH 7.0. The fractions exhibiting enzymic activity were pooled and dialyzed against 10 mM phosphate buffer, pH 7.0. Purification of NR from Chlorella, as well as isolation of the fragments generated from the reaction of this protein with MRP, was achieved by methods described previously (Solomonson et al., 1984). Treatment with Proteinases
Purified substrate proteins were incubated at 28OC in 0.04 Tris-HC1 buffer, pH 6.8, with the chosen proteinases. The reactions were terminated at the desired time by denaturing them with buffer containing 2% (w/v) SDS, 5% (v/v) 2mercaptoethanol, and 10% (v/v) glycerol and placing them in a boiling water bath for 2.5 min. Substrate pretreatment with SDS involved incubation of the substrate with 0.1% SDS (w/v) for 10 min at 28OC prior to the addition of proteinase. Peptides were then separated by SDS-PAGE (Laemmli, 1970), visualized by silver staining (Wray et al., 1981), or transferred to PVDF membrane for microsequence analysis. M
RESULTS AND DlSCUSSlON Characterization of M R P
MRP was routinely purified from corn root homogenates. Purity was confirmed by silver staining after SDS-PAGE. With this technique only one protein band was observed (data not shown). Final preparations contained 3.5% of the maximum observed activity. Fresh frozen roots (450 g) yielded 20 pg of MRP protein. The specific activity of this preparation with NR as a substrate was 0.17 units/mg of protein. This recovery was similar to that described by Shannon and Wallace (1979). A unit of MRP activity is defined as that which inactivates 1 unit of NR/min at 28OC. A unit of NR activity is defined as that which produces 1 pmol of N02-/min at 28OC. MRP can partially hydrolyze a wide range of proteins, in addition to the NR protein (Table I). However, some substrates are cleaved much more efficiently than others. For example, cleavage of NR is about 10 times faster than the cleavage of BSA even after treatment of BSA with 0.1% SDS. Using the model protein BSA as a substrate for MRP, we have shown that by altering its structure by pretreatment with SDS, the peptide map is dramatically altered relative to that of nondenatured BSA (Fig. 1). Both with and without SDS, BSA is cleaved, indicating that MRP recognizes the primary structure of the substrate molecule. Microsequence analysis of the BSA (A) (66 kD) and degradation products B (35 kD) and C (30 kD) yielded N-terminal sequences of Asp-
Characterization of a Maize Root Proteinase BSA
BSA+MRP BSA+SDS +MRP
417
MRP
66 kD
45 36 29 24
0
20
40
60
80
100
120
TIME OF CPY REACTION (MIN) Figure 1. Blot for microsequence analysis of BSA. SDS-PAGE (13%) of MRP-treated BSA was transferred to PVDF for microsequence analysis. The membrane was stained with Coomassie brilliant blue. Each lane contained 16 ^g of BSA, with or without pretreatment with 0.1% SDS and/or 1.3 Mg of MRP as indicated. The reaction was allowed to proceed for 6 h. The N-terminal sequence of bands A, B, and C were Asp-Thr-His-Lys-Ser-GIn, Asp-Thr-His-Lys-SerGln, and Asp-Glu-Ser-His-Ala-Gly, respectively.
Thr-His-Lys-Ser-Gly (N terminus of BSA), Asp-Thr-His-LysSer-GIn, and Asp-Glu-Ser-His-Ala-Gly (N terminus of new fragment), respectively. Each lane contained 240 pmol of BSA. Quantities of amino acids detected during sequencing were in the range of 10 pmol. Comparison of the results with that of the established sequences of BSA (Fasman, 1976) shows that one of the major cleavage sites of this substrate is Ala55-Asp56. The sequence Asp-Glu-Ser-His-Ala-Gly is only found once in the entire BSA sequence, starting at amino acid 56. Peptides from the C-terminal region of BSA were more fully hydrolyzed, and the resultant fragments, which were too small to be detected, were not sequenced. Other fragments from the BSA + MRP treatment (Fig. 1) did not
-MRP
+MRP
200 kD 116 96 66 45 24
Figure 3. Chlorella NR large fragment C-terminal analysis. The reaction contained 80 ^M large fragment and 0.8 JIM carboxypeptidase Y. An aliquot was removed at each time point and proteins were precipitated with an equal volume of methanol. A portion of the supernatant containing free amino acids was removed, derivatized with OPA, and injected into the HPLC apparatus for amino acid analysis. The C-terminal sequence was determined based on the relative rate of release of amino acids. •, Ala; x, Val; A, Leu; +, lie.
