Hydrophobic binding domains of rat intestinal maltase–glucoamylase

1986 ◽  
Vol 64 (8) ◽  
pp. 782-787 ◽  
Author(s):  
L. Lee ◽  
G. Forstner
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phospholipid Bilayer ◽  
Phospholipid Vesicles ◽  
Mercury Vapour ◽  
Radioactive Label ◽  
Binding Domains ◽  
Extraction And Purification ◽  
Mercury Vapour Lamp ◽  
Hydrophobic Binding

Rat intestinal microvillus maltase–glucoamylase was isolated by detergent extraction and purification in the presence of protease inhibitors as previously described and incorporated into phospholipid vesicles. After purification of the vesicles on Scphadex G-50, maltase was labelled with 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]T1D) by photolysis using a water-jacketed mercury vapour lamp with a saturated CuSO4 filter. The labelled enzyme was extracted with acetone, resuspended in 1% Triton X-100, reincorporated into phospholipid vesicles, and digested with activated papain to release the hydrophilic polar head of the enzyme from the vesicle bilayer. Vesicle-bound and free enzyme components were separated on Scpharose 4B. Ninety percent of the enzymatic activity was free, while a similar percentage of radioactive label remained with the vesicles in keeping with the separation of an active polar headpiece from a labelled apolar peptide in the lipid bilayer. The vesicle fractions were subjected to chromatography on Sephadex LH-60 with cthanol – formic acid (7:3) as the eluant. A single radioactive peak (14 kilodaltons (kDa)) was separated from labelled lipid. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the peak showed a radioactive doublet of 26–28 kDa, possibly representing a dimer. No other labelled peptides were found. These results suggest that detergent-solubilized maltase–glucoamylase is inserted into the phospholipid bilayer via an apolar peptide with a minimum molecular mass of 14 kDa. The peptide probably represents a terminal anchor segment of the 145-kDa subunit which is converted to 130 kDa when the membrane-bound enzyme is solubilized by papain.

scholarly journals The susceptibility of muscle phosphorylases a and b to digestion by a neutral proteinase from rat intestinal muscle. Comparison with the effects produced by pancreatic trypsin and chymotrypsin

1978 ◽  
Vol 175 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Ian T. Carney ◽  
Robert J. Beynon ◽  
John Kay ◽  
Nigel Birket
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Pyridoxal Phosphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Covalent Modification ◽  
Protective Effects ◽  
Radioactive Label ◽  
Initial Event ◽  
Enzyme Degradation

1. Phosphorylase b was inactivated three times more rapidly than phosphorylase a by a neutral, trypsin-like proteinase from rat intestinal muscle. Digestion of phosphorylase a produced a modified form which was deactivated by AMP. Removal of the pyridoxal phosphate cofactor increased the rate of inactivation of the b form by about 3-fold but the subceptibility of apophosphorylase a was no different from the holo form. 2. The extent of proteolysis of both holoenzyme forms, as guaged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was limited and similar digestion patterns were obtained in both cases. 3. With 32P-labelled phosphorylase a as substrate, the initial event in the inactivation was the release of a trichloroacetic acid-soluble peptide from the N-terminus of the enzyme, leaving the original 100000 subunit form essentially unchanged. Subsequent proteolysis was restricted, producing derivatives of mol.wt. 85000, 70000 and 65000, none of which contained any radioactive label. 4. By treatment of inactivated phosphorylase b with carboxypeptidase B, it was shown that the intestinal muscle proteinase had cleaved approximately 3 -Lys-X and 3 -Arg-X bonds in the polypeptide. 5. The protective effects of various allosteric modulators of phosphorylase on the inactivation of the a and b forms were generally in agreement with the known roles of the modifiers. Glucose increased the susceptibility of phosphorylase a. 6. Inactivation of phosphorylase b by trypsin and chymotrypsin also resulted in limited proteolysis but, in both cases, the digestion patterns obtained on sodium dodecyl sulphate/polyacrylamide gels were different from each other and from the pattern obtained with the intestinal muscle proteinase. 7. Inactivation of phosphorylase b by the muscle proteinase is about 100 times more rapid than the effects produced by trypsin or chymotrypsin when the activities are compared on an equimolar basis. 8. Consideration is given to regulation of the rate of enzyme degradation intracellularly by modulation of the conformation and susceptibility of the enzyme via factors such as covalent modification, allosteric ligands and state of aggregation.

scholarly journals Monoclonal antibody against the N-terminal end of human plasma fibronectin

1983 ◽  
Vol 215 (1) ◽  
pp. 147-151 ◽  
Author(s):  
T Vartio ◽  
E M Salonen ◽  
G De Petro ◽  
S Barlati ◽  
V Miggiano ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Plasma ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cathepsin G ◽  
Heparin Binding ◽  
Plasma Fibronectin ◽  
Binding Domains ◽  
Immunoblotting Assay

Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin.

