Purification and partial characterization of a rat kidney aldehyde dehydrogenase that oxidizes retinal to retinoic acid

1993 ◽  
Vol 71 (1-2) ◽  
pp. 85-89 ◽  
Author(s):  
Jean Labrecque ◽  
Pangala V. Bhat ◽  
André Lacroix
Keyword(s):  
Molecular Weight ◽  
Retinoic Acid ◽  
Vitamin A ◽  
Aldehyde Dehydrogenase ◽  
Rat Kidney ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Size Exclusion

A NAD-dependent aldehyde dehydrogenase (EC 1.2.1.3) which catalyzes the oxidation of retinal to retinoic acid was purified to homogeneity from rat kidney by using Affi-Gel blue affinity chromatography and chromatofocusing, followed by Mono-Q anion-exchange chromatography. The apparent molecular weight of the native enzyme determined by size-exclusion fast protein liquid chromatography was 140 000. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a subunit molecular weight of 53 000. The isoelectric point as measured by chromatofocusing was 8.5. The enzyme also catalyzed the oxidation of acetaldehyde, but showed much lower Km value for the retinal substrate. We suggest that aldehyde dehydrogenase found in the kidney may be a specific retinal dehydrogenase, involved in vitamin A metabolism.Key words: aldehyde dehydrogenase, vitamin A, retinal, retinoic acid, kidney.

Purification and some properties of β-glucosidase from the ectomycorrhizal fungus Pisolithus tinctorius strain SMF

1993 ◽  
Vol 39 (1) ◽  
pp. 125-129 ◽  
Author(s):  
Weiguo Cao ◽  
Don L. Crawford
Keyword(s):  
Molecular Weight ◽  
Ethylenediaminetetraacetic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Ectomycorrhizal Fungus ◽  
Pisolithus Tinctorius ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
A Cell

A cell-associated β-glucosidase was purified 152-fold to homogeneity from the ectomycorrhizal fungus Pisolithus tinctorius strain SMF. The apparent molecular weight of the native protein, as determined by size exclusion chromatography, was approximately 450 000. A single band with a molecular weight of 150 000 was obtained after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Thus, the native enzyme may consist of three monomers. The pI of the enzyme was determined to be 3.8 by isoelectric focusing. The enzyme had an optimal pH of 4.0 and an optimal temperature for activity of 65 °C. It showed a high substrate specificity toward aryl-β-glucosides, such as p-nitrophenyl β-D-glucopyranoside (PNPG), and β-1,6 glucosidic linkages. Cellobiose was hydrolyzed at about two-thirds the rate of PNPG. The Km for hydrolysis of PNPG was 0.87 mM. Strong inhibitors of the enzyme were aluminum, copper, ethylenediaminetetraacetic acid (EDTA), deoxynojirimycin, gluconic acid, and SDS. Calcium, manganese, and p-hydroxymercuribenzoic acid reduced the activity to a lesser extent. Potassium, mercury, cobalt, dithiothreitol, and glucosamine had no effect on activity. Enzyme activity was slightly increased to 112% in the presence of 1% glycerol. The enzyme was more stable under acidic conditions than under alkaline conditions.Key words: Pisolithus, ectomycorrhizal, β-glucosidase, purification.

Variation in goat fibre protein

1989 ◽  
Vol 40 (3) ◽  
pp. 675 ◽  
Author(s):  
DJ Tucker ◽  
AHF Hudson ◽  
A Laudani ◽  
RC Marshall ◽  
DE Rivett
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Wool Fibre ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Cashmere Goats

The proteins from a range of cashmere, mohair, angoratcashmere crossbred and wool fibre samples were extracted at pH 8 with 8 M urea containing dithiothreitol, and were then radiolabelled by S-carboxymethylation using iodo(2-14C) acetate. The proteins from each sample were examined by two dimensional polyacrylamide gel electrophoresis in which the separation in the first dimension was according to charge at pH 8.9 and in the second dimension according to apparent molecular weight in the presence of sodium dodecyl sulfate. After electrophoresis the proteins were detected by fluorography. Protein differences in keratin samples from some individual goats existed, although the overall protein patterns were similar. None of the differences were consistent with any one goat fibre type. The protein patterns obtained for fibre samples from individual cashmere goats showed some differences when compared to those found for commercial blends from the same country of origin, indicating that blending can mask any animal-to-animal variation. While the electrophoretic technique does not unequivocally distinguish between cashmere, mohair and angora/cashmere crossbred fibres it does differentiate between wool and goat fibres.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

scholarly journals Purification and subunit structure of legumin of Vicia faba L. (broad bean)

