scholarly journals Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions

1977 ◽  
Vol 164 (3) ◽  
pp. 541-547 ◽  
Author(s):  
Ian H. Fraser ◽  
Sailen Mookerjea
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Column Chromatography ◽  
High Speed ◽  
Affinity Column ◽  
High Molecular Weight Fraction ◽  
Serum Enzyme ◽  
Membrane Enzyme ◽  
Membrane Bound ◽  
High Speed Supernatant

1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn2+ that could not be replaced by Ca2+, Mg2+, Zn2+ or Co2+, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent Km for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2–5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-ε-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the ε-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.

scholarly journals Studies on the purification and properties of UDP-galactose glycoprotein galactosyltransferase from rat liver and serum

1976 ◽  
Vol 156 (2) ◽  
pp. 347-355 ◽  
Author(s):  
I H Fraser ◽  
S Mookerjea
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
High Speed ◽  
Broad Band ◽  
Chromatographic Technique ◽  
Appreciable Effect ◽  
Serum Enzyme ◽  
Membrane Bound ◽  
Triton X ◽  
High Speed Supernatant

1. Rat liver microsomal preparations incubated with 200mM-NaCl at either 0 or 30 degrees C released about 20-30% of the membrane-bound UDP-galactose-glycoprotein galactosyl-transferase (EC 2.4.1.22) into a ‘high-speed’ supernatant. The ‘high-speed’ supernatant was designated the ‘saline wash’ and the galactosyltransferase released into this fraction required Triton X-100 for activation. It was purified sixfold by chromatography on Sephadex G-200, and appeared to have a higher molecular weight than the soluble serum enzyme. 2. Rat serum galactosyltransferase was purified 6000-7000-fold by an affinity-chromatographic technique using a column of activated Sepharose 4B coupled with α-lactalbumin. The purified enzyme ran as a single broad band on polacrylamide gels and contained no sialytransferase, N-acetylglucosaminyltransferase and UDP-galactose pyrophosphatase activities. 3. The highly purified enzyme had properties similar to those of both soluble and membrane-bound galactosyltransferase. It required 0.1% Triton X-100 for stabilization, but lost activity on freezing. The enzyme had an absolute requirement for Mn2+, not replaceable by Ca2+, Mg2+, Zn2+ or Co2+. It was active over a wide pH range (6-8) and had a pH optimum of 6.8. The apparent Km for UDP-galactose was 12.5 × 10(-6) M. α-Lactalbumin had no appreciable effect on UDP-galactose-glycoprotein galactosyltransferase, but it increased the specificity for glucose rather than for N-acetylglucosamine, thus modifying the enzyme to a lactose synthetase. 4. The possibility of a conversion of higher-molecular-weight liver enzyme into soluble serum enzyme is discussed, especially in relation to the elevated activities of this and other glycosyltransferases in patients with liver diseases.

Anreicherung und Charakterisierung einer „Transhydrogenase-Aktivität“ und der 17 β-Hydroxysteroid-Oxidoreduktase aus der Cytoplasma-Fraktion der Leber von weiblichen Ratten / The Enrichment and Characterisation of Cytoplasmatic “Transhydrogenase-activity” and 17 β-hydroxysteroid-oxidoreductase from Rat Liver

1972 ◽  
Vol 27 (8) ◽  
pp. 981-988
Author(s):  
Kunhard Pollow ◽  
Barbara Pollow
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Isoelectric Focusing ◽  
Isoelectric Point ◽  
Column Chromatography ◽  
Enzymatic Activities ◽  
Kinetic Constants ◽  
Gel Chromatography ◽  
Ammonium Sulphate Precipitation

The cytoplasmatic fraction of rat liver contains both 17 β-hydroxysteroid-oxidoreductase and a “transhydrogenase-activity”, which catalyses the transfer of hydrogen from the 17 β-position of estradiol-17 β to the 17-position of 4-androstene-3,17-dione. The 17 β-hydroxysteroid-oxidoreductase was purified 718-fold and the “transhydrogenase-activity” 264-fold by ammonium sulphate precipitation, gel chromatography with Sephadex G-200, column chromatography on DEAE-Sephadex and isoelectric focusing. The two enzymic activities could not be separated. The characteristics of the two enzymatic activities give some evidence that the “transhydrogenase-activity” is identical with the already known 17 β-hydroxysteroid-oxidoreductase.Isoelectric focusing of the chromatographycally enriched 17 β-enzyme gave an isoelectric point at 5,2. The 17 β-enzyme has a molecular weight of 62 — 65 000 as determined by mobility on Sephadex G-200 superfine.The kinetic constants for both the 17 β-enzyme and the “transhydrogenase-activity” were determined.

Characterization of cytosolic female rat liver receptors of the lactogenic type

1990 ◽  
Vol 123 (2) ◽  
pp. 231-237
Author(s):  
M. Emtner ◽  
P. Roos
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Sodium Dodecyl ◽  
Human Growth ◽  
Structural Features ◽  
Female Rat ◽  
Membrane Bound

Abstract. Some properties of cytosolic receptors of the lactogenic type from female rat liver were studied and compared with those of membrane-bound (microsomal) receptors. The association constant between the cytosolic receptors and human growth hormone was 2.2 l/nmol, which was not significantly different from the value obtained for the microsomal receptors (3.6 l/nmol). Since unlabelled hGH and human prolactin, but not bovine growth hormone, displaced [125I]hGH bound to receptors from both sources, the cytosolic receptors, like the microsomal receptors, must be lactogenic. Furthermore, the cytosolic receptors were recognized by a monoclonal antibody raised against microsomal receptors from female rat liver. However, covalent cross-linking of cytosolic receptors to [125I]hGH and subsequent sodium dodecyl sulphate electrophoresis gave a single band corresponding to a molecular weight of 42 200 (after subtraction of the molecular weight of hGH), which differs significantly (p<0.01) from the values determined for the two distinct bands given by the microsomal fraction. Moreover, upon molecular sieve chromatography the receptor activity in the two fractions appeared at significantly (p<0.05) different elution volumes. These results show that the cytosolic and microsomal receptors have some structural features in common but are definitely not identical.

