scholarly journals Soyuretox, an Intrinsically Disordered Polypeptide Derived from Soybean (Glycine Max) Ubiquitous Urease with Potential Use as a Biopesticide

2019 ◽  
Vol 20 (21) ◽  
pp. 5401 ◽  
Author(s):  
Karine Kappaun ◽  
Anne H. S. Martinelli ◽  
Valquiria Broll ◽  
Barbara Zambelli ◽  
Fernanda C. Lopes ◽  
...  
Keyword(s):  
Glycine Max ◽  
Plant Protection ◽  
Sodium Dodecyl ◽  
Membrane Integrity ◽  
Amino Acid Sequences ◽  
Intrinsically Disordered ◽  
Sds Micelles ◽  
Ubiquitous Urease ◽  
Insect Immune Response ◽  
Induced Aggregation

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.

scholarly journals A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 1964-1969 ◽  
Author(s):  
F Flug ◽  
R Espinola ◽  
LX Liu ◽  
C SinQuee ◽  
R DaRosso ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Polymorphism ◽  
Glycoprotein Iiia ◽  
Polymerase Chain ◽  
Induced Aggregation

Abstract We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105–129 and 452- 428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67- Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.

Differential roles of two DDX17 isoforms in the formation of membraneless organelles

2020 ◽  
Vol 168 (1) ◽  
pp. 33-40
Author(s):  
Yuya Hirai ◽  
Eisuke Domae ◽  
Yoshihiro Yoshikawa ◽  
Keizo Tomonaga
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Rna Helicase ◽  
Intracellular Distribution ◽  
Amino Acid Sequences ◽  
Nucleolar Localization ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Dead Box ◽  
Additional Amino Acid

Abstract The RNA helicase, DDX17 is a member of the DEAD-box protein family. DDX17 has two isoforms: p72 and p82. The p82 isoform has additional amino acid sequences called intrinsically disordered regions (IDRs), which are related to the formation of membraneless organelles (MLOs). Here, we reveal that p72 is mostly localized to the nucleoplasm, while p82 is localized to the nucleoplasm and nucleoli. Additionally, p82 exhibited slower intranuclear mobility than p72. Furthermore, the enzymatic mutants of both p72 and p82 accumulate into the stress granules. The enzymatic mutant of p82 abolishes nucleolar localization of p82. Our findings suggest the importance of IDRs and enzymatic activity of DEAD-box proteins in the intracellular distribution and formation of MLOs.

scholarly journals The N-Terminal Region of Soybean PM1 Protein Protects Liposomes during Freeze-Thaw

2020 ◽  
Vol 21 (15) ◽  
pp. 5552
Author(s):  
Liyi Chen ◽  
Yajun Sun ◽  
Yun Liu ◽  
Yongdong Zou ◽  
Jianzi Huang ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Membrane Fusion ◽  
Terminal Region ◽  
Freezing Stress ◽  
Disordered Proteins ◽  
Distribution Analysis ◽  
Protective Function ◽  
Freeze Thaw ◽  
Intrinsically Disordered ◽  
Induced Aggregation

Late embryogenesis abundant (LEA) group 1 (LEA_1) proteins are intrinsically disordered proteins (IDPs) that play important roles in protecting plants from abiotic stress. Their protective function, at a molecular level, has not yet been fully elucidated, but several studies suggest their involvement in membrane stabilization under stress conditions. In this paper, the soybean LEA_1 protein PM1 and its truncated forms (PM1-N: N-terminal half; PM1-C: C-terminal half) were tested for the ability to protect liposomes against damage induced by freeze-thaw stress. Turbidity measurement and light microscopy showed that full-length PM1 and PM1-N, but not PM1-C, can prevent freeze-thaw-induced aggregation of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes and native thylakoid membranes, isolated from spinach leaves (Spinacia oleracea). Particle size distribution analysis by dynamic light scattering (DLS) further confirmed that PM1 and PM1-N can prevent liposome aggregation during freeze-thaw. Furthermore, PM1 or PM1-N could significantly inhibit membrane fusion of liposomes, but not reduce the leakage of their contents following freezing stress. The results of proteolytic digestion and circular dichroism experiments suggest that PM1 and PM1-N proteins bind mainly on the surface of the POPC liposome. We propose that, through its N-terminal region, PM1 functions as a membrane-stabilizing protein during abiotic stress, and might inhibit membrane fusion and aggregation of vesicles or other endomembrane structures within the plant cell.

