scholarly journals Developmental pattern of rat intestinal brush-border enzymic proteins along the villus–crypt axis

1979 ◽  
Vol 178 (2) ◽  
pp. 407-413 ◽  
Author(s):  
Patricia M. Simon ◽  
Michèle Kedinger ◽  
Francis Raul ◽  
Jacques F. Grenier ◽  
Katy Haffen
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Band 3 ◽  
Developmental Pattern ◽  
Simultaneous Increase ◽  
Cell Compartments ◽  
Crypt Cells

At various postnatal stages, intestinal epithelial cells were isolated sequentially from villus tip to crypt base by successive EDTA treatments. According to the localization of marker enzymic activities, isolated cells were pooled into three cell compartments: villus (V), lower villus and upper crypt (VC) and crypt (C). Purified brush-border-membrane proteins were separated by 7.5%-polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Enzymic activities could be assigned to some protein bands: maltase/glucoamylase (protein band 3), sucrase–isomaltase (protein bands 3 and 6), lactase (protein band 5) and alkaline phosphatase (region of protein bands 8 and 9). The findings suggest the following. (1) Sucrase–isomaltase activities appeared in compartment C at 17 days with a simultaneous increase of the pre-existing protein band 3 and appearance of a well-defined protein band in position 6; the enzymic complex remained still present in the crypt cells until adulthood. From the day 21 onwards, sucrase–isomaltase was detected in compartments VC and V. (2) Lactase was only present in the three cell compartments until day 21; at this developmental stage its activity completely disappeared from compartment C, in spite of the persistence of a weak protein band. (3) Alkaline phosphatase activity could be detected as a single peak corresponding to protein band 9 in all three cell compartments until day 21; thereafter it was replaced by two peaks of activity showing a less precise correlation with the well-defined protein bands 8 and 9. In the crypt cells of the adult rat, however, the preweaning situation, which was regularly observed, is an unexpected phenomenon. (4) Maltase and glucoamylase did not display any marked qualitative or quantitative modifications either along the villus–crypt axis or during the period of postnatal development studied. Evidence is given from the present data that each brush-border enzyme investigated has a specific developmental pattern.

scholarly journals Characterization of brush borders purified in iso-osmotic medium and microvillar membranes subfractionated from mouse small intestine

1981 ◽  
Vol 196 (3) ◽  
pp. 669-673 ◽  
Author(s):  
M Fujita ◽  
H Ohta ◽  
T Uezato
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gamma Glutamyl Transpeptidase ◽  
Electrophoretic Patterns ◽  
Mouse Small Intestine ◽  
Brush Borders ◽  
Typical Morphology ◽  
Glutamyl Transpeptidase

Brush borders free of nuclei were isolated by repeated homogenization and centrifugation in iso-osmotic medium. They showed typical morphology under electron microscopy. The mean recovery and enrichment of alkaline phosphatase activity in the brush-border fraction were 50% and 17.5-fold respectively. gamma-Glutamyl transpeptidase showed a close parallelism with alkaline phosphatase and sucrase in subcellular distribution. Microvillar membranes were purified from isolated brush borders; they showed a further enrichment for alkaline phosphatase and were composed of homogeneous vesicles. Both brush-border and microvillar-membrane preparations were analysed for contamination by basolateral and endoplasmic-reticular membranes. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the microvillar-membrane preparation in six different systems revealed approx. 40 components in the mol.wt. range 15 000-232 000. They were grouped into seven major classes on the basis of molecular weight and electrophoretic patterns.

scholarly journals Weissellicin 110, a Newly Discovered Bacteriocin from Weissella cibaria 110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand

2007 ◽  
Vol 73 (7) ◽  
pp. 2247-2250 ◽  
Author(s):  
Sirinat Srionnual ◽  
Fujitoshi Yanagida ◽  
Li-Hsiu Lin ◽  
Kuang-Nan Hsiao ◽  
Yi-sheng Chen
Keyword(s):  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Proteinase K ◽  
Mass Spectrometry Analysis ◽  
Fermented Fish ◽  
Spectrometry Analysis ◽  
Weissella Cibaria ◽  
Fish Product