High molecular weight soluble neutral maltase-glucoamylases in the intestine of the suckling rat

1990 ◽  
Vol 68 (9) ◽  
pp. 1103-1111 ◽  
Author(s):  
L. Lee ◽  
G. Forstner
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Suckling Rats ◽  
Adult Rat ◽  
Lentil Lectin ◽  
Hydrophobic Binding ◽  
High Mannose ◽  
Sepharose 4B

Previous work from our laboratory has shown that the intestine of the suckling rat, unlike adult rat intestine, contains abundant quantities of at least two soluble neutral maltase-glucoamylases. These enzymes are related antigenically to membrane-bound maltase-glucoamylase, which predominates in adult intestine, but are either more easily solubilized or occupy a different cellular locus. To study the soluble enzymes further, we attempted their isolation from the intestine of 11-day-old suckling rats. Initial attempts were complicated by proteolytic degradation, despite the addition of phenylmethylsulfonyl fluoride, N-ethylmaleimide, leupeptin, pepstatin, and EDTA to buffers used for homogenization and column chromatography. Addition of aprotinin, amastatin, bestatin, and phosphoramidone resulted, however, in the isolation of two stable, high molecular weight maltases (HM1 and HM2). Both enzymes eluted before a papain-solubilized membrane-derived maltase-glucoamylase on Sepharose 4B and were separable by DE-52 and Sepharose 6B – Tris affinity columns. They were further purified on a lentil lectin – Sepharose 4B column. Substrate specificities were almost the same and characteristic of maltase-glucoamylases. Hydrophobic binding properties and pH optima of HM1 and HM2 were also similar. HM1 was resolved by sodium dodecyl sulfate – polyacrylamide gel electrophoresis into approximately equal portions of an endo-β-N-acetylglucosaminidase H sensitive enzyme of molecular weight (MW) 200 000 and an endo-β-N-acetylglucosaminidase H resistant but endo-β-acetylglucosaminidase F sensitive enzyme of MW 400 000. In contrast, most of HM2 consisted of a doublet of MW 200 000 – 210 000 that was endo-β-N-acetylglucosaminidase H sensitive. The intestine of the suckling rat, therefore, contains two soluble maltase–glucoamylase fractions, with a major portion of high mannose rather than complex oligosaccharides. In HM1, it is possible that the high mannose derivative is transformed during trafficking into the complex endo-β-N-acetylglucosaminidase H resistant form. HM2 appears to undergo little oligosaccharide processing.Key words: soluble maltase-glucoamylases, suckling rats.

scholarly journals Binding of Porphyromonas gingivalisFimbriae to Proline-Rich Glycoproteins in Parotid Saliva via a Domain Shared by Major Salivary Components

1998 ◽  
Vol 66 (5) ◽  
pp. 2072-2077 ◽  
Author(s):  
Atsuo Amano ◽  
Satoshi Shizukuishi ◽  
Hiroshi Horie ◽  
Shigenobu Kimura ◽  
Ichijiro Morisaki ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Human Saliva ◽  
Parotid Saliva ◽  
Binding Domains ◽  
Molecular Sizes ◽  
Overlay Assay

ABSTRACT Porphyromonas gingivalis, a putative periodontopathogen, can bind to human saliva through its fimbriae. We previously found that salivary components from the submandibular and sublingual glands bind to P. gingivalis fimbriae and that acidic proline-rich protein (PRP) and statherin function as receptor molecules for fimbriae. In this study, we investigated the fimbria-binding components in parotid saliva. Fractionated human parotid saliva by gel-filtration chromatography was immobilized onto nitrocellulose membranes for the overlay assay following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The salivary components on the membrane were allowed to interact with fimbriae purified fromP. gingivalis ATCC 33277, and the interacted fimbriae were probed with anti-fimbria antibodies. The fimbriae were shown to bind to two forms of proline-rich glycoproteins (PRGs) as well as to acidic PRPs and statherin. Moreover, fimbriae bound to several components of smaller molecular size which appeared to be acidic PRP variants and basic PRPs. Fimbriae bound strongly to the purified PRGs adsorbed onto hydroxyapatite (HAP) beads. In contrast, PRGs in solution failed to inhibit the fimbrial binding to the immobilized PRGs on the HAP beads. These findings suggest that the appearance of binding site(s) of PRGs can be ascribed to their conformational changes. We previously identified the distinct segments within PRP and statherin molecules that are involved in fimbrial binding. The peptides analogous to the binding regions of PRP and statherin (i.e., PRP-C and STN-C) markedly inhibit the binding of fimbriae to PRP and statherin immobilized on the HAP beads, respectively. The PRP-C significantly inhibited the binding of fimbriae to PRG-coated HAP beads as well as to PRP on HAP beads. The peptide did not affect the binding of fimbriae to statherin, whereas the STN-C showed no effect on the fimbrial binding to PRPs or PRGs. In the overlay assay, the PRP-C clearly diminished the interactions between the fimbriae and the various salivary components, including PRPs, the PRGs, and the components with smaller molecular sizes but not statherin. These results strongly suggest that fimbriae bind to salivary components (except statherin) via common peptide segments. It is also suggested that fimbriae bind to saliva through the two distinct binding domains of receptory salivary components: (i) PRGs and PRPs and (ii) statherin.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