1974 ◽  
Vol 141 (2) ◽  
pp. 413-418 ◽  
Author(s):  
David J. Wright ◽  
Donald Boulter
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Broad Bean ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Isoelectric Precipitation ◽  
Molecular Weights

Zonal isoelectric precipitation was shown to be an effective method for the preparation of legumin which was homogeneous as judged by ultracentrifugation and polyacrylamide-gel electrophoresis. The subunit structure of legumin was investigated by preparative sodium dodecyl sulphate–polyacrylamide-gel electrophoresis and ion-exchange chromatography in urea. Five distinct subunits, of which two were acidic (α) and had a molecular weight of 37000, and three were basic (β) with molecular weights of 20100, 20900 and 23800, were identified. The α and β subunits were present in equimolar amounts in the legumin molecule and, in view of this and molecular-weight considerations, an α6β6 subunit model was proposed for legumin.

scholarly journals HEMICELLULOSE APPARENT MOLECULAR SIZE CHANGES DURING SOFTENING OF PEACH FRUIT

HortScience ◽  
1994 ◽  
Vol 29 (7) ◽  
pp. 737d-737
Author(s):  
Supreetha Hegde ◽  
Niels Maness
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Apparent Molecular Weight ◽  
Weight Fraction ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Neutral Sugar ◽  
Fruit Softening ◽  
Peach Fruit ◽  
Cell Wall Polymers

Peach fruit softening appears to be associated with changes in cell wall polymers, particularly pectins and hemicelluloses. To determine changes of cell wall polymers associated with peach fruit softening, we conducted sequential extractions of pectin and hemicellulose from softening fruit. A more tightly bound hemicellulose fraction contained considerable amounts of pectin associated sugars. This fraction was separated into charged and neutral fractions, using anion exchange chromatography, and then fractionated into two apparent molecular weight classes by size exclusion chromatography. Virtually all of the charged fraction eluted in the higher apparent molecular weight fraction. The neutral sugar fraction segregated into both apparent molecular weight size classes, with a redistribution from the large to the small size class during softening. This redistribution was accompanied by changes in neutral sugar composition. A possible relationship between changes in this fraction and fruit softening will be discussed. Supported by USDA grant 92-34150-7190 and the Oklahoma Agricultural Experiment Station.

scholarly journals Characterization of a Novel β-Galactosidase fromBifidobacterium adolescentis DSM 20083 Active towards Transgalactooligosaccharides

2000 ◽  
Vol 66 (4) ◽  
pp. 1379-1384 ◽  
Author(s):  
Katrien M. J. Van Laere ◽  
Tjakko Abee ◽  
Henk A. Schols ◽  
Gerrit Beldman ◽  
Alphons G. J. Voragen
Keyword(s):  
Size Exclusion Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Extract ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Bifidobacterium Adolescentis ◽  
Exclusion Chromatography

ABSTRACT This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highestV max values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.

scholarly journals Purification and characterization of subcomponent C1q of the first component of mouse complement

1981 ◽  
Vol 193 (2) ◽  
pp. 621-629 ◽  
Author(s):  
K Yonemasu ◽  
T Sasaki
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weight Range ◽  
Covalently Linked

1. Mouse C1q, a subcomponent of the first component of complement, has been purified in a highly haemolytically active form by a combination of precipitation with EGTA, ion-exchange chromatography and gel filtration. Yields ranged from 3 to 5 mg/200 ml of serum, and the activity of final preparations was in the range of 2 × 10(13)-4 × 10(13) C1q effective molecules/mg. 2. The molecular weight of mouse C1q was 439 500 +/- 1586, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. Mouse C1q was shown to be composed of non-covalently linked subunits, all being in the molecular-weight range 45 000-46 000, and three covalently linked chains each having a molecular weight of approx. 23 000 as determined on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by using non-covalently and covalently linked subunits of human C1q as markers with known molecular weights calculated theoretically previously [Porter & Reid (1978) Nature (London) 275, 699-704]. 4. Mouse C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approx. 9% carbohydrate. The absorption coefficient and nitrogen content of C1q were also determined.

