scholarly journals On the presence of two non-specific nucleotide-sugar-hydrolysing enzymes in rat liver

1984 ◽  
Vol 220 (1) ◽  
pp. 95-103 ◽  
Author(s):  
H G Muilerman ◽  
A M Lasthuis ◽  
G J M Hooghwinkel ◽  
W Van Dijk
Keyword(s):  
Rat Liver ◽  
Sodium Dodecyl ◽  
Rat Tissues ◽  
Nucleotide Sugar ◽  
Nitrophenyl Phosphate ◽  
Molecular Masses ◽  
Almost All ◽  
Triton X ◽  
Enzyme I ◽  
Specific Nucleotide

Evidence is presented for the occurrence of two different non-specific nucleotide-sugar hydrolases in rat liver and other rat tissues. These two enzymes (I and II) were separated by chromatography on a 5′-AMP-aminohexyl-Sepharose column. Enzyme I is most probably identical with phosphodiesterase I (EC 3.1.4.1). Enzyme II appeared to be identical with an enzyme described in literature as ‘CMP-sialic acid hydrolase’ [Kean & Bighouse (1974) J. Biol. Chem. 249, 7813-7823], since almost all activity with CMP-N-acetylneuraminate as substrate was recovered in this enzyme fraction. CMP-N-acetylneuraminate was a poor substrate for Enzyme I, whereas deoxythymidine-5′-p-nitrophenyl phosphate and all nucleoside-diphosphosugars tested were good substrates for both Enzyme I and II. Therefore it is suggested that CMP-N-acetylneuraminate is used as an additional substrate to discriminate between the activities of Enzyme I and II in homogenates or membrane preparations. The various substrates appeared to be competitive inhibitors of each other, suggesting that, in each enzyme preparation, only one enzyme is responsible for the hydrolysis of the various substrates. The dissimilar properties of the two enzymes are substantiated by studying the subunit molecular masses (Enzyme I, 125 kDa; Enzyme II, 50-55 kDa), the sensitivity towards Triton X-100, Sarkosyl and sodium dodecyl sulphate and towards trypsin treatment. It is discussed whether the alpha-N-acetylglucosamine phosphodiesterase described by Varki & Kornfeld [(1981) J. Biol. Chem. 256, 9937-9943] is identical with one of the nucleotide-sugar hydrolases described here.

A monoclonal antibody to lactogenic receptors from female rat liver

1989 ◽  
Vol 120 (3) ◽  
pp. 401-407 ◽  
Author(s):  
M. Emtner ◽  
J. Brandt ◽  
U. Johansson ◽  
B. Jouper ◽  
L. Fryklund ◽  
...  
Keyword(s):  
Rat Liver ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Structural Similarity ◽  
Male Rat ◽  
Receptor Interaction ◽  
Rat Tissues ◽  
Female Rat ◽  
Sds Electrophoresis

ABSTRACT Affinity-purified lactogenic receptors from female rat liver microsomal membranes were used to raise antibodies in female Balb/c mice. Mouse spleen and myeloma cells were fused and hybridoma-derived monoclonal antibodies (Mabs) were produced by in-vitro cell culture. Mab from a selected clone was sequentially purified by chromatography on a thiophilic gel and on agarose-bound protein A. The Mab was found to be of IgG1 subclass and of κ type. The Mab recognized membrane-bound and solubilized (by the detergent heptaoxyethylene lauryl ether; G3707) receptors as well as receptors purified by affinity chromatography and subsequent sodium dodecyl sulphate (SDS) electrophoresis from female rat liver. The Mab bound to receptors from several other female rat tissues, such as ovary, kidney and adrenal, whereas there was no binding to liver receptors from cow and rabbit. Displacement experiments showed that the Mab was specific for a lactogenic type of receptor, in agreement with the finding that the Mab did not recognize receptors from male rat liver. The Mab also bound to cytoplasmic receptors (present in the supernatant after centrifugation at 100 000 g) from female rat liver, suggesting a structural similarity between the cytoplasmic and the microsomal receptors. Analysis of purified receptors by SDS electrophoresis and subsequent Western blot with 125I-labelled Mab as a probe showed one band corresponding to an Mr of 45 500 ± 2500 (n=5). The same band was obtained with 125I-labelled human GH, showing that the Mab binds to the unit which accommodates the hormone-binding site. The binding sites are, however, not identical since the binding of Mab to the receptor did not inhibit the hormone–receptor interaction. Whether this binding unit represents the intact receptor molecule or a part of it cannot be concluded. Journal of Endocrinology (1989) 120, 401–407

scholarly journals Preparation and properties of a complex from rat liver of polyribosomes with components of the cytoskeleton