The Recovery of Factor VIII from Fresh-Frozen, Indated and Outdated Human Plasma

1976 ◽  
Vol 36 (01) ◽  
pp. 071-077 ◽  
Author(s):  
Daniel E. Whitman ◽  
Mary Ellen Switzer ◽  
Patrick A. McKee
Keyword(s):  
Factor Viii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
The United States ◽  
Fresh Frozen Plasma ◽  
Red Cross ◽  
Random Samples ◽  
Fresh Frozen ◽  
Factor Viii Concentrates ◽  
Frozen Plasma

SummaryThe availability of factor VIII concentrates is frequently a limitation in the management of classical hemophilia. Such concentrates are prepared from fresh or fresh-frozen plasma. A significant volume of plasma in the United States becomes “indated”, i. e., in contact with red blood cells for 24 hours at 4°, and is therefore not used to prepare factor VIII concentrates. To evaluate this possible resource, partially purified factor VIII was prepared from random samples of fresh-frozen, indated and outdated plasma. The yield of factor VIII protein and procoagulant activity from indated plasma was about the same as that from fresh-frozen plasma. The yield from outdated plasma was substantially less. After further purification, factor VIII from the three sources gave a single subunit band when reduced and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results indicate that the approximately 287,000 liters of indated plasma processed annually by the American National Red Cross (ANRC) could be used to prepare factor VIII concentrates of good quality. This resource alone could quadruple the supply of factor VIII available for therapy.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

Majra Honey Abrogated the Normal and Cancer Cells Proliferation Inhibition by Juniperus procera Extract and Extract/Honey Generated AgNPs

2020 ◽  
Vol 20 (8) ◽  
pp. 970-981
Author(s):  
Hamed A. Ghramh ◽  
Essam H. Ibrahim ◽  
Mona Kilnay
Keyword(s):  
Anticancer Activity ◽  
Cancer Cells ◽  
Biological Activities ◽  
Sugar Content ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bioactive Molecules ◽  
Sds Page ◽  
Splenic Cells ◽  
Juniperus Procera

Background: Juniperus procera and Majra honey are well-known as a folk medicine in many countries. Objectives: This work aimed to study the immunomodulatory effects after mixing Majra honey, J. procera water leaves extract and silver Nanoparticles (AgNPs) on immune or cancer cells. Methods: Juniperus procera water leaves extract and 20% Majra honey were prepared. Both the extract and honey were used separately to synthesize AgNPs. AgNPs were characterized using UV/Vis spectrophotometry and electron microscopy. Bioactive molecules in honey and the extract were explored using Fourier Transform Infrared (FT-IR) spectroscopy. Protein profile of honey was explored using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and honey sugar content was determined using High- Performance Liquid Chromatography (HPLC). Biological activities of honey and the extract were tested. Results: The results demonstrated the ability of the extract/honey to produce AgNPs in a spherical shape. The extract/honey contained many functional groups. SDS-PAGE of Majra honey showed many protein bands. HPLC revealed honey is of good quality and no external additives are added to it. The extract and extract+ AgNPs inhibited the growth of normal rat splenic cells while honey stimulated it. The extract+honey turned stimulatory to the splenic cells’ growth and significantly diminished the inhibitory potential of the extract containing AgNPs. Both the extract and honey have antimicrobial activities, this potential increased in the presence of AgNPs. Honey and Honey+AgNPs inhibited HepG2 cancer cell proliferation while Hela cell growth inhibited only with honey+AgNPs. Conclusion: Both honey and the extract have antibacterial and immunomodulatory potentials as well as the power to produce AgNPs. Majra honey alone showed anticancer activity against HepGe2 cells, but not against Hela cells, and when contained AgNPs had anticancer activity on both cell lines. Mixing of Majra honey with J. procera extract showed characterized immunomodulatory potentials that can be described as immunostimulant.

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