scholarly journals Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of Bombyx mori, That Binds to Cry1A Toxins

2004 ◽  
Vol 70 (8) ◽  
pp. 4604-4612 ◽  
Author(s):  
Delwar M. Hossain ◽  
Yasuyuki Shitomi ◽  
Kenta Moriyama ◽  
Masahiro Higuchi ◽  
Tohru Hayakawa ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Brush Border ◽  
Membrane Vesicle ◽  
Gel Filtration ◽  
Resistance Mechanism ◽  
Binding Assays ◽  
Toxin Resistance ◽  
Cry Toxin ◽  
Triton X

ABSTRACT We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and Kd constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.

scholarly journals Neutral maltase/glucoamylase from rabbit renal cortex

1989 ◽  
Vol 261 (1) ◽  
pp. 43-47 ◽  
Author(s):  
B Pereira ◽  
S Sivakami
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Renal Cortex ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Rabbit Kidney ◽  
Purification Procedure ◽  
Glucoamylase Activity ◽  
Maltase Activity ◽  
Triton X

Maltase activity (EC 3.2.1.20) was solubilized from rabbit kidney brush-border membrane by using 1.0% Triton X-100 and purified 230-fold with an overall recovery of 30%. The purification procedure makes use of heat precipitation, chromatography on DE-52 DEAE-cellulose and gel filtration on Sephacryl S-300. Rabbit kidney brush border exhibited glucoamylase activity with a maltase/glucoamylase ratio of 1.5:1 to 2.0:1. During purification the maltase and glucoamylase activities behaved identically. The Mr of the complex is 590,000, and it appears to be composed of eight identical subunits linked by disulphide bridges.

scholarly journals Purification and characterization of two forms of microsomal carbonyl reductase in guinea pig liver

1984 ◽  
Vol 223 (3) ◽  
pp. 697-705 ◽  
Author(s):  
S Usui ◽  
A Hara ◽  
T Nakayama ◽  
H Sawada
Keyword(s):  
Carbonyl Compounds ◽  
Gel Filtration ◽  
Carbonyl Reductase ◽  
Cofactor Specificity ◽  
Major Form ◽  
Aldehydes And Ketones ◽  
Minor Form ◽  
Triton X ◽  
Guinea Pig Liver

Two forms of microsomal carbonyl reductase, solubilized in Triton X-100, were purified to homogeneity from the liver of male guinea pigs, primarily by affinity, DEAE-Sephacel, gel-filtration and hydroxyapatite chromatography. The major form was a tetrameric glycoprotein of single subunits of Mr 32000 and a pI value of 7.0; another minor form was a monomeric protein with Mr 34000 and a pI value of 7.8. The enzymes were immunologically distinct. Although the enzymes showed similar substrate specificity for exogenous aldehydes and ketones and apparently absolute cofactor specificity for NADPH, their specificity for natural carbonyl compounds differed. The major form irreversibly reduced 5 alpha- and 5 beta-dihydrotestosterones, menadione and lauryl aldehyde with low Km values of 10-70 microM, whereas the minor form not only reduced 17-oxosteroids, of which 3 alpha-hydroxy-5 beta-androstan-17-one was the best substrate, but also oxidized 17-hydroxysteroids in the presence of NADP+. The two forms of carbonyl reductase also exhibited different sensitivity to heavy metal ions, dicoumarol, tetramethyleneglutaric acid, phenobarbitone and corticosteroids.

scholarly journals Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

1995 ◽  
Vol 308 (3) ◽  
pp. 983-989 ◽  
Author(s):  
I N Fleming ◽  
S J Yeaman
Keyword(s):  
Phosphatidic Acid ◽  
Rat Liver ◽  
Lysophosphatidic Acid ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Sds Page ◽  
Chromatography Columns ◽  
Triton X ◽  
Second Peak

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

scholarly journals Solubilization and characterization of pellet-associated human brain α-L-fucosidase activity

1985 ◽  
Vol 229 (3) ◽  
pp. 679-685 ◽  
Author(s):  
R L Hopfer ◽  
J A Alhadeff
Keyword(s):  
Human Brain ◽  
Isoelectric Focusing ◽  
Human Liver ◽  
Gel Filtration ◽  
Compelling Evidence ◽  
Supernatant Fluid ◽  
Ph Optimum ◽  
Triton X ◽  
Soluble Supernatant

