ABSTRACT
Amylases are an important family of enzymes involved in insect carbohydrate metabolism that are required for the survival of insect larvae. For this reason, enzymes from starch-dependent insects are targets for insecticidal control. PF2 (Olneya tesota) is a lectin that is toxic to Zabrotes subfasciatus (Coleoptera: Bruchidae) larvae. In this study, we evaluated recognition of the PF2 lectin to α-amylases from Z. subfasciatus midgut and the effect of PF2 on α-amylase activity. PF2 caused a decrease of total amylase activity in vitro. Subsequently, several α-amylase isoforms were isolated from insect midgut tissues using ion exchange chromatography. Three enzyme isoforms were verified by an in-gel assay for amylase activity; however, only one isoform was recognized by antiamylase serum and PF2. The identity of this Z. subfasciatus α-amylase was confirmed by liquid chromatography-tandem mass spectrometry. The findings strongly suggest that a glycosylated α-amylase isoform from larval Z. subfasciatus midgut interacts with PF2, which interferes with starch digestion.
Subject(s)
Coleoptera/physiology , Digestive System/drug effects , Fabaceae/chemistry , Lectins/metabolism , Plant Proteins/metabolism , alpha-Amylases/biosynthesis , Animals , Digestive System/metabolism , Fabaceae/metabolism , Larva/drug effects , Larva/physiology , Plant Lectins , alpha-Amylases/antagonists & inhibitorsABSTRACT
Adherence to the gastrointestinal tract is a key element desirable for many of the proposed beneficial health effects of probiotic bacteria. The aims of this study were to determine the amounts of adhesion of 3 Lactobacillus salivarius strains (Lb6, Lb9, and Lb10) to porcine small intestinal mucins and to determine whether adhesion is a function of lectin-like activities. Dot and Western blot assays were performed to investigate bacterial adhesion. Several carbohydrates and glycoproteins were evaluated to determine whether they interfered with adhesion of the Lactobacillus strains to intestinal mucins and to determine whether they had lectin-like activities. The Lb9 and Lb10 strains had greater association with piglet mucins than did those from 22- to 24-wk-old finishing pigs (P = 0.021 and 0.037, respectively), whereas the Lb6 strain adhered to both (P = 0.138). Western blot assays showed that bacterial adhesion detected piglet mucosa from the duodenum, jejunum, and ileum. In finishing pigs, the adhesion was variable throughout the gastrointestinal tract. Galactose and mannose diminished the interaction of the Lb9 and Lb10 strains in intestinal mucosa (P = 0.028 and 0.026, respectively), whereas pig gastric mucin reduced the adhesion of the Lb6 strain (P = 0.013). Adhesion of the Lb9 and Lb10 strains to intestinal mucosa was less after protease treatment (P = 0.023 and 0.018, respectively), which indicates that proteins are needed for the Lb9 and Lb10 strains to recognize mucin. The Lb6 strain also demonstrated diminished adhesion after periodate treatment (P = 0.038). From these results, we suggest that the nature of the bacterial lectin-like substance is a surface protein that loosely binds to the bacterial cell surface. All the tested strains adhered to specific targets in the small intestinal mucosa of piglets, and the bacteria had lectin-like proteins involved in this adhesion.
Subject(s)
Carbohydrates/chemistry , Lactobacillus/chemistry , Mucins/chemistry , Peptidylprolyl Isomerase/chemistry , Animals , Bacterial Adhesion/physiology , Immunoblotting , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Intestine, Small/metabolism , NIMA-Interacting Peptidylprolyl Isomerase , SwineABSTRACT
Alpha amylase inhibitor from Palo Fierro seeds (alphaAI-PF) was purified using affinity chromatography on a fetuin-fractogel column followed by anionic exchange chromatography. AlphaAI-PF has a molecular mass of 77kDa with two subunits (15.8 and 17.4 kDa), it is nonglycosylated and has pI of 4.7. AlphaAI-PF inhibited porcine pancreatic alpha-amylase (PPA) (1,4-alpha-D-glucan glucanohydrolase; EC 3.2.1.1), but was almost devoid of inhibitory activity on alpha-amylase extracts from Zabrotes subfasciatus (ZSA). Analysis of alphaAI-PF peptides showed a high homology to alphaAI-1 from Phaseolus vulgaris that also inhibits PPA.
Subject(s)
Fabaceae/chemistry , Seeds/chemistry , alpha-Amylases/antagonists & inhibitors , Amino Acid Sequence , Dose-Response Relationship, Drug , Molecular Sequence DataABSTRACT
We are interested in identifying potential protein interactors of MADS domain transcription factors during Arabidopsis thaliana flower development. We based our biochemical search on a conserved motif in the MADS domain that includes putative phosphatase and phosphorylation sites that may mediate protein interactions. An affinity column with this motif and a few surrounding hypervariable amino acids derived from the AGAMOUS sequence was prepared and used to isolate potential interactors from floral crude extracts. Only two proteins were specifically bound to the affinity column. The first corresponds to a carpel specific storage protein, VSP1, that presents acid phosphatase activity, and the second is a novel leucine-rich repeat protein that we have named FLOR1. Coimmunoprecipitation, two-hybrid yeast, and affinity column assays show that the FLOR1-VSP1 complex interacts with AGAMOUS and that this transcription factor directly interacts with FLOR1. This is the first assay to show an interaction between plant MADS domain factors and non-MADS proteins.