Article

1998 ◽  
Vol 76 (2) ◽  
pp. 171-182 ◽  
Author(s):  
Fengde Xi ◽  
L Ross Barclay
Keyword(s):  
Methyl Linoleate ◽  
Antioxidant Activities ◽  
Lignin Content ◽  
Synergistic Effects ◽  
Sodium Dodecyl ◽  
Ascorbyl Palmitate ◽  
Semiquinone Radical ◽  
Semiquinone Radicals ◽  
Sds Micelles

The antioxidant activities, kinh, of catechol, 1, 4-tert-butylcatechol, 2, and 3,5-di-tert-butylcatechol (DTBC), 3, determined by the inhibited oxygen-uptake method during peroxidation of styrene initiated by AIBN are 55.0 x 104, 88.4 x 104, and 149 x 104 M-1s-1, respectively, and the stoichiometric factors (n) were 2.1-2.3. A decrease by 50-fold in kinh for 3 and a drop of 1.1-1.4 in n observed during inhibited peroxidation of methyl linoleate in aqueous sodium dodecyl sulfate (SDS) initiated by di-tert-butylhyponitrile (DBHN) is attributed to hydrogen bonding by water on the antioxidant and on the intermediate di-tert-butylsemiquinone radical, 5, formed in the inhibition step. Combinations of ascorbyl palmitate with 3 exhibited cooperative (not synergistic) antioxidant effects during inhibited peroxidation of styrene in solution. Combinations of ascorbic acid with 3 exhibited synergistic effects during inhibited peroxidation of methyl linoleate initiated by DBHN in SDS micelles. A profile of the effect of concentration of ascorbate on this synergism indicates a mole of 3 is regenerated per mole of ascorbate. The thiols, homocysteine, and polyethylene glycol thiol (polythiol) also exhibited synergistic effects with DTBC in this inhibition. Either ascorbate or polythiol rapidly reduces di-tert-butyl-ortho-quinone (DTBQ), 4, to 3 in methanol or in SDS micelles, and the combination of 3 + ascorbate acted as an efficient inhibitor in this medium. The esr studies indicate the semiquinone radical, 5, produced photochemically from 3 or spontaneously from 3 + 4 in solution, to be very persistent at room temperature. A pathway, mediated by 5, is proposed to account for the cooperative and synergistic effects observed and for the additional combination effect discovered when the three inhibitors: 3, ascorbate, and a thiol are used in the SDS medium. Combinations of such antioxidants are expected to be useful for inhibition of yellowing of pulps and paper with high lignin content, and to be significant in the in vivo reductions of ortho-quinones and semiquinone radicals formed during oxidations of various biomolecules.Key words: catechols, ascorbate, thiols, radicals, antioxidants.

Relationship between semen quality and seminal plasma cholesterol, triacylglycerols and proteins in the domestic cat

2019 ◽  
Vol 22 (10) ◽  
pp. 882-889 ◽  
Author(s):  
María F García ◽  
Romina Nuñez Favre ◽  
María C Stornelli ◽  
Ramiro Rearte ◽  
María C García Mitacek ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Morphology ◽  
Semen Quality ◽  
Sperm Count ◽  
Sodium Dodecyl ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Domestic Cat ◽  
Sds Page ◽  
The Relationship