The pellet-associated portion of human brain alpha-L-fucosidase (which represents approx. 20% of the homogenate activity) was solubilized with 0.5% (w/v) Triton X-100, characterized with regard to several properties and compared with the corresponding properties of the soluble supernatant-fluid enzyme in an attempt to find a second alpha-L-fucosidase in human brain. The solubilized and soluble alpha-L-fucosidase activities exhibited complete stability after storage at 2-4 degrees C for up to 29 days, comparable thermostability after preincubation at 50 degrees C, comparable apparent Km values (0.07-0.08 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside, comparable hydrophobicity, comparable isoelectric-focusing profiles (six major forms, with pI values between 4.5 and 5.8) and comparable immunoprecipitation curves (with the IgG fraction of antisera prepared against human liver alpha-L-fucosidase). Differences in three properties were found between solubilized and soluble alpha-L-fucosidase activities: the solubilized activity was less stable to storage at −20 degrees C, had a 0.5-pH-unit neutral shift in its pH optimum (6.0) and had smaller Mr forms after gel filtration on Sephadex G-200. The overall results indicate that the pellet-associated and soluble portions of human brain alpha-L-fucosidase are quite similar in most of their properties. Thus there is still no compelling evidence for the existence of a second mammalian alpha-L-fucosidase.

Vitamin D-dependent, particulate calcium-binding activity and intestinal calcium transport

1975 ◽  
Vol 229 (5) ◽  
pp. 1198-1204 ◽  
Author(s):  
S Kowarski ◽  
D Schachter
Keyword(s):  
Vitamin D ◽  
Brush Border ◽  
Calcium Binding ◽  
Protein Complexes ◽  
Gel Filtration ◽  
Binding Activity ◽  
Particulate Fraction ◽  
Small Intestinal Mucosa ◽  
Mucosal Cells ◽  
Triton X

A vitamin D-dependent calcium-binding activity of relatively high molecular weight has been identified in the particulate fraction of rat small intestinal mucosa. The Ca-binding activity is sedimented at 140,000 X g after treatment of the mucosal particulate fraction with Triton X-114. Intestinal brush-border suspensions can also be used as starting material. The Ca-binding component is inactivated by heat and repeated freeze-thawing and consists of one or more protein complexes in the range of 0.5-1.0 million mol wt as indicated by gel filtration. The Ca-binding activity correlates positively with known features of the intestinal Ca transport mechanism, as demonstrated by studies of the distribution in the small intestine and the effects of vitamin D, dietary Ca, cycloheximide treatment, and rat age. It is suggested that the component might function in the transit of Ca across the brush-border surface to the cytosol of intestinal mucosal cells.

scholarly journals Cry1Ac and Vip3Aa proteins fromBacillus thuringiensistargeting Cry toxin resistance inDiatraea flavipennellaandElasmopalpus lignosellusfrom sugarcane

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2866 ◽  
Author(s):  
Ana Rita Nunes Lemes ◽  
Camila Soares Figueiredo ◽  
Isis Sebastião ◽  
Liliane Marques da Silva ◽  
Rebeka da Costa Alves ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Binding Assays ◽  
Toxin Resistance ◽  
Transgenic Sugarcane ◽  
New Genes ◽  
Future Production ◽  
Cry Toxin ◽  
Biological Potential ◽  
Elasmopalpus Lignosellus ◽  
Competition Assays

The biological potential of Vip and Cry proteins fromBacillusis well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity againstDiatraea flavipennellaandElasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed inEscherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50values. Toxins fromE. coliwere purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV ofD. flavipennellaandE. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control ofD. flavipennellaandE. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests.

SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF THYROID MEMBRANE TSH BINDING PROTEINS

1979 ◽  
Vol 92 (3) ◽  
pp. 512-521 ◽  
Author(s):  
B. Czarnocka ◽  
J. Nauman ◽  
G. Adler ◽  
W. Kiełczyński
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Soluble Proteins ◽  
The Other ◽  
High Affinity ◽  
Triton X ◽  
Maximal Capacity

ABSTRACT Crude plasma membranes obtained from bovine thyroids were found to possess one class of high affinity, low capacity binding sites for TSH with average association constant (Ka) of 1.301 × 109 m−1 and maximal capacity 8.76 × 10−10 m/mg of protein. Treatment of crude membranes fraction with 0.1 % Triton X-100 and the subsequent sonication in ultrasonic disintegrator resulted in solubilization of membranes proteins with mean recovery of 40.0 ± 6.2 %. Soluble proteins retained the property to bind [125I]TSH, but the binding of the hormone was decreased. The removal of the detergent from the solubilizate by gel filtration on Sephadex LH-20 increased the binding of TSH well above that demonstrated for crude thyroid membranes. The chromatography of soluble proteins on Ultrogel AcA-44 revealed the presence of two TSH binding proteins, one with the molecular weight (m.w.) above 130 000 daltons and the other with the m.w. approximately 30 000 daltons. The electrofocusing of solubilizate on Ampholine resulted in two protein peaks, one at pH 4.0–4.1 and the other at pH 4.4–4.6. The latter peak was shown to bind [125I]TSH specifically. The present results have confirmed the heterogeneous character of solubilized TSH receptor preparation and have shown that the hormone binding sites belong to acid proteins.

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