Subject(s)
AGAMOUS Protein, Arabidopsis/metabolism , Acid Phosphatase/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Endopeptidases/chemistry , Endopeptidases/metabolism , Leucine/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Repetitive Sequences, Amino Acid , AGAMOUS Protein, Arabidopsis/chemistry , AGAMOUS Protein, Arabidopsis/genetics , Acid Phosphatase/chemistry , Acid Phosphatase/genetics , Acid Phosphatase/isolation & purification , Amino Acid Motifs , Amino Acid Sequence , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/isolation & purification , Base Sequence , Blotting, Western , Carrier Proteins/isolation & purification , Chromatography, Affinity , Cloning, Molecular , Endopeptidases/isolation & purification , Macromolecular Substances , Membrane Proteins/isolation & purification , Molecular Sequence Data , Organ Specificity , Plant Structures/chemistry , Plant Structures/genetics , Plant Structures/growth & development , Plant Structures/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , RNA, Plant/metabolism , Sequence Alignment , Two-Hybrid System TechniquesABSTRACT
OBJECTIVES: The origin of the elevation of serum immunoglobulin A1 (IgA1) in Type 2 diabetes mellitus (DM) is unsettled. The aim of this study was to address the carbohydrate changes of serum IgA1 from patients with Type 2 diabetes mellitus, as a possible cause of the elevation. DESIGN AND METHODS: IgA1 was purified from sera of 6 DM patients and 4 healthy matched controls by using highly acetylated-Sepharose 6B, anti-IgA-agarose, and jacalin-agarose columns, and further separated into jacalin low-affinity, medium, and high-affinity fractions. Hinge and Fc fragments from native IgA1 were obtained and analyzed by using Sambucus nigra, Maackia amurensis, Arachis hypogaea, Erythrina cristagalli, and Ricinus communis lectins. RESULTS: The jacalin high-affinity fraction, mostly constituted by macromolecular IgA1, was more abundant in DM patients than in controls and also more reactive to Sambucus nigra, and Maackia amurensis lectins. CONCLUSIONS: Macromolecular serum IgA1 from DM patients is hypersialylated and this probably contributes to the high level of IgA1 in DM patients.
Subject(s)
Diabetes Mellitus, Type 2/blood , Immunoglobulin A/blood , Aged , Carbohydrates/blood , Case-Control Studies , Chromatography, Agarose , Electrophoresis, Polyacrylamide Gel , Female , Humans , Immunoglobulin A/chemistry , Immunoglobulin A/isolation & purification , Lectins/metabolism , Male , Middle Aged , Protein Binding , Time FactorsABSTRACT
One of the few sources of long-chain n-3 polyunsaturated fatty acids is fish oil, but considerable variation may exist according to species and season. In this study, the fatty acid profiles of sardine oils from Sardinops sagax caeruleus of the Gulf of California, Mexico, were evaluated in three seasonal catch periods. Oil quality was also evaluated by peroxide and free acid values. The most abundant fatty acids found in the oils were palmitic acid (19.3%), oleic acid (14.3%), eicosapentaenoic acid (EPA, 20.4%), and docosahexaenoic acid (DHA, 12.2%). There was no significant difference in the composition and quality among the six reduction plants where the samples were obtained. However, a significant difference in the proportion of EPA and DHA in one of the catch seasons analyzed was observed.
Subject(s)
Fatty Acids/analysis , Fish Oils/chemistry , Seasons , Chromatography, Gas , Fish Oils/standards , Quality ControlABSTRACT
1. Resolution of the fraction of sandbar shark (Carcharhinus plumbeus) serum that was soluble in 50% saturated ammonium sulfate by gel-immobilized metal-affinity chromatography allowed the isolation of a novel disulfide-bonded heterodimer of intact mass 70 kDa. 2. Following reduction, the molecule could be resolved into two chains of apparent mass 36 and 24 kDa. 3. The molecules were glycoproteins as determined by an observed reduction in molecular weight following enzymatic glycosylation. 4. The two separate chains were related to one another on the basis of amino-acid composition analysis and by comparison of the N-terminal amino acids (seven out of 10 identities). 5. The exact relationship of this molecule to characterized heterodimers of higher vertebrates is unknown. 6. Cross-linked agarose-acetate was synthesized and proved to be an efficient concentrating agent and also a hydrophobic interaction adsorbant.
Subject(s)
Blood Proteins/isolation & purification , Sharks/blood , Amino Acid Sequence , Amino Acids/analysis , Animals , Blood Proteins/chemistry , Chromatography, Affinity/methods , Glycoproteins/blood , Molecular Sequence Data , Molecular Weight , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
Intact plants of the halophilic species Mesembryanthemum crystallinum were induced to exhibit Crassulacean acid metabolism by irrigation with nutrient solution containing 500 millimolar NaCl. During the induction period, the extractable activity of phosphoenolpyruvate carboxylase (PEPcase) increased approximately 40-fold. This increase was linearly correlated with a mass increase of PEPcase protein as measured by single radial immunodiffusion. De novo synthesis of PEPcase protein was shown by immunoprecipitation of the newly synthesized, radioactively labeled protein in leaf discs from salt-treated plants. Nontreated plants were characterized by a low level of the enzyme and low rates of PEPcase synthesis. Synthesis of this enzyme in leaf discs was correlated with the concentration of NaCl in the nutrient solution during growth.