Objectives The current study aimed to evaluate the relationship between specific seminal plasma components – cholesterol (CHOL), triacylglycerols (TAG) and total protein (PROT) concentrations – and semen quality in cats. A further aim was to determine the relationship between specific seminal protein bands and semen quality. Methods Thirteen toms, 2–5 years of age, were included. Semen collection was performed by electroejaculation every 4 weeks. Fifty-eight ejaculates were assessed for motility, velocity, volume, sperm concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology. Samples were divided into two groups: good semen quality (GSQ) and poor semen quality (PSQ). After evaluation, seminal plasma was separated from the sperm by centrifugation and stored at −20°C. CHOL, TAG and PROT concentrations were then assessed and seminal plasma protein profile was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results Seminal plasma CHOL and TAG concentrations, motility, velocity, sperm concentration, total sperm count and sperm morphology were significantly higher in GSQ cats compared with PSQ cats ( P <0.01). Moreover, seminal plasma SDS-PAGE analysis showed an identifiable extra band exclusively in the GSQ group. Conclusions and relevance Data obtained in this study showed that seminal plasma CHOL and TAG concentrations and specific protein bands could be used to improve semen evaluation in toms. In this sense, the 14 kDa protein band could be a valuable marker for semen quality in the cat and should be further investigated. However, more studies are necessary to determine its relationship with fertility.

Electropherogram pattern similarity of seed proteins from 21 different soybean (Glycine max) varieties

1977 ◽  
Vol 55 (16) ◽  
pp. 2245-2250 ◽  
Author(s):  
Clifton F. Savoy
Keyword(s):  
Glycine Max ◽  
Seed Protein ◽  
Sodium Dodecyl ◽  
Seed Proteins ◽  
Plant Proteins ◽  
Relative Mobility ◽  
Molecular Weights ◽  
Pattern Similarity ◽  
Protein Components ◽  
Phosphate Detergent

Soybean (Glycine max) seed protein has been characterized using a phosphate-detergent (sodium dodecyl sulfate) polyacrylamide gel electrophoretic system, which has been extensively tested on plant proteins. The same general densitometer electropherogram pattern as regards numbers and kinds of protein components resolved was observed for all soybean varieties tested, and one pattern is presented along with appropriate descriptive characterizations (numbers, molecular weights, relative mobility, and light absorption at 597 nm) to aid in distinguishing the components. Quantitative differences, however, of individual components may occur.

Evolution of tissue damage in compressive spinal cord injury in rats

1987 ◽  
Vol 66 (4) ◽  
pp. 595-603 ◽  
Author(s):  
Hideaki Iizuka ◽  
Hirotaka Yamamoto ◽  
Yuzo Iwasaki ◽  
Teiji Yamamoto ◽  
Hidehiko Konno
Keyword(s):  
Spinal Cord ◽  
Tissue Damage ◽  
Spinal Cord Injuries ◽  
Sodium Dodecyl ◽  
Membrane Integrity ◽  
Secondary Degeneration ◽  
Content Type ◽  
Cell Membrane Integrity ◽  
Cord Injury ◽  
Mechanical Insult

✓ The evolution of tissue damage in compressive spinal cord injuries in rats was studied using an immunohistochemical technique and by sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) analysis. The rupture of small vessels accompanied by intense tissue permeation of serum components in and around the hemorrhagic foci appeared to be immediate consequences of the mechanical insult. The loss of cell membrane integrity in neural elements became evident within 1 hour after injury as shown by the diffuse albumin-immunoreactivity of the cytoplasm. At the site of mechanical insult, approximately 30% of the neurofilament proteins were degraded within 1 hour, and 70% of them were lost within 4 hours after injury. A large number of cells positive for glial fibrillary acidic protein were found to demarcate the injured tissue within 1 hour after injury. The progression of tissue damage largely subsided within 48 hours. One week after, injury, severe degeneration of the ascending tracts in the posterior funiculus was shown clearly by axon staining and less convincingly by myelin staining. Secondary degeneration of the corticospinal tract in distal segments remained inconspicuous for up to 3 months.

scholarly journals Purification and Characterization of a Novel Mannitol Dehydrogenase from a Newly Isolated Strain of Candida magnoliae

2003 ◽  
Vol 69 (8) ◽  
pp. 4438-4447 ◽  
Author(s):  
Jung-Kul Lee ◽  
Bong-Seong Koo ◽  
Sang-Yong Kim ◽  
Hyung-Hwan Hyun
Keyword(s):  
Substrate Specificity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Amino Acid Sequences ◽  
Size Exclusion ◽  
Mannitol Dehydrogenase ◽  
High Substrate ◽  
Candida Magnoliae

ABSTRACT Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k cat = 823 s−1, K m = 28.0 mM, and k cat /K m = 29.4 mM−1 s−1) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.

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