‘Binding’ of [14C]phenol to rat liver high-speed supernatant

1980 ◽  
Vol 8 (1) ◽  
pp. 117-118
Author(s):  
H. PAUL A. ILLING ◽  
ESTHER S. A. HOUSE
Keyword(s):  
Rat Liver ◽  
High Speed ◽  
High Speed Supernatant

scholarly journals Studies on the meta and para O-sulphation of the catechol compound 3,4-dihydroxybenzoic acid by rat liver sulphotransferase in vitro

1980 ◽  
Vol 191 (1) ◽  
pp. 133-138 ◽  
Author(s):  
E J M Pennings ◽  
G M J Van Kempen
Keyword(s):  
Chemical Synthesis ◽  
Rat Liver ◽  
High Speed ◽  
Helix Pomatia ◽  
Dihydroxybenzoic Acid ◽  
High Speed Supernatant ◽  
Optimal Ph

The enzymic meta and para O-sulphation of 3,4-dihydroxybenzoic acid was investigated in vitro with a dialysed high-speed supernatant from rat liver. The O-sulphated products were identified by comparison with the reference compounds. The chemical synthesis and identification of the reference O-sulphate esters is described in detail. The sulphotransferase activity of the dialysed supernatant from rat liver towards 3,4-dihydroxybenzoic acid was 580 pmol of 3-O-sulphate and 120 pmol of 4-O-sulphate formed/min per mg of protein at the optimal pH of 7.4. The meta/para ratio of O-sulphation was independent of pH, time of incubation, concentration of enzyme and presence of dithiothreitol. The O-sulphate esters of 3,4-dihydroxybenzoic acid were found to be good substrates for the arylsulphatase reaction at pH 5.6. The arylsulphatase activity of a dialysed preparation from rat liver was 4.0 nmol of 3-O- and 5.7 nmol of 4-O-sulphate ester hydrolysed/min per mg of protein, respectively. Arylsulphatase from Helix pomatia had an activity of 620 pmol of 3-O-sulphate and of 16.6 nmol of 4-O-sulphate ester hydrolysed/min per unit (mumol/h) of sulphatase.

Purification and stabilization of galactosyl transferase from serum and lysolecithin extracted microsomes

1980 ◽  
Vol 58 (10) ◽  
pp. 878-884 ◽  
Author(s):  
Ian H. Fraser ◽  
Patricia Wadden ◽  
Sailen Mookerjea
Keyword(s):  
Enzyme Activity ◽  
High Speed ◽  
Specific Activity ◽  
Active Form ◽  
Liver Microsomes ◽  
Affinity Column ◽  
Chromatographic Technique ◽  
Rat Liver Microsomes ◽  
Galactosyl Transferase ◽  
Membrane Enzyme

Rat liver microsomes treated with increasing concentrations of lysolecithin released, after a brief lag, progressively increasing amounts of UDPgalactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. A second extraction of the microsomes with lysolecithin (8 mM) resulted in a total release of about 45% enzyme. The specific activity of the enzyme in the second extract was 12 times higher than that of the first extract. The galactosyl-transferase present in the extract was purified 417-fold by an affinity column chromatographic technique using a column of activated Sepharose 4B coupled with α-lactalbumin. During purification, the column and elution buffers required 0.1% lysolecithin to keep the enzyme in active form. For purposes of comparison the soluble serum galactosyltransferase was also purified by identical techniques, which also required 0.1% lysolecithin in column and elution buffers to prevent the loss of enzyme activity. The pure serum and membrane galactosyltransferase contained no sialyltransferase and ran as a double band on polyacrylamide gels (molecular weight 63 000–64 000). The pure enzyme had an absolute requirement for Mn2+, not replaceable by Cu2+, Mg2+, Zn2+, and Co2+. The enzymes were active over a wide pH range, with optimum pH of 6.5. The apparent Km's for UDPgalactose for the serum and membrane enzymes were 12.05 and 11.8 μM, respectively. The specific activities of these two purified enzymes were also remarkably similar, 3.99 × 106 for serum and 3.84 × 106 for membrane enzyme. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose (DSG-fetuin). The enzyme activity with DSG-fetuin acceptor was inhibited to a lesser extent by α-lactalbumin.Effect of various additives on the stability of purified membrane and serum galactosyltransferase was studied at 0–5 °C and at −20 °C up to 60 days. At both temperatures, albumin was found to be the best stabilizer. Ammonium sulfate was a good stabilizer for the serum but not for the membrane enzyme. Glycerol showed some stabilizing effect for both enzymes. EDTA, p-methylbenzenesulfonyl fluoride, N-acetylglucosamine-Mn2+, and water did not offer any stabilization of the pure enzyme.

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