1983 ◽  
Vol 216 (1) ◽  
pp. 215-226 ◽  
Author(s):  
A Adams ◽  
E G Fey ◽  
S F Pike ◽  
C J Taylorson ◽  
H A White ◽  
...  
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Cytoskeletal Proteins ◽  
Deoxyribonuclease I ◽  
Microsomal Membranes ◽  
Cytoskeletal Elements ◽  
Triton X

Gel filtration with 1% agarose (Bio-Gel A-150m) separates polyribosomes bound to microsomal membranes from ‘free’ polyribosomes when these fractions are prepared by standard centrifugal techniques. However, when polyribosomes contained in an unfractionated postmitochondrial supernatant are run on an identical column, over 90% of the total polyribosomes are present as aggregates, designated ‘membrane-cytomatrix’, which are eluted in the column void volume. Polyribosomes are not released from these aggregates on removal of microsomal phospholipids by treatment of postmitochondrial supernatant with 1% Triton X-100, a neutral detergent. The aggregates are disrupted by the usual ultracentrifugation techniques used in subcellular fractionation. After treatment of membrane-cytomatrix with Triton X-100 to remove phospholipids and membrane proteins, 58% of the polyribosomes still remain associated with protein-containing complexes in the form of a cytomatrix and are not ‘free’. Preparations of both membrane-cytomatrix and cytomatrix are capable of sustained protein synthesis. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the cytoskeletal proteins actin and myosin are present in the cytomatrix. Incubation of cytomatrix preparations with the actin-depolymerizing agent deoxyribonuclease I caused release of the polyribosomes. Polyribosome release by deoxyribonuclease I was prevented by prior incubation with phalloidin, which is known to stabilize F-actin. Thus polyribosomes are associated with cytoskeletal elements in rat liver, and this association is dependent on polymeric forms of actin.

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.

scholarly journals Partial Purification of Integral Membrane Antigenic Proteins fromTrypanosoma evansiThat Display Immunological Cross-Reactivity withTrypanosoma vivax

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Norma P. Velásquez ◽  
Rocío E. Camargo ◽  
Graciela L. Uzcanga ◽  
José Bubis
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Trypanosoma Evansi ◽  
Partial Purification ◽  
Limiting Factor ◽  
Dodecyl Sulfate ◽  
Causative Agents ◽  
Molecular Masses ◽  
Triton X

Trypanosoma evansiandTrypanosoma vivax, which are the major causative agents of animal trypanosomosis in Venezuela, have shown a very high immunological cross-reactivity. Since the production ofT. vivaxantigens is a limiting factor as this parasite is difficult to propagate in experimental animal models, our goal has been to identify and isolate antigens fromT. evansithat cross-react withT. vivax. Here, we used the VenezuelanT. evansiTEVA1 isolate to prepare the total parasite lysate and its corresponding cytosolic and membranous fractions. In order to extract theT. evansiintegral membrane proteins, the particulate portion was further extracted first with Triton X-100, and then with sodium dodecyl sulfate. After discarding the cytosolic and Triton X-100 solubilized proteins, we employed sedimentation by centrifugation on linear sucrose gradients to partially purify the sodium dodecyl sulfate-solubilized proteins from the Triton X-100 resistant particulate fraction ofT. evansi. We obtained enriched pools containing polypeptide bands with apparent molecular masses of 27 kDa, 31 kDa, and 53 kDa, which were recognized by anti-T. vivaxantibodies from experimentally and naturally infected bovines.

Labelling of Proteins by Isolated Rat Liver Nuclei

1972 ◽  
Vol 50 (2) ◽  
pp. 190-199 ◽  
Author(s):  
K. M. Anderson ◽  
M. Slavik ◽  
O. P. Elebute
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Nuclear Protein ◽  
Sucrose Solution ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rat Liver Nuclei ◽  
Teleological Argument ◽  
Liver Nuclei ◽  
Triton X

Rat liver nuclei, isolated in hypertonic sucrose solution and washed with Triton X-100, incorporate radioactive amino acids into hot trichloroacetic acid insoluble materials.Optical and biochemical evidence of nuclear purity is presented. The temperature-dependent incorporation continued for 20–30 min, and was proportional to the concentrations of both nuclear protein between 0.5–1.5 mg/ml, and radioactive amino acid. The radioactive product was degraded by pronase, and a number of inhibitors reduced incorporation, but only if present at [Formula: see text]. Proteins extracted from labelled nuclei and microsomes and examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis at pH 7.2 exhibited different patterns of radioactivity. This provides further support for the concept of protein synthesis intrinsic to rat liver nuclei.A teleological argument for the function of nuclear protein synthesis is discussed.

