Two subtilisin‐like proteases from soybean

Abstract

Two subtilisin-like proteases (SLP) were identified in soybean (Glycine max [L.] Merr.). The first, SLP-1, was localized in seed coats early in seed development, but became undetectable with anti-SLP-1 antibodies as seed fill progressed. A partial purification of SLP-1 was achieved using a two step chromatographic procedure. NH2-terminal sequence analysis of the partially purified enzyme permitted primers to be designed that were used to amplify cDNA encoding SLP-1. A genomic clone encoding SLP-1 was also obtained. Characterization of the cDNA and partially purified SLP-1 revealed the initial translation product was an 82 694 MW precursor. After removal of a signal peptide, the mature protein was formed by removal of an NH2-terminal propeptide. A COOH-terminal peptide also appeared to be removed from some of the protease molecules. DNA blot analysis suggested that at least one additional SLP gene was present in soybean. The second gene, SLP-2, was subsequently cloned and characterized. Although the coding regions for SLP-1 and SLP-2 were homologous, their promoters were quite divergent. RT-PCR revealed that SLP-2 message was found in the mature plant and in cotyledons of germinating seeds. Although SLP-2 mRNA could be identified in developing seeds, the message was at least an order of magnitude less abundant than that for SLP-1, and it was mis-spliced such that a chain termination event would preclude obtaining a product. As with SLPs from other organisms, the functions of the soybean proteases are unknown. However, SLP-1 is one of only a few proteins from soybean seed coats that have been described.

Sequential Development of Cysteine Proteinase Activities and Gene Expression during Somatic Embryogenesis in Carrot

“…The crude enzyme solution (300 g of protein) was loaded into a well of the stacking gel, and proteins were separated at 100 V at 4 C. After PAGE, the gels were rinsed with 1 M sodium acetate buffer, pH 5.4, containing 30 mM 2-ME for a few minutes and overlaid on a gelatin plate containing 1% (w/v) swine skin gelatin (Type I from Sigma), 8% (w/v) acrylamide, 0.1% (w/v) tetramethylenediamine (TEMED), and 0.1% ammonium persulfate. The PAGE gel-gelatin plate sandwich was incubated for between 14 and 48 h at 37 C. Subsequently, the gelatin plate was soaked in 1% (w/v) amido black 10B in 7% (v/v) acetic acid. After destaining with 7% acetic acid, clear bands were present where the gelatin had been digested by the proteinases.…”

“…For example, asparaginyl endopeptidase as a cysteine proteinase has been found in several developing legume seeds including jackbean, soybean, mung bean, peanut, and kidney bean. 8) Other cysteine proteinases or several subtilisin-like serine proteinases and their mRNAs also have differential developmental patterns during seed maturation in tung, 9) Vicia, 10,11) soybean, [12][13][14] and peanut. 15) Many of these enzymes must be stored in protein bodies and degrade storage proteins during/after germination.…”

Identification and Purification of Metalloprotease from Dry Grass Pea (Lathyrus sativus L.) Seeds

“…Numerous reports including our previous data supported that the proteases are responsible for protein degradation [5][6][7][8][9][10]. With renewed interest, there has been proliferation of reports in the last decade concerning purification and characterization of these proteases from leguminous and nonleguminous seeds [2,[11][12][13][14][15][16]. Exploration for existence of valuable proteases as well as understanding the appropriate physiological role of such proteases in plants is still an open area of investigation.…”

A Combined Proteome and Transcriptome Analysis of Developing Medicago truncatula Seeds

“…3B and 4) matched one gene sequence accessible in the BAC clone mth2-12j18 (GenBank AC146561.9). The predicted molecular masses and pI values of the polypeptides correspond with co-and post-translational modifications of a prepro-enzyme (65). The transcript and protein spots peaked in the prestorage phase and were not detected in other tissues (Fig.…”