ABSTRACT Weissella cibaria 110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from W. cibaria 110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.

scholarly journals Increased membrane binding of erythrocyte catalase in hereditary spherocytosis and in metabolically stressed normal cells

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 113-123 ◽  
Author(s):  
DW Allen ◽  
S Cadman ◽  
SR McCann ◽  
B Finkel
Keyword(s):  
Molecular Weight ◽  
Hereditary Spherocytosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane Binding ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Normal Cells ◽  
Calcium Dependent ◽  
Subunit Molecular Weight ◽  
Erythrocyte Catalase

Abstract Normal red blood cell (RBC) membranes were compared with (1) RBC membranes from six patients with hereditary spherocytosis (HS), (2) normal membranes after hemolysis of the RBC in the presence of calcium, or (3) normal membranes after incubation of RBC for 24 hr in phosphate- buffered saline containing calcium without added glucose. When compared with normal controls, polyacrylamide gel electrophoresis with sodium dodecyl sulfate (PAGE SDS) of all three preparations showed an increase in membrane binding of globin and protein band 4.5 (60,000 molecular weight). In an attempt to identify band 4.5, 14 enzymes were assayed in the RBC membranes. Of these, catalase and lactate dehydrogenase were increased in membranes from HS RBC and from normal cells exposed to calcium. Only catalase, however, was present in sufficient quantity and had the correct subunit molecular weight on PAGE SDS and calcium- dependent membrane binding to account for an appreciable portion of 4.5. Caralase was further identified with a component of band 4.5 by double immunodiffusion using a specific anti-catalase antibody.

scholarly journals The Nα-acetylenkephalin carboxypeptidase activity of N-acetyltyrosine deacetylase from monkey kidney. Purification, characterization and substrate specificity

1983 ◽  
Vol 209 (2) ◽  
pp. 471-479 ◽  
Author(s):  
S T George ◽  
A S Balasubramanian
Keyword(s):  
Amino Acids ◽  
Gel Electrophoresis ◽  
High Speed ◽  
Metal Chelate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Protein Band ◽  
Monkey Kidney ◽  
Hydrophobic Amino Acids

N alpha-Acetylenkephalin carboxypeptidase was co-purified with N-acetyltyrosine deacetylase from monkey kidney. Almost 90% of the activity from the homogenate was recovered in a high-speed supernatant without the use of detergents. The crucial steps in the purification were Cibacron Blue F3GA-Sepharose chromatography (involving negative and positive binding sequentially) and metal chelate affinity chromatography. The purified enzyme showed three bands on gel electrophoresis under non-denaturing conditions. All the three bands exhibited both N-acetyltyrosine deacetylase and N-acetylenkephalin carboxypeptidase activity, indicating their co-migration, Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence and absence of 2-mercaptoethanol gave a single protein band of mol.wt. 34 000. The native enzyme was a dimer of mol.wt. 66 000 as observed on Bio-Gel P-300 gel filtration. The carboxypeptidase removed two amino acids from the C-terminal end of either N-acetyl[Met5]- or N-acetyl[Leu5]-enkephalin. Non-acetylated enkephalins were less active as substrates. Peptides with their carboxy end blocked were inactive as substrates. Models suggested for carboxypeptidase A [Hartsuck & Lipscomb (1971) Enzymes 3, 1-56] support the idea that the kidney N-acetylated aromatic amino acid deacetylase or acylase III [Endo (1978) Biochim. Biophys. Acta 523, 207-217] can act as a carboxypeptidase on peptides having hydrophobic amino acids at the C-terminal end.

