Proteases catalyzing vicilin cleavage in developing pea (Pisum sativum L.) seeds.

Legume species differ in whether or not the 7S globulins stored in seeds undergo proteolytic processing during seed development, while preserving the bicupin structure and trimeric assembly necessary for accumulation and packing into protein storage vacuoles. Two such cleavage sites have been documented for the vicilins in pea cotyledons: one in the linker region between the two cupin domains, and another in an exposed loop in the C-terminal cupin. In this report, we explain the occurrence of vicilin cleavage in developing pea by showing that the storage vacuoles are already acidified before germination, in contrast to soybean and peanut where acidification occurs only after germination.

Transfer Zymography.

The technique described here, transfer zymography, was developed to overcome two limitations of conventional zymography. When proteolytic enzymes are resolved by nonreducing SDS-PAGE into a polyacrylamide gel with copolymerized protein substrate, the presence of the protein substrate can result in anomalous, often slower, migration of the protease and an estimated mass higher than its actual mass. A further drawback is that the presence of a high background of substrate protein interferes with proteomic analysis of the protease band by excision, tryptic digestion, and LC-MS/MS analysis.

Role of vacuolar membrane proton pumps in the acidification of protein storage vacuoles following germination.

During soybean (Glycine max (L.) Merrill) seed development, protease C1, the proteolytic enzyme that initiates breakdown of the storage globulins β-conglycinin and glycinin at acidic pH, is present in the protein storage vacuoles (PSVs), the same subcellular compartments in seed cotyledons where its protein substrates accumulate. Actual proteolysis begins to be evident 24 h after seed imbibition, when the PSVs become acidic, as indicated by acridine orange accumulation visualized by confocal microscopy.

Proteolysis of the peanut allergen Ara h 1 by an endogenous aspartic protease.

The 7S and 11S globulins of peanuts are subjected to proteolysis two days after seed imbibition, with Ara h 1 and the arachin acidic chains being among the first storage proteins to be mobilized. Proteolytic activity was greatest at pH 2.6-3 and is inhibited by pepstatin A, characteristic of an aspartic protease.

College Students' Views of Work-Life Balance in STEM Research Careers: Addressing Negative Preconceptions.

In career discussions, female undergraduates said that if they were to attend graduate school in science, technology, engineering, and mathematics (STEM) and were to follow a career based on their research training, they would have to give up having a family. A subsequent survey showed that many students, both men and women, thought work-life balance would be more difficult to achieve in a STEM research path than in other professions they were considering. Their views of STEM research being less family-friendly were more pronounced on issues of parental leaves and caring for children than finding a spouse/partner and landing two jobs in the same locality.

Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases.

Filtering of MS/MS data for peptide identification.

Background: The identification of proteins based on analysis of tandem mass spectrometry (MS/MS) data is a valuable tool that is not fully realized because of the difficulty in carrying out automated analysis of large numbers of spectra. MS/MS spectra consist of peaks that represent each peptide fragment, usually b and y ions, with experimentally determined mass to charge ratios. Whether the strategy employed is database matching or De Novo sequencing, a major obstacle is distinguishing signal from noise.

Seed storage proteins as a system for teaching protein identification by mass spectrometry in biochemistry laboratory.

Mass spectrometry (MS) has become an important tool in studying biological systems. One application is the identification of proteins and peptides by the matching of peptide and peptide fragment masses to the sequences of proteins in protein sequence databases. Often prior protein separation of complex protein mixtures by 2D-PAGE is needed, requiring more time and expertise than instructors of large laboratory classes can devote.

The PA domain is crucial for determining optimum substrate length for soybean protease C1: structure and kinetics correlate with molecular function.

A subtilisin-like enzyme, soybean protease C1 (EC 3.4.21.

Mobilization of seed protein reserves.

The mobilization of seed storage proteins upon seed imbibition and germination is a crucial process in the establishment of the seedling. Storage proteins fold compactly, presenting only a few vulnerable regions for initial proteolytic digestion. Evolutionarily related storage proteins have similar three-dimensional structure, and thus tend to be initially cleaved at similar sites.

Electrophoretic transfer protein zymography.

Zymography detects and characterizes proteolytic enzymes by electrophoresis of protease-containing samples into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing a copolymerized protein substrate. The usefulness of zymography for molecular weight determination and proteomic analysis is hampered by the fact that some proteases exhibit slower migration through a gel that contains substrate protein. This article introduces electrophoretic transfer protein zymography as one solution to this problem.

Phytochrome A mediates rapid red light-induced phosphorylation of Arabidopsis FAR-RED ELONGATED HYPOCOTYL1 in a low fluence response.

