Silvia Valdés-Rodríguez

Digestive amylase from the larger grain borer, Prostephanus truncatus Horn

A combination of ion-exchange chromatography, preparative electrophoresis and gel filtration chromatography allowed a 1209-fold purification of one of the two major digestive alpha-amylases from larvae of the larger grain borer, Prostephanus truncatus Horn. The purified enzyme showed a molecular mass of 60.2 kDa, an isoelectric point of 4.7 and an optimal pH for activity of 6.0. The enzyme was heat labile and it was recognized by proteinaceous inhibitors from amaranth seeds (Amaranthus hypochondriacus), whereas extracts from maize (Zea mays) and tepary bean (Phaseolus acutifolius) produced very low inhibition. When the enzyme was measured at different stages of development, maximal activity was found in the second instar larvae. Activity drastically decreased to a very low level during the pupae stage and increased again at the adult stage. A zymogram of the different developmental stages showed two main bands of alpha-amylase activity, which almost disappeared at the pupae stage to increase again during the adult stage, revealing a new, smaller band. This new band may be required for a better adaptation of the adult insect to its new environment.

α-amylases from three species of stored grain coleoptera and their inhibition by wheat and corn proteinaceous inhibitors☆

Abstract Two α-amylase isozymes were isolated from adults of both the rice weevil, Sitophilus oryzae , and the red flour beetle, Tribolium castaneum , and a single α-amylase from adults of the yellow mealworm, Tenebrio molitor . All of the purified enzymes had similar amino acid compositions as well as physical and chemical properties. The apparent molecular masses ranged from 53 to 58 kDa. Circular dichroism measurements revealed approx. 33% α-helical content. V max and K m values ranged from 1.33 to 5.98 mM min −1 mg −1 and 0.76 to 5.57 mg ml −1 , respectively, using starch as the substrate. An α-amylase inhibitor from wheat (WRP-25) inhibited all five enzymes, whereas an inhibitor from corn inhibited only enzymes from the red flour beetle and yellow mealworm.

Purification, Characterization, and Complete Amino Acid Sequence of a Trypsin Inhibitor from Amaranth (Amaranthus hypochondriacus) Seeds

A protein proteinase inhibitor was purified from a seed extract of amaranth (Amaranthus hypochondriacus) by precipitation with (NH4)2SO4, gel-filtration chromatography, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. It is a 69-amino acid protein with a high content of valine, arginine, and glutamic acid, but lacking in methionine. The inhibitor has a relative molecular weight of 7400 and an isoelectric point of 7.5. It is a serine proteinase inhibitor that recognizes chymotrypsin, trypsin, and trypsin-like proteinase activities extracted from larvae of the insect Prostephanus truncatus. This inhibitor belongs to the potato-I inhibitor family, showing the closest homology (59.5%) with the Lycopersicum peruvianum trypsin inhibitor, and (51%) with the proteinase inhibitor 5 extracted from the seeds of Cucurbita maxima. The position of the lysineaspartic acid residues present in the active site of the amaranth inhibitor are found in almost the same relative position as in the inhibitor from C. maxima.

Identification of avocado (Persea americana) root proteins induced by infection with the oomycete Phytophthora cinnamomi using a proteomic approach.

Avocado root rot, caused by Phytophthora cinnamomi, is the most important disease that limits avocado production. A proteomic approach was employed to identify proteins that are upregulated by infection with P. cinnamomi. Different proteins were shown to be differentially expressed after challenge with the pathogen by two-dimensional (2-D) gel electrophoresis. A densitometric evaluation of protein expression indicated differential regulation during the time-course analyzed. Some proteins induced in response to the infection were identified by standard peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of the 400 protein spots detected on 2-D gels, 21 seemed to change in abundance by 3 hours after infection. Sixteen proteins were upregulated, 5 of these were only detected in infected roots and 11 showed an increased abundance. Among the differentially expressed proteins identified are homologs to isoflavone reductase, glutathione S-transferase, several abscisic acid stress-ripening proteins, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase, cysteine synthase and quinone reductase. A 17.3-kDa small heat-shock protein and a glycine-rich RNA-binding protein were identified as downregulated. Our group is the first to report on gene induction in response to oomycete infection in roots from avocado, using proteomic techniques.

Recombinant amaranth cystatin (AhCPI) inhibits the growth of phytopathogenic fungi.