In vitro cellulolytic activity of the plant pathogen Clavibacter michiganensis subsp. sepedonicus

1995 ◽  
Vol 41 (10) ◽  
pp. 877-888 ◽  
Author(s):  
Debra Baer ◽  
Neil C. Gudmestad
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Cellulolytic Activity ◽  
Clavibacter Michiganensis ◽  
Endoglucanase Activity ◽  
Clavibacter Michiganensis Subsp ◽  
Cellulose Substrates ◽  
Key Words Cellulase

The activity of four Clavibacter michiganensis subsp. sepedonicus strains against various cellulose substrates was investigated. Sixty-seven Clavibacter michiganensis subsp. sepedonicus strains grew well on media amended with carboxymethylcellulose, 64 strains produced zones of hydrolysis. Endoglucanase activity was optimal at 37 °C and pH 6.0 against carboxymethylcellulose incorporated in plate assays. Zymogram and sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed the presence of a protein band corresponding to the cellulolytic activity in the molecular weight (MW) range of approximately 28 000. Protein bands in the same range were detected in five Clavibacter michiganensis subsp. sepedonicus strains. Studies on crude enzyme extracts of Clavibacter michiganensis subsp. sepedonicus strain N-1-1 revealed that p-nitrophenyl β-D-cellobioside (pNPC) was hydrolyzed, with optimal activity at 37 °C and pH 7.0.Key words: cellulase, endo-1,4-β-glucanase (EC 3.2.1.4), Corynebacterium sepedonicum, Solanum tuberosum.

scholarly journals Characterization of the Vaccinia Virus A35R Protein and Its Role in Virulence

2006 ◽  
Vol 80 (1) ◽  
pp. 306-313 ◽  
Author(s):  
Rachel L. Roper
Keyword(s):  
Vaccinia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Normal Growth ◽  
Protein Band ◽  
Mutant Virus ◽  
Translation System ◽  
Wild Type ◽  
Infected Cells

ABSTRACT The vaccinia virus A35R gene is highly conserved among poxviruses and encodes a previously uncharacterized hydrophobic acidic protein. Western blotting with anti-A35R peptide antibodies indicated that the protein is expressed early in infection and resolved as a single sharp band of ∼23 kDa, slightly higher than the 20 kDa predicted from its sequence. The protein band appeared to be the same molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whether expressed in an in vitro transcription/translation system without microsomes or expressed in infected cells, suggesting that it was not glycosylated. A mutant virus with the A35R gene deleted (vA35Δ) formed wild-type-sized plaques on all cell lines tested (human, monkey, mouse, and rabbit); thus, A35R is not required for replication and does not appear to be a host range gene. Although the A35R protein is hydrophobic, it is unlikely to be an integral membrane protein, as it partitioned to the aqueous phase during TX-114 partitioning. The protein could not be detected in virus-infected cell supernatants. A35R localized intracellularly to the virus factories, where the first stages of morphogenesis occur. The vA35Δ mutant formed near-normal levels of the various morphogenic stages of infectious virus particles and supported normal acid-induced fusion of virus-infected cells. Despite normal growth and morphogenesis in vitro, the vA35Δ mutant virus was attenuated in intranasal challenge of mice compared to wild-type and A35R rescue virus. Thus, the intracellular A35R protein plays a role in virulence. The A35R has little homology to any protein outside of poxviruses, suggesting a novel virulence mechanism.

scholarly journals Increased membrane binding of erythrocyte catalase in hereditary spherocytosis and in metabolically stressed normal cells

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 113-123
Author(s):  
DW Allen ◽  
S Cadman ◽  
SR McCann ◽  
B Finkel
Keyword(s):  
Molecular Weight ◽  
Hereditary Spherocytosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane Binding ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Normal Cells ◽  
Calcium Dependent ◽  
Subunit Molecular Weight ◽  
Erythrocyte Catalase

Normal red blood cell (RBC) membranes were compared with (1) RBC membranes from six patients with hereditary spherocytosis (HS), (2) normal membranes after hemolysis of the RBC in the presence of calcium, or (3) normal membranes after incubation of RBC for 24 hr in phosphate- buffered saline containing calcium without added glucose. When compared with normal controls, polyacrylamide gel electrophoresis with sodium dodecyl sulfate (PAGE SDS) of all three preparations showed an increase in membrane binding of globin and protein band 4.5 (60,000 molecular weight). In an attempt to identify band 4.5, 14 enzymes were assayed in the RBC membranes. Of these, catalase and lactate dehydrogenase were increased in membranes from HS RBC and from normal cells exposed to calcium. Only catalase, however, was present in sufficient quantity and had the correct subunit molecular weight on PAGE SDS and calcium- dependent membrane binding to account for an appreciable portion of 4.5. Caralase was further identified with a component of band 4.5 by double immunodiffusion using a specific anti-catalase antibody.