Determination of the Molecular Weight and the Hydrodynamic Properties of a Polypeptide from the Thylakoid Membrane by Sedimentation, Diffusion and Binding Measurements in Dodecyl Sulphate Solutions

1975 ◽  
Vol 30 (9-10) ◽  
pp. 615-621 ◽  
Author(s):  
Hans Craubner ◽  
Friederike Koenig ◽  
Georg H. Schmid
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Equilibrium Dialysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Partial Specific Volume ◽  
Hydrodynamic Properties ◽  
Lamellar System ◽  
Self Diffusion

The molecular weight and hydrodynamic properties of a polypeptide isolated from the lamellar system of Antirrhinum chloroplasts were determined in sodium dodecyl sulphate solution by measurement of sedimentation velocity, diffusion and effective partial specific volume. The polypeptide fraction exhibits a molecular weight of 25 000 which agrees with the apparent molecular weight found by polyacrylamide gel electrophoresis. The molecular weight of the polypeptidesodium dodecyl sulphate micelle was 54 000, with a friction ratio of 1.6 which indicates an effective asymmetric hydrodynamic shape. For binding measurements self-diffusion equilibrium dialysis with dodecyl [35S] sulphate was used. In this case, dialysis equilibrium was reached within about 10 hours, in contrast to the dialysis with initial concentration differences which requires much longer times. A binding value of δD = 1.15g sodium dodecyl sulphate per g polypeptide was obtained which corresponds to a molar binding ratio of 100 mol dodecyl sulphate bound per mol of polypeptide. After the removal of dodecyl sulphate the polypeptide is present in an aggregated state. In phosphate buffers of pH 6.8 and 7.5 the aggregates preponderantly have sedimentation coefficients of 11.7 and 6.8 Svedberg units respectively. Assuming equivalent spheres the molecular weights were calculated to be 340 000 and 150 000.

scholarly journals Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes

1975 ◽  
Vol 151 (3) ◽  
pp. 685-697 ◽  
Author(s):  
M Letarte-Muirhead ◽  
A N Barclay ◽  
A F Williams
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Lentil Lectin ◽  
Polypeptide Chains

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.

scholarly journals Platelets contain proteins immunologically related to red cell spectrin and protein 4.1

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 52-59
Author(s):  
GE Davies ◽  
CM Cohen
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Cell Protein ◽  
Immunoperoxidase Staining ◽  
Red Cell ◽  
Protein 4.1 ◽  
Platelet Protein

Human platelets were tested for the presence of proteins immunologically cross-reactive with red cell spectrin and protein 4.1. As assessed by indirect immunofluorescence microscopy, platelets were specifically reactive with affinity-purified rabbit antisera against red cell spectrin and protein 4.1. The immunoreactive platelet constituents were further analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, followed by electrophoretic transfer to nitrocellulose paper and immunoperoxidase staining. We found that whole platelets, membranes, and cytoskeletal preparations isolated by Triton X-100 extraction contain small amounts of proteins reacting with anti-spectrin or anti-protein 4.1 antiserum. The immunoreactive spectrin-like platelet protein has an apparent molecular weight of 240,000 and comigrates with the alpha-subunit of red cell spectrin. The major immunoreactive protein 4.1-like constituent has an apparent molecular weight of 78,000, which is slightly less than that of red cell protein 4.1. We conclude that platelets contain a spectrin- like protein which, by analogy with red cell spectrin, may have a role in membrane-cytoskeletal attachment. The properties and function of the platelet protein 4.1-like constituent are not yet known.

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