scholarly journals A study of highly purified mucolipidosis III urinary N-acetyl-β-D-hexosaminidase B

1982 ◽  
Vol 204 (2) ◽  
pp. 557-563 ◽  
Author(s):  
S Hirani ◽  
L Little ◽  
A L Miller
Keyword(s):  
Ricinus Communis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Genetic Defect ◽  
Sodium Dodecyl ◽  
Acid Hydrolases ◽  
Enzyme Preparations ◽  
Normal Enzyme ◽  
Considerable Homology ◽  
Protein Components ◽  
Normal Urine

Highly purified N-acetyl-beta-D-hexosaminidase B from normal urine and urine of a patient with mucolipidosis III was used to determine whether it has undergone any of the alterations associated with this genetic defect. Examination by sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed that both the enzyme preparations contained protein components with apparent Mr values of 55 000 and 28 000. No differences in the binding and apparent KI (50%) to concanavalin A of the normal and mucolipidosis III enzymes were detected. However, the patient's N-acetyl-beta-D-hexosaminidase B had a slightly greater affinity for the lectin from Ricinus communis than did the normal enzyme. Two-dimensional tryptic peptide maps of the corresponding normal and the patient's N-acetyl-beta-D-hexosaminidase B subunits showed considerable homology. These results indicate that N-acetyl-beta-D-hexosaminidase b does not undergo the significant carbohydrate alterations characteristic of other acid hydrolases in mucolipidosis III.

scholarly journals Mucolipidosis III β-N-acetyl-d-hexosaminidase A. Purification and properties

1982 ◽  
Vol 207 (3) ◽  
pp. 421-428 ◽  
Author(s):  
Barry C. Kress ◽  
Shirish Hirani ◽  
Hudson H. Freeze ◽  
Laureen Little ◽  
Arnold L. Miller
Keyword(s):  
Structural Changes ◽  
Ricinus Communis ◽  
Sodium Dodecyl ◽  
Compositional Analysis ◽  
Acid Hydrolases ◽  
Complex Type ◽  
Apparent Homogeneity ◽  
Hexosaminidase A ◽  
Normal Enzyme ◽  
High Mannose

Mucolipidosis III acid hydrolases possess an altered carbohydrate recognition marker needed for their lysosomal localization. As a result of this alteration, a portion of these enzymes is secreted from the cell to the extracellular spaces. The structural changes that may have occurred to one of these secreted enzymes, β-N-acetyl-d-hexosaminidase A (EC 3.2.1.52) were investigated. Normal and mucolipidosis III urinary β-N-acetyl-d-hexosaminidase A were purified to apparent homogeneity by using affinity [Sepharose-2-acetamido-N-(ε-aminocaproyl)-2-deoxy-β- d-glucopyranosylamine] and ion-exchange (DEAE- and CM-cellulose) chromatography. Sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis showed that both enzymes had similar subunit patterns consisting of apparent mol.wts. of 68000, 60000–58000, 55000 and 29000. Differences, however, were noted in the relative proportions of the protein bands where the normal urinary β-N-acetyl-d-hexosaminidase A contained predominantly the smaller subunits, whereas the mucolipidosis III enzyme had a predominance of the larger subunits. The binding of mucolipidosis III β-N-acetyl-d-hexosaminidase A to Ricinus communis lectin and concanavalin A with and without endo-β-N-acetyl-d-glucosaminidase H treatment indicated that the mutation leads to a modification of a portion of the normally occurring high-mannose-type oligosaccharide units to the complex-type. This was further supported by carbohydrate compositional analysis, which revealed a mannose/galactose ratio of 2.1 for the mucolipidosis III β-N-acetyl-d-hexosaminidase A compared with a ratio of 3.5 for the normal enzyme. Our results indicate that as a result of their inability to be properly localized to the lysosome the majority of the mucolipidosis III lysosomal hydrolase high-mannose oligosaccharide units are further processed to the complex-type before secretion of predominantly higher-molecular-weight subunits from the cell.

scholarly journals Fractionation of the proteins of plant microbodies

1974 ◽  
Vol 144 (3) ◽  
pp. 559-566 ◽  
Author(s):  
R H Brown ◽  
J M Lord ◽  
M J Merrett
Keyword(s):  
Membrane Proteins ◽  
Ricinus Communis ◽  
Osmotic Shock ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Weight One ◽  
Protein Components

1. Glyoxysomes and peroxisomes have been isolated from dark- and light-grown seedlings of pumpkin (Cucurbita pepo) by sucrose-density-gradient centrifugation. 2. Pumpkin microbodies and castor-bean (Ricinus communis) glyoxysomes may be fractionated, by a combination of osmotic shock and treatment with KCl, into three distinct groups of proteins: readily soluble (matrix enzymes), solubilized in the presence of KCl (membrane-bound enzymes) and relatively insoluble (membrane ‘ghost’ proteins). 3. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis of ‘ghost’ fractions indicated that the membrane proteins were generally of low molecular weight; one gel band (mol.wt. 27000–28000) was common to all three microbodies. 4. Although there were major differences in the soluble protein components of pumpkin glyoxysomes and peroxisomes, electrophoresis of the pumpkin microbody ‘ghosts’ indicated that the membrane proteins were similar, four main components being common to each class of microbody (monomer molecular weights 42000, 34000, 27000 and 17000).