scholarly journals Specific protein synthesis in isolated rat testis leydig cells. Influence of luteinizing hormone and cycloheximide

1977 ◽  
Vol 162 (2) ◽  
pp. 341-346 ◽  
Author(s):  
F H A Janszen ◽  
B A Cooke ◽  
H J van der Molen
Keyword(s):  
Protein Synthesis ◽  
Luteinizing Hormone ◽  
Leydig Cell ◽  
Half Life ◽  
Leydig Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Specific Protein ◽  
Rat Testis

The effect of luteinizing hormone (luteotropin) and cycloheximide on specific protein synthesis in rat testis Leydig cells has been investigated. Proteins were labelled with either I114C]leucine, [3H]leucine or [35S]methionine during incubation with Leydig-cell suspensions in vitro. Total protein was extracted from the cells and separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. No detectable increase in the synthesis of specific proteins could be observed after incubation of Leydig cells with luteinizing hormone for up to 1 h. However, after a 2h incubation period, an increase in [35S]methionine incorporation was observed in a protein with an apparent mol.wt. of 21000 (referred to as ‘protein 21’). When, after labelling of this protein with [35S]-methionine, Leydig cells were incubated for another 30min with cycloheximide, no decrease in radioactivity of this protein band was observed, indicating that it does not have a short half-life. However, another protein band was detected, which after incubation with cycloheximide disappeared rapidly, the reaction following first-order kinetics, with a half-life of about 11 min. This protein, with an apparent mol.wt. of 33000 (referred to as “protein 33”), was found to be located in the particulate fraction of the Leydig cell, and could not be demonstrated in other rat testis-cell types or blood cells. No effect of luteinizing hormone on molecular weight, subcellular localization or half-life of protein 33 was observed. A possible role for protein 33 and protein 21 in the mechanism of action of luteinizing hormone on testosterone production of Leydig cells is discussed.

scholarly journals Purification and properties of a β-1,6-glucanase from Penicillium brefeldianum

1984 ◽  
Vol 223 (3) ◽  
pp. 707-714 ◽  
Author(s):  
G P Schep ◽  
M G Shepherd ◽  
P A Sullivan
Keyword(s):  
High Pressure ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Dry Weight ◽  
Protein Band ◽  
Purification And Properties ◽  
Freeze Dried ◽  
Beta Glucosidase

An inducible endo-beta-1,6-glucanase was purified from Penicillium brefeldianum by DEAE-cellulose, Bio-Gel P-150 and high-pressure liquid chromatography. The final preparation was essentially free from beta-1,3-glucanase and beta-glucosidase activities. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one protein band with an Mr of 44000. The Vmax. and Km values were calculated to be 624 units (mumol/min)/mg and 2.78 mg/ml respectively. The glucanase had lytic activity against mycelial cells of the yeast Candida albicans. The yield of purified beta-1,6-glucanase from 100 mg dry weight of freeze-dried culture filtrate varied from 60 to 180 units.

Evaluation of wheat endosperm protein fingerprints as indices of Udon-noodle making quality

2001 ◽  
Vol 52 (9) ◽  
pp. 919
Author(s):  
H. Nakamura
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Protein Band ◽  
Eating Quality ◽  
Endosperm Protein ◽  
Wheat Lines ◽  
Noodle Quality

Hexaploid wheat (Triticum aestivum) endosperm protein fingerprints were used to determine the indices of Japanese soft Udon-noodle quality. The endosperm proteins of Japanese Udon wheat lines were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis to determine differences between them in protein composition in two soil environments. The differences between the lines included differences in the composition of the endosperm with regard to the 53 kDa protein band or high-molecular-weight glutenin subunit (HMW-GS) 2* and in the sensory viscoelasticity score of cooked noodles, which is related to eating-quality in Japanese Udon wheats. One line (Kanto 107) showed variation for the presence of the 53 kDa and HMW-GS 2* bands between environments.

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