give definitive sequence results due to the presence of multiple peptides or low protein concentrations. Chlorella NR was chosen as another substrate for MRP because it was relatively easy to purify and because it was known that there were two major reaction products, a large and a small fragment (Solomonson et al., 1984). The disadvantage of this substrate is that the entire amino acid sequence of Chlorella NR is not yet known. NR proteins from other higher plant sources are difficult to purify to homogeneity; however, in many cases the amino acid sequences have been deduced from known nucleotide sequences (DanielVedele et al., 1989). Microsequence analysis of the blots of the type shown in Figure 2 yielded no data for the N terminus of either the native NR (band A) or the large fragment (band B), a result that suggests that this protein may have a blocked N terminus. The small fragment (band C) created by the action of MRP had a unique N-terminal sequence of AlaAla-Ala-Pro-Glu. Each lane contained 100 pmol of Chlorella NR and quantities of amino acids detected during microse-
Table II. Cleavage specificity of MRP Bold denotes sequences determined by microsequence analysis. Boc, N-t-butoxycarbonyl; ONp, p-nitrophenyl ester. Substrate
Alanine esters Figure 2. Blot for microsequence analysis of NR. SDS-PAGE (11.5%) of Chlorella NR treated with MRP and transferred to PVDF for microsequence analysis. The membrane was stained with Coomassie brilliant blue. Each lane contained 11 ^g of Chlorella NR with or without 0.2 ^g of MRP as indicated. The reaction was allowed to proceed for 3 h. Bands A and B did not yield any sequence data. The N-terminal sequence of band C was Ala-AlaAla-Pro-Glu.
Insulin (3 chain BSA
Chlorella NR 'Battand Wallace, 1989.
Cleavage Site
1 Boc-A-ONpa 1 -H-L-V-E-A-L-Y-L-V-C-' 1 -L-T-C-V-A-D-E-S-HI A-A-A-A-P-E-
Goodfellow et al.
418
Plant Physiol. Vol. 101, 1993
a specificity that is unique relative to trypsin, chymotrypsin, and elastase. 66 kD
5
—— * ... >-
45 36 29 24 20 14
Figure 4. Peptide MRP of BSA. Silver-stained 15% SDS-PACE peptide map of BSA. 1, + Chymotrypsin; 2, + trypsin; 3, + elastase; 4, + MRP; and 5, substrate (BSA alone). Each lane contained 4 ^8 of BSA with or without 0.16 Mg of MRP, 0.4 ftg of elastase, 0.4 jig of
trypsin, or 0.4 ^g of chymotrypsin as indicated. Reactions were terminated after 16 h for MRP, 1 h for elastase, 1.5 h for trypsin, and 30 min for chymotrypsin. Arrows indicate the positions of the proteinases.
quence analysis were in the range of 10 pmol. The amino acid sequence deduced from nucleotide sequences of maize NR also contains a similar sequence, Ala-Ala-Ala-Pro-Gly, which is localized in the relatively variable hinge region of the protein (Daniel-Vedele et al., 1989). We suspect that this sequence is also in the hinge region of Chlorella NR. To complete the picture with this substrate, we examined the C terminus of the large fragment by the carboxypeptidase Y reaction. The results show that Ala is the new C-terminal amino acid (Fig. 3). Thus, once again MRP cleaves such that the newly created fragment has a C-terminal Ala. As a control for this experiment, the C terminus of BSA was also determined, and the expected result of Ala was obtained (data not shown). Because MRP-treated maize NR yields different size fragments than does MRP-treated Chlorella NR (Poulle et al., 1987), it is likely that these two substrates are cleaved at different Ala residues. Table II summarizes the data on the cleavage site of MRP with each substrate. It is clear that this endopeptidase shows a preference for hydrolysis of peptide bonds containing the small hydrophobic Ala residue on the N-terminal side of the targeted bond and has no apparent specificity for the amino acid on the C-terminal side of the targeted bond.
