scholarly journals Exploring the Extracellular Macromolecular Composition of Crude Extracts of Penicillium rubens Strain 212 for Elucidation Its Mode of Action as a Biocontrol Agent

2020 ◽  
Vol 6 (3) ◽  
pp. 131
Author(s):  
Maria Carreras ◽  
Eduardo A. Espeso ◽  
Alba Gutierrez-Docio ◽  
Silvia Moreno-Fernandez ◽  
Marin Prodanov ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Protein Fraction ◽  
Biocontrol Agent ◽  
Solid Phase ◽  
High Molecular Mass ◽  
Systemic Resistance ◽  
Genome Database ◽  
Tomato Plants ◽  
Crude Extracts ◽  
Proteomic Techniques

Penicillium rubens strain 212 (PO212) acts as an inducer of systemic resistance in tomato plants. The effect of crude extracellular extracts of PO212 on the soil-borne pathogen Fusarium oxysporum f. sp. lycopersici has been evaluated. Evidence of the involvement of soluble, thermo-labile, and proteinase-inactivated macromolecules present in PO212 crude extracts in the control of Fusarium vascular disease in tomato plants was found. Proteomic techniques and the availability of the access to the PO212 genome database have allowed the identification of glycosyl hydrolases, oxidases, and peptidases in these extracellular extracts. Furthermore, a bioassay-guided fractionation of PO212 crude extracellular extracts using an integrated membrane/solid phase extraction process was set up. This method enabled the separation of a PO212 crude extracellular extract of seven days of growth into four fractions of different molecular sizes and polarities: high molecular mass protein fraction >5 kDa, middle molecular mass protein fraction 5–1 kDa, low molecular mass metabolite fraction, and nutrients from culture medium (mainly glucose and minerals). The high and middle molecular mass protein fractions retained disease control activity in a way similar to that of the control extracts. Proteomic techniques have allowed the identification of nine putatively secreted proteins in the high molecular mass protein fraction matching those identified in the total crude extracts. Therefore, these enzymes are considered to be potentially responsible of the crude extracellular extract-induced resistance in tomato plants against F. oxysporum f. sp. lycopersici. Further studies are required to establish which of the identified proteins participate in the PO212’s action mode as a biocontrol agent.

High molecular mass kininogen inhibits cathepsin G-induced platelet activation by forming a complex with cathepsin G

2001 ◽  
Vol 2 (6) ◽  
pp. 371-377 ◽  
Author(s):  
Tarek E Selim ◽  
Hayam R Ghoneim ◽  
Hassan A Abdel Ghaffar ◽  
Robert W Colman ◽  
Raul A Dela Cadena
Keyword(s):  
Molecular Mass ◽  
Platelet Activation ◽  
High Molecular Mass ◽  
Cathepsin G

scholarly journals Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 453
Author(s):  
Sebastian Estrada-Gómez ◽  
Leidy Johana Vargas-Muñoz ◽  
Cesar Segura Latorre ◽  
Monica Maria Saldarriaga-Cordoba ◽  
Claudia Marcela Arenas-Gómez
Keyword(s):  
Enzymatic Activity ◽  
Molecular Mass ◽  
Evolutionary Relationship ◽  
Venom Gland ◽  
Duplication Event ◽  
High Molecular Mass ◽  
Amino Acid Sequences ◽  
Secretory Proteins ◽  
Phospholipases A2 ◽  
Kunitz Type

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

Phosphoenolpyruvate Carboxykinase in C4 Plants: Its Role and Regulation

1997 ◽  
Vol 24 (4) ◽  
pp. 459 ◽  
Author(s):  
Robert P. Walker ◽  
Richard M. Acheson ◽  
László I. Técsi ◽  
Richard C. Leegood
Keyword(s):  
Molecular Mass ◽  
Malic Enzyme ◽  
Phosphoenolpyruvate Carboxykinase ◽  
C4 Plants ◽  
Panicum Maximum ◽  
C4 Plant ◽  
Cam Plants ◽  
Crude Extracts ◽  
High Concentrations ◽  
Regulatory Properties

Some of the recent findings which revise our view of the role and regulation of phosphoenolpyruvate carboxykinase (PEPCK) in C4 plants are discussed. Evidence is presented that PEPCK is present at appreciable activities in the bundle-sheath of some NADP-malic enzyme-type C4 plants, such as maize, but it was not detectable in NAD-malic enzyme-type C4 plants. PEPCK is rapidly inactivated in crude extracts of leaves of the C4 plant, Panicum maximum. This inactivation could be prevented by high concentrations of dithiothreitol or by the inclusion of ADP or ATP, suggesting the involvement of thiols at the active site. PEPCK is also subject to rapid proteolysis in crude extracts of a range of C4 plants, resulting in cleavage to a smaller (62 kDa) form. This can be reduced by extraction at high pH and by the inclusion of SDS, but it means that intact PEPCK has never been purified from a C4 plant. The molecular mass of PEPCK varies considerably in C4 plants, unlike C3 and CAM plants in which it is usually 74 kDa. PEPCK is phosphorylated during darkness (and reversed by light) in some C4 plants with PEPCK of a larger molecular mass, such as Panicum maximum (71 kDa), but it was not phosphorylated in the PEPCK-type C4 plant, Sporobolus pyramidalis (69 kDa). The known regulatory properties of PEPCK are discussed in relation to its role in C4 photosynthesis, in particular its sensitivity to regulation by adenylates and by Mn2+.

scholarly journals Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears

2002 ◽  
Vol 366 (2) ◽  
pp. 511-520 ◽  
Author(s):  
Benjamin L. SCHULZ ◽  
David OXLEY ◽  
Nicolle H. PACKER ◽  
Niclas G. KARLSSON
Keyword(s):  
Molecular Mass ◽  
Peptide Mass Fingerprinting ◽  
High Molecular Mass ◽  
Tear Fluid ◽  
Protein Variant ◽  
Peptide Mass ◽  
Composite Gel ◽  
Molecular Masses ◽  
Protein Components ◽  
Sialyl Lea

Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex0→2NeuAc1→xHexxHexNAcx(-ol), x = 1–6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Lea epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.

High-Molecular-Mass Receptors for Ammodytoxin in Pig Are Tissue-Specific Isoforms of M-Type Phospholipase A2 Receptor

2001 ◽  
Vol 289 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Nina Vardjan ◽  
Nicholas E Sherman ◽  
Jože Pungerčar ◽  
Jay W Fox ◽  
Franc Gubenšek ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Molecular Mass ◽  
High Molecular Mass ◽  
Tissue Specific ◽  
Phospholipase A2 Receptor

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

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