scholarly journals Bis(β-lactosyl)-[60]fullerene as novel class of glycolipids useful for the detection and the decontamination of biological toxins of the Ricinus communis family

2014 ◽  
Vol 10 ◽  
pp. 1504-1512 ◽  
Author(s):  
Hirofumi Dohi ◽  
Takeru Kanazawa ◽  
Akihiro Saito ◽  
Keita Sato ◽  
Hirotaka Uzawa ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Protein Interactions ◽  
Colloidal Solution ◽  
Ricinus Communis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cycloaddition Reaction ◽  
Azide Group ◽  
Red Color ◽  
Sds Page

Glycosyl-[60]fullerenes were first used as decontaminants against ricin, a lactose recognition proteotoxin in the Ricinus communis family. A fullerene glycoconjugate carrying two lactose units was synthesized by a [3 + 2] cycloaddition reaction between C60 and the azide group in 6-azidohexyl β-lactoside per-O-acetate. A colloidal aqueous solution with brown color was prepared from deprotected bis(lactosyl)-C60 and was found stable for more than 6 months keeping its red color. Upon mixing with an aqueous solution of Ricinus communis agglutinin (RCA120), the colloidal solution soon caused precipitations, while becoming colorless and transparent. In contrast, a solution of concanavalin A (Con A) caused no apparent change, indicating that the precipitation was caused specifically by carbohydrate–protein interactions. This notable phenomenon was quantified by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results were discussed in terms of detection and decontamination of the deadly biological toxin in the Ricinus communis family.

scholarly journals A method for the direct demonstration of the lectin-binding components of the human erythrocyte membrane

1976 ◽  
Vol 153 (2) ◽  
pp. 265-270 ◽  
Author(s):  
M J A Tanner ◽  
D J Anstee
Keyword(s):  
Ricinus Communis ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Erythrocyte Membrane ◽  
Individual Pattern ◽  
Direct Demonstration ◽  
Staining Techniques ◽  
Membrane Components

1. A method which allows the characterization of lectin-binding components is described. This method should be useful in defining the nature and heterogeneity of these components in cell membranes. 2. The method, which we have used on erythrocyte “ghosts”, involves the fixation of “ghost” components after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and incubation with purified 125I-labelled lectins. 3. Each of the four lectins used shows an individual pattern of reactivity towards “ghosts” components. Band 3, the major membrane-penetrating glycoprotein, is bound by the lectins from Ricinus communis and Phaseolus vulgaris (phytohaemagglutinin) and by concanavalin A. The major erythrocyte sialoglycoprotein is bound by the lectins from R. communis, P. vulgaris and Maclura aurantiaca. 4. Three of the lectins displays binding for other membrane components, some of which are not demonstratable by conventional protein- and carbohydrate-staining techniques.

scholarly journals Identification of the Nucleocapsid, Tegument, and Envelope Proteins of the Shrimp White Spot Syndrome Virus Virion

2006 ◽  
Vol 80 (6) ◽  
pp. 3021-3029 ◽  
Author(s):  
Jyh-Ming Tsai ◽  
Han-Ching Wang ◽  
Jiann-Horng Leu ◽  
Andrew H.-J. Wang ◽  
Ying Zhuang ◽  
...  
Keyword(s):  
White Spot Syndrome Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Envelope Proteins ◽  
White Spot ◽  
Structural Proteins ◽  
Nucleocapsid Proteins ◽  
Tegument Proteins ◽  
White Spot Syndrome ◽  
Protein Components

ABSTRACT The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.

Cell wall associations of some antigenic proteins of slime-forming, encapsulated Streptococcus cremoris from the fermented milk product "viili"

1986 ◽  
Vol 32 (2) ◽  
pp. 176-178 ◽  
Author(s):  
Raili Forsén ◽  
Teuvo Hentunen ◽  
Kaua Valkonen ◽  
Sirpa Kontusaari
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Cell Walls ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fermented Milk ◽  
Milk Product ◽  
Streptococcus Cremoris ◽  
Protein Components

Cell walls were isolated from mechanically disrupted cells of the slime-forming, encapsulated Streptococcus cremoris strains T5 and MLS96 by using sucrose gradient centrifugation as the last purification step. This cell wall isolation procedure was developed to obtain cell wall associated protein components. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed several polypeptide bands; the 50 kiloDalton band was major in strain T5 cell walls and the 26 and 30 kiloDalton bands were major in strain MLS96 cell walls. Both strains contained five antigenic polypeptides with molecular radius (Mr) values of 40, 47, 50, 54, and 70 kiloDaltons as analysed by immunoblotting and autoradiography. The polypeptides of strain MLS96 with molecular mass of 40 and 70 kiloDaltons reacted most strongly with homologous anti-whole cell serum. In addition, antigenic polypeptides with molecular mass of 100 and 160 kiloDaltons were also detected in strain T5.