Comparison of Cleavage Site to Other Ser Endopeptidases The activity of MRP for production of new peptides was compared with that of other Ser endopeptidases using BSA as a substrate (Fig. 4). The time of the reaction for each proteinase was optimized such that the substrate protein was not completely hydrolyzed. Each of the limited action proteinases cleaved BSA in a distinct manner, resulting in different polypeptide products. These results show that MRP has
CONCLUSIONS Previous investigations with model substrates indicated that MRP catalyzes the scission of a peptide (or ester) bond on the carboxyl side of Ala residues (Batt and Wallace, 1989). These findings suggested that MRP was similar to elastase. Further investigation was required to determine the specificity of cleavage of MRP with large protein substrates and to compare MRP with other Ser proteinases. The products of MRP-mediated degradation of two proteins, BSA and Chlorella NR, were isolated and subjected to analysis to identify the amino acid sequences on either side of the cleavage site. These results confirm the fact that MRP cleaves on the carboxy side of an Ala residue, and also that the action of MRP is not influenced by the nature of the amino acid residue on the other side of the targeted bond. The high degree of specificity exhibited by MRP should render it useful in the characterization of the primary structures of proteins. MRP has been shown to hydrolyze NR in vitro. However, further research is required to established whether this proteinase actually has a role in regulating the levels of the NR protein in vivo. Received July 15, 1992; accepted October 15, 1992. Copyright Clearance Center: 0032-0889/93/101/0415/05. LITERATURE CITED
Batt, R, Wallace W (1989) Characteristics of the active site and substrate specificity of a maize root endopeptidase. Biochim Biophys Acta990: 109-112 Daniel-Vedele F, Dorbe M-F, Caboche M, Rouze P (1989) Cloning and analysis of the tomato nitrate reductase-encoding gene: protein domain structure and amino acid homologies in higher plants. Gene 85: 371-380 Fasman GD, ed (1976) Handbook of Biochemistry and Molecular Biology, Vol 3, Proteins. CRC Press, Cleveland, p 497 Kubo Y, Ogura N, Nakagawa H (1988) Limited proteolysis of the nitrate reductase from spinach leaves. J Biol Chem 263: 19684-19689 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 Long DM, Oaks A (1990) Stabilization of nitrate reductase in maize roots by chymostatin. Plant Physiol 93: 846-850 Martin B, Svendsen I, Ottesen M (1977) Use of carboxypeptidase Y for carboxy-terminal sequence determination in proteins. Carlsberg Res Commun 42: 99-102 Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262: 10035-10038 Poulle M, Oaks A, Bzonek P, Goodfellow VJ, Solomonson LP (1987) Characterization of nitrate reductases from corn leaves (Zea mays cv W64AxW182E) and Chlorella vulgaris. Plant Physiol 85: 375-378 Shannon JD, Wallace W (1979) Isolation and characterisation of peptide hydrolases from the maize root. Eur J Biochem 102: 399-408 Solomonson LP, Barber MJ, Robbins AP, Oaks A (1986) Functional domains of assimilatory NADH:nitrate reductase from Chlorella.} Biol Chem 261: 11290-11294 Solomonson LP, Howard WD, Yamaya T, Oaks A (1984) Mode of
Characterization of a Maize Root Proteinase action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase. Arch Biochem Biophys 233: 469-474 Wallace W (1973) A nitrate reductase inactivating enzyme from the maize root. Plant Physiol 52: 197-201 Wallace W (1975) Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora. Biochim Biophys Acta 377: 239-250 Wallace W, Oaks A (1986) Role of proteinases in the regulation of nitrate reductase. Zn MJ Dalling, ed, Plant Proteolytic Enzymes, Vol. 2. CRC Press, Boca Raton, FL, pp 81-89
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Winspear MJ, Oaks A (1983) Automated pre-column amino acid analyses by reverse phase high performance liquid chromatography. J Chromatqgr 270: 378-382 Wray W, Boulikas T, Wray VI’, Hancock R (1981) Silver staining of proteins in polyacrylamide gels. Ana1 Biochem 118: 197-203 Yamaya T, Oaks A, Boesel IL (1980a) Characteristics of nitrate reductase-inactivating proteins obtained from corn roots and rice cell cultures. Plant Physiol 65: 141-145 Yamaya T, Solomonson LP, Oaks A (1980b) Action of corn and rice-inactivating proteins on a purified nitrate reductase from Chlorella vulgavis. Plant Physiol 65: 146-150