Phytochrome A (phyA) is the primary photoreceptor for mediating the far-red high irradiance response in Arabidopsis thaliana. FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its homolog FHY1-LIKE (FHL) define two positive regulators in the phyA signaling pathway. These two proteins have been reported to be essential for light-regulated phyA nuclear accumulation through direct physical interaction with phyA.

Applicability of multigene family-specific antibodies toward studies of the subtilases in Arabidopsis thaliana.

Polyclonal antibodies were made to two synthetic peptides with sequences patterned after conserved regions in a multigene family of 56 subtilisin-related proteolytic enzymes in Arabidopsis thaliana. GST-fusion proteins encompassing full-length or partial cDNAs bearing putative epitope regions were cloned and expressed in Escherichia coli. Immunoblots showed that the antibodies bound 12 of 13 fusion proteins tested.

Protein storage vacuole acidification as a control of storage protein mobilization in soybeans.

Soybean protease C1 (EC 3.4.21.

Light-responsive subtilisin-related protease in soybean seedling leaves.

Protease C1 (E.C. 3.

New serine carboxypeptidase in mung bean seedling cotyledons.

Two serine carboxypeptidases (EC 3.4.16.

Cleavage specificity of the subtilisin-like protease C1 from soybean.

The cleavage specificity of protease C1, isolated from soybean (Glycine max (L.) Merrill) seedling cotyledons, was examined using oligopeptide substrates in an HPLC based assay. A series of peptides based on the sequence Ac-KVEKEESEEGE-NH2 was used, mimicking a natural cleavage site of protease C1 in the alpha subunit of the storage protein beta-conglycinin.

Effect of embryonic axis removal and exogenous calcium on carboxypeptidase I of mung bean seedling cotyledons.

Removal of the embryonic axis prevents the normal decline of carboxypeptidase (Cpase) I in mung bean seedling cotyledons. Cpase I activity and protein, the latter manifested on western blots, almost completely disappear about 24 h before the cotyledon abscises. Of the 3 proteolytic enzyme patterns, only that of Cpase I can be restored by an exogenous supply of 10 mM CaCl2 in the agar growth medium.

Protease C2, a cysteine endopeptidase involved in the continuing mobilization of soybean beta-conglycinin seed proteins.

The protease that degrades the beta subunit of the soybean (Glycine max (L.) Merrill) storage protein beta-conglycinin was purified from the cotyledons of seedlings grown for 12 days. The enzyme was named protease C2 because it is the second enzyme to cleave the beta-conglycinin storage protein, the first (protease C1) being one that degrades only the alpha' and alpha subunits of the storage protein to products similar in size and sequence to the remaining intact beta subunit.

An acidic amino acid-specific protease from germinating soybeans.

The degradation of the beta-conglycinin protein reserves in soybean seeds during germination and early growth begins with the proteolysis of its alpha and alpha' subunits by an enzyme called Protease C1. In the pathway, a number of proteolytic intermediates are produced and subsequently degraded. Determination of the N-terminal sequences of these intermediates provides insight regarding the requirements of the cleavage sites.

Characterization of a soybean beta-conglycinin-degrading protease cleavage site.

Protease C1, an enzyme from soybean (Glycine max [L.] Merrill cv Amsoy 71) seedling cotyledons, was previously determined to be the enzyme responsible for the initial degradation of the alpha' and alpha subunits, but not the beta subunit, of beta-conglycinin storage protein. The sizes of the proteolytic products generated by the action of protease C1 suggest that the cleavage sites on the alpha' and alpha subunits of beta-conglycinin may be located in their N-terminal domain, which is not found in the beta subunit of beta-conglycinin.

Characterization of the Major Protease Involved in the Soybean beta-Conglycinin Storage Protein Mobilization.

Protease C1, the protease responsible for the initial degradation of the alpha' and alpha subunits of the soybean beta-conglycinin storage protein (Glycine max [L.] Merrill), has been purified. The enzyme was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of 70,000 and a pH optimum of 3.

Isolation and Partial Characterization of a Subtilisin Inhibitor from the Mung Bean (Vigna radiata).

The subtilisin inhibitor (MBSI-A) from the mung bean (Vigna radiata (L.) Wilczek) seed has been purified to homogeneity. MBSI-A consists of a single polypeptide chain of 119 residues, with a high content of glutamic acid/glutamine, aspartic acid/asparagine, valine, threonine, and proline (19, 12, 10, 9, and 8 residue percent, respectively).

Survey of the Proteolytic Activities Degrading the Kunitz Trypsin Inhibitor and Glycinin in Germinating Soybeans (Glycine max).

The cotyledons of the soybean (Glycine max [L.] Merrill cv Amsoy 71) were examined for proteolytic activities capable of degrading soybean seed proteins. Three distinct activities were identified that attack the native Kunitz soybean trypsin inhibitor of Amsoy 71, Ti(a).