Electrophoretic Analysis of Polypeptides of Plasma Membranes from Bovine Pituitary Gland

1974 ◽  
Vol 52 (7) ◽  
pp. 620-630
Author(s):  
André Lemay ◽  
Fernand Labrie
Keyword(s):  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Electrophoretic Pattern ◽  
Apparent Molecular Weight ◽  
Electrophoretic Analysis ◽  
Polyacrylamide Gel ◽  
Protein Components ◽  
Membrane Components

Purified plasma membranes from bovine hypophyseal tissue have been fractionated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under various conditions of pH and acrylamide concentrations. The best separation of protein components is achieved at a concentration of 7.5% acrylamide and at pH 7.1. Under these conditions, the electrophoretic pattern consistently shows 36 protein bands ranging in molecular weights from 250 000 to 15 000. Only one broad band, having an apparent molecular weight of 150 000, stains for glycoproteins by the period acid – Schiff technique. After electrophoresis on a two-dimensional polyacrylamide gel system using disc gels containing urea and Triton X-100 in the first dimension and SDS in the second dimension, approximately 45 different protein components can be identified. Less than 12% of the membrane proteins are solubilized by washing the membranes with 1 M KCl or NH4Cl. Denaturating agents like urea and lithium 3,4-diiodosalycilate solubilize 55–60% of membrane components. Adenohypophyseal plasma membranes show an eleetrophoretic pattern completely different from that obtained with membranes isolated from the intermediate or posterior pituitary lobes.

Membranes of chromaffin granules. Isolation and partial characterization of two proteins

1971 ◽  
Vol 122 (3) ◽  
pp. 298.1-304 ◽  
Author(s):  
Heide Hörtnagl ◽  
H. Winkler ◽  
J. A. L. Schöpf ◽  
W. Hohenwallner
Keyword(s):  
Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Major Protein ◽  
Chromaffin Granules ◽  
Protein Components ◽  
Acid Gel ◽  
Total Membrane

Membranes of chromaffin granules were isolated from the adrenal glands of four different species. The solubilized membrane proteins could be resolved into several bands by polyacrylamide-gel electrophoresis (alkaline and acid gel systems). Two major protein components appeared to be common to the chromaffin granule membranes of ox, horse, pig and man. The various membrane proteins of bovine chromaffin granules were separated by filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. Two major membrane proteins (A and B) were obtained in purified form. Treatment of protein A with 2-mercaptoethanol before electrophoresis resulted in two more rapidly migrating subunits, whereas protein B was unaffected by mercaptoethanol treatment. The amino acid compositions of the two purified proteins were determined. They are very similar to that of the total membrane proteins but significantly different from that of the chromogranins, the soluble proteins of chromaffin granules.

Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE

1990 ◽  
Vol 64 (3) ◽  
pp. 175-179 ◽  
Author(s):  
M. Itoh ◽  
S. Sato ◽  
A. Moriyama ◽  
M. Sasaki
Keyword(s):  
Proteinase Inhibitor ◽  
Cysteine Proteinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Clonorchis Sinensis ◽  
Sodium Dodecyl ◽  
Heating Process ◽  
Cysteine Proteinase Inhibitor ◽  
Electrophoretic Patterns ◽  
Sds Page ◽  
Protein Components

ABSTRACTSince Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinensis, when their extracts were treated with E-64.

scholarly journals Identity of purified monoacylglycerol lipase, palmitoyl-CoA hydrolase and aspirin-metabolizing carboxylesterase from rat liver microsomal fractions. A comparative study with enzymes purified in different laboratories

1985 ◽  
Vol 232 (2) ◽  
pp. 479-483 ◽  
Author(s):  
R Mentlein ◽  
R K Berge ◽  
E Heymann
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Monoacylglycerol Lipase ◽  
Enzyme Preparations ◽  
Nitrophenyl Phosphate ◽  
Liver Microsomal Fraction ◽  
Coa Hydrolase ◽  
Rat Liver Cytosol

Two purified carboxylesterases that were isolated from a rat liver microsomal fraction in a Norwegian and a German laboratory were compared. The Norwegian enzyme preparation was classified as palmitoyl-CoA hydrolase (EC 3.1.2.2) in many earlier papers, whereas the German preparation was termed monoacylglycerol lipase (EC 3.1.1.23) or esterase pI 6.2/6.4 (non-specific carboxylesterase, EC 3.1.1.1). Antisera against the two purified enzyme preparations were cross-reactive. The two proteins co-migrate in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Both enzymes exhibit identical inhibition characteristics with Mg2+, Ca2+ and bis-(4-nitrophenyl) phosphate if assayed with the two substrates palmitoyl-CoA and phenyl butyrate. It is concluded that the two esterase preparations are identical. However, immunoprecipitation and inhibition experiments confirm that this microsomal lipase differs from the palmitoyl-CoA hydrolases of rat liver cytosol and mitochondria.

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