A metalloendoproteinase from leaves of soybean (Glycine max) has been purified 1160-fold to electrophoretic homogeneity. The native protein is monomeric with a molecular mass of 15 kilodaltons as estimated by gel filtration and 19 kilodaltons as estimated by denaturing gel electrophoresis. The enzyme has a pH optima of 8.0 to 9.0 using Azocoll as substrate. The proteolytic activity is susceptible to metal chelating agents and the inactivated enzyme can be restored to 69% of original activity by the addition of ZnC12. Western analysis shows that a fraction of the soybean metalloendoproteinase is present within the extracellular space of older leaves. Soybean metalloendoproteinase 1 is the Azocollase A activity first described by Ragster and Chrispeels (Plant Physiol 64: 857-862;).An understanding of proteolytic processes and the enzymes involved is paramount to a thorough knowledge of metabolism at both the cellular and tissue levels. Proteolytic enzymes are categorized into four classes based on catalytic mechanisms. These classes are serine, cysteine, aspartic acid, and metalloproteinases (1). Although the number of reports describing plant proteolytic activities has increased substantially over the last decade, few proteinases have been purified to homogeneity or studied in molecular detail (9,15,18). Most plant proteolytic activities examined appear to fall into the cysteine or aspartic acid classes. Those proteinases involved with the metabolism of seed storage proteins and senescence have received the most attention (8,22). With the exception of a few reports (3,19,25) the metallo and cysteine classes of proteinases, respectively. Their findings have aided us in further characterization of the Azocollase A activity, including the purification to homogeneity of this enzyme. We provide additional evidence that the enzyme is a metalloendoproteinase, which we have termed SMEPl. The proteinase is most abundant in maturing leaves and appears to be localized extracellularly. This is the first report of purification to homogeneity of a metalloendoproteinase from a plant. MATERIALS AND METHODS Plant MaterialSoybean plants (Glycine max var Williams 82) were grown in environmental growth chambers under a 16 h photoperiod. Seeds were obtained from Mid-Wood Inc. (Bowling Green, OH). Enzymatic AssayDetermination of SMEPI activity was accomplished by addition of 5 to 100 ,uL of protein sample to 1 mL of 25 mm Tris, pH 9.0, containing 1 mg (dry weight) of Azocoll (Calbiochem). The reaction mixtures were allowed to shake (250 rpm) for 1 h at 37°C in an orbital shaker. Mixtures contained a saturating amount of substrate and the amount of enzyme was adjusted so that rates ofhydrolysis were linear with respect to time. Reaction mixtures were briefly centrifuged and the supernatant fluids monitored for an increase in absorbance at 520 nm. Units of activity are defined as A optical density 520 nm/h. Protein DeterminationProtein concentrations were determined by the Bio-Rad assay system (5) according to the manufacturer'...

Progeny of two transgenic tobacco (Nicotiana tabacum L.) lines that expressed an activated form of maize (Zea mays L.) ribosome-inactivating protein (RIP) had varying resistance to the insect species tested. A subset of R(2) plants from the two lines appeared to be more resistant to larvae of the cigarette beetle, Lasioderma serricorne (F.), and the tobacco hornworm, Manduca sexta (L.) than the wild type plants. Progeny (R(3)) of the more resistant R(2) plants were tested more extensively for insect resistance. Resistance to the corn earworm, Helicoverpa zea (Boddie), was most consistent, with significantly decreased feeding often accompanied by increased mortality and reduced weights of survivors fed on leaf disks of the two transgenic lines compared to the wild type. The amount of damage by H. zea was significantly inversely correlated with levels of RIP. Resistance of RIP-producing plants to H. zea was greater than expected on the basis of prior in vitro results using diet-incorporated maize RIP. The R(3) transgenic plant leaf disks were also often more resistant to feeding by larvae of L. serricorne compared to wild type plants. Although reduced feeding by M. sexta was noted when they were fed leaf disks from transgenic compared to wild type plants the first day of exposure, differences were not significant. This information provides further support for maize RIP having a role in resistance to maize-feeding insects.

A novel zinc endoproteinase has been sequenced and characterized from soybean leaves (Glycine max var Williams 82) and has been designated as Protein Identification Resource accession No. A41820 SMEP1 (soybean metalloendoproteinase 1). Comparison of the primary amino acid sequence with other zinc proteinases revealed the enzyme to be a new member of the matrix metalloproteinase (MMP) family of enzymes. SMEP was found to have MMP cleavage specificity toward peptide substrates and the enzyme is specifically inhibited by naturally occurring tissue inhibitors of MMPs through a high-affinity interaction (inhibitor concentration resulting in an approximate 50% decrease in enzyme activity = 23 x 10-molar). of SMEP1 and further characterization of the plant protein that places the enzyme within the MMP family. MATERIALS AND METHODSChemical Sequencing SMEP1 was purified for sequencing as previously described (10) followed by HPLC purification in a Polypore RP column (2.1 x 30 mm, Brownlee) using a linear gradient of acetonitrile/0.1% TFA (8-80%) over 30 min. Reduction and alkylation were performed by incubating the protein in 6 M guanidine-HCL, 1 M Tris, 20 mm DTT (Calbiochem), 10 mm EDTA at pH 8.6 for 1 h at 370C under nitrogen followed by addition of 4-vinylpyridine (Sigma) to 50 mi. All sequence-grade proteinase (Boehinger Mannheim, Indianapolis, IN) digestions, except pepsin (Pierce Chemical Co., Rockford, IL), were performed at room temperature over 16 to 19 h under conditions recommended by the suppliers and as previously described (9). For Asp-N digests, the protein was pretreated by incubation with 4 M urea for 30 min at 500C. Digestion with immobilized pepsin was performed in 0.1S% formic acid with the addition of a few beads for 15 min. Reduction of methionine sulfoxide was performed using 750 mm DTT for 48 h at 370C. Automated Edman degradations were performed using the ABI 477A liquid pulse sequencer and an ABI 120A PTH analyzer. Mass spectra of the intact SMEP1 protein or peptide fragments were collected on a Sciex API-III mass spectrometer equipped with ion-spray inlet. Spectra were scanned from m/z 300 to m/z 2200 repetitively at 5 s per scan and several spectra were averaged for molecular mass calculations. Cleavage SpecificityDigests were conducted with 100 /LM substrate and 20 nm enzyme using the buffer conditions of Stack and Gray (19). Hydrolyses were initiated by the addition of SMEP1 to a solution of DNP-Pro-Leu-Gly-Leu-Trp-Ala-d-Arg-NH2 (100 'tM) in 100 ,L of assay buffer. The reaction was incubated at 370C and hydrolysis proceeded for 2.5 h. Aliquots were removed during the hydrolysis and the reaction quenched www.plantphysiol.org on April 3, 2019 -Published by Downloaded from