scholarly journals Studies on the structure-bound sedimentability of some rat liver lysosome hydrolases

1971 ◽  
Vol 122 (3) ◽  
pp. 363-371 ◽  
Author(s):  
F. M. Baccino ◽  
G. A. Rita ◽  
Maria Franca Zuretti
Keyword(s):  
Acid Phosphatase ◽  
Rat Liver ◽  
High Speed ◽  
Particulate Material ◽  
The Other ◽  
Acid Proteinase ◽  
Freezing And Thawing ◽  
Clear Cut ◽  
Almost All ◽  
Triton X

1. Lysosome-rich fractions from rat liver were subjected to several disruptive procedures: osmotic lysis or freezing and thawing in different media, shearing forces in a high-speed blender, treatment with Triton X-100. 2. The soluble and particulate phases were then separated by high-speed centrifugation and assayed for their content of acid phosphatase, β-galactosidase, β-N-acetylglucosaminidase, acid proteinase, acid ribonuclease, acid deoxyribonuclease and protein. 3. The degree of elution of these hydrolases appeared to depend on both the enzyme species and the treatment. The resulting patterns of solubilization were rather complex, so that a clear-cut discrimination between soluble and structure-bound enzymes could not always be traced. 4. Although only β-galactosidase was readily solubilizable after all treatments, acid proteinase could also be extensively eluted from the sedimentable material in the presence of EDTA and acid phosphatase was fully extracted by Triton X-100. On the other hand, considerable proportions of the other activities could not be solubilized by any of the procedures used. 5. In other experiments, the adsorbability of hydrolases on subcellular structures was investigated by measuring the partition between sedimentable particles and soluble fraction of solubilized enzymes added to ‘intact’ liver homogenates. 6. Large proportions of acid proteinase, ribonuclease and deoxyribonuclease, and almost all of β-N-acetylglucosaminidase, were found to be adsorbed on the particulate material.

Solubility and adsorption behaviors of chlorophenols in the presence of surfactant

1997 ◽  
Vol 35 (7) ◽  
pp. 123-130 ◽  
Author(s):  
J. C. Liu ◽  
P. S. Chang
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Critical Micelle Concentration ◽  
Sodium Dodecyl ◽  
Adsorption Behaviors ◽  
Brij 35 ◽  
Important Index ◽  
Dodecyl Sulfate ◽  
Triton X

The solubility of chlorophenols as affected by surfactant was investigated. Three kinds of surfactant, sodium dodecyl sulfate, Triton X-100, and Brij 35, were utilized. The solubilization of chlorophenols by surfactant follows the order of 2,4,6-trichlorophenol > 2,4-dichlorophenol > 2,6-dichlorophenol > 2-chlorophenol; and the critical micelle concentration is an important index. The adsorption reactions of 2,4-dichlorophenol and 2,4,6- trichlorophenol onto hydrous montmorillonite in the presence of surfactant were examined. The presence of surfactant decreased the adsorption of chlorophenols significantly. The roles of hydrophobicity of chlorophenols in solubilization and adsorption behaviors are discussed.

Different submicellar solubilization mechanisms revealed by 1H NMR and 2D diffusion ordered spectroscopy (DOSY)

2020 ◽  
Vol 22 (19) ◽  
pp. 11075-11085
Author(s):  
Mengjian Wu ◽  
Zhaoxia Wu ◽  
Shangwu Ding ◽  
Zhong Chen ◽  
Xiaohong Cui
Keyword(s):  
Nmr Spectroscopy ◽  
Sodium Dodecyl Sulfate ◽  
1H Nmr ◽  
Sodium Dodecyl ◽  
Butyl Methacrylate ◽  
H Nmr ◽  
Molecular Scale ◽  
Two Systems ◽  
Dodecyl Sulfate ◽  
Triton X

Different submicellar solubilization mechanisms of two systems, Triton X-100/tetradecane and sodium dodecyl sulfate (SDS)/butyl methacrylate, are revealed on the molecular scale by 1H NMR spectroscopy and 2D diffusion ordered spectroscopy (DOSY).

Fractionation of rat liver mitochondrial components after short treatments with triton x-100

1982 ◽  
Vol 14 (10) ◽  
pp. 933-940 ◽  
Author(s):  
M.C. Barbero ◽  
E. Rial ◽  
J.J. Otamendi ◽  
J.I.G. Gurtubay ◽  
F.M. Goni
Keyword(s):  
Rat Liver ◽  
Triton X
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