Phytocystatins are cysteine proteinase inhibitors from plants implicated in defense mechanisms against insects and plant pathogens. We have previously characterized an amaranth cystatin cDNA and analyzed its response to different kinds of abiotic stress [37]. In order to characterize amaranth cystatin, the coding sequence was expressed in Escherichia coli using the pQE-2 vector. Recombinant cystatin was predominantly found in the soluble fraction of the cell extract. Large amounts (266 mgL(-1)) of pure recombinant protein were obtained by affinity chromatography in a single step of purification. The amaranth cystatin with a pI 6.8 and an apparent 28 kDa molecular mass inhibited papain (E.C.3.4.22.2) (Ki 115 nM), ficin (E.C.3.4.22.3) (Ki 325 nM) and cathepsin L (E.C.3.4.22.15) (Ki 12.7 nM) but not stem bromelain (E.C.3.4.22.32), and cathepsin B (E.C.3.4.22.1) activities, in colorimetric assays. Furthermore, it was able to arrest the fungal growth of Fusarium oxysporum, Sclerotium cepivorum and Rhyzoctonia solani. It was further demonstrated that recombinant AhCPI is a weak inhibitor of the endogenous cysteine proteinase activities in the fungal mycelium. These findings contribute to a better understanding of the amaranth cystatin activity and encourage further studies of this protein.

Accelerated Identification of Proteins by Mass Spectrometry by Employing Covalent Pre-Gel Staining with Uniblue A

Background The identification of proteins by mass spectrometry is a standard method in biopharmaceutical quality control and biochemical research. Prior to identification by mass spectrometry, proteins are usually pre-separated by electrophoresis. However, current protein staining and de-staining protocols are tedious and time consuming, and therefore prolong the sample preparation time for mass spectrometry. Methodology and Principal Findings We developed a 1-minute covalent pre-gel staining protocol for proteins, which does not require de-staining before the mass spectrometry analysis. We investigated the electrophoretic properties of derivatized proteins and peptides and studied their behavior in mass spectrometry. Further, we elucidated the preferred reaction of proteins with Uniblue A and demonstrate the integration of the peptide derivatization into typical informatics tools. Conclusions and Significance The Uniblue A staining method drastically speeds up the sample preparation for the mass spectrometry based identification of proteins. The application of this chemo-proteomic strategy will be advantageous for routine quality control of proteins and for time-critical tasks in protein analysis.

Identification of B6T173 (ZmPrx35) as the prevailing peroxidase in highly insect-resistant maize (Zea mays, p84C3) kernels by activity-directed purification

Plant peroxidases (PODs) are involved in diverse physiological processes, including defense against pathogens and insects. Contrary to their biological importance, only very few plant PODs have been proven on protein level, because their low abundance makes them difficult to detect in standard proteomics work-flows. A statistically significant positive correlation between POD activity and post-harvest insect resistance has been found for maize (Zea mays, p84C3) kernels. In combining activity-directed protein purification, genomic and proteomic tools we found that protein B6T173 (ZmPrx35) is responsible for the majority of the POD activity of the kernel. We successfully produced recombinant ZmPrx35 protein in Escherichia coli and demonstrate both, in vitro activity and the presence of a haem (heme) cofactor of the enzyme. Our findings support the screening for insect resistant maize variants and the construction of genetically optimized maize plants.

Multifunctional amaranth cystatin inhibits endogenous and digestive insect cysteine endopeptidases: A potential tool to prevent proteolysis and for the control of insect pests.

In a previous study, the amaranth cystatin was characterized. This cystatin is believed to provide protection from abiotic stress because its transcription is induced in response to heat, drought, and salinity. It has also been shown that recombinant amaranth cystatin inhibits bromelain, ficin, and cysteine endopeptidases from fungal sources and also inhibits the growth of phytopathogenic fungi. In the present study, evidence is presented regarding the potential function of amaranth cystatin as a regulator of endogenous proteinases and insect digestive proteinases. During amaranth germination and seedling growth, different proteolytic profiles were observed at different pH levels in gelatin‐containing SDS‐PAGE. Most of the proteolytic enzymes detected at pH 4.5 were mainly inhibited by transepoxysuccinyl‐leucyl amido(4‐guanidino)butane (E‐64) and the purified recombinant amaranth cystatin. Furthermore, the recombinant amaranth cystatin was active against insect proteinases. In particular, the E‐64‐sensitive proteolytic digestive enzymes from Callosobruchus maculatus, Zabrotes subfasciatus, and Acanthoscelides obtectus were inhibited by the amaranth cystatin. Taken together, these results suggest multiple roles for cystatin in amaranth, specifically during germination and seedling growth and in the protection of A. hypochondriacus against insect predation. Amaranth cystatin represents a promising tool for diverse applications in the control of insect pest and for preventing undesirable proteolytic activity.