scholarly journals Identification and partial purification of a unique phenolic steroid sulphotransferase in rat liver cytosol

1984 ◽  
Vol 224 (3) ◽  
pp. 947-953 ◽  
Author(s):  
Y Sugiyama ◽  
A Stolz ◽  
M Sugimoto ◽  
J Kuhlenkamp ◽  
T Yamada ◽  
...  
Keyword(s):  
Rat Liver ◽  
Organic Anion ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Male Rat ◽  
Partial Purification ◽  
Ph Optimum ◽  
Liver Cytosol ◽  
Bile Acid Binding ◽  
Rat Liver Cytosol

Phenolic steroid sulphotransferase activity for both oestradiol and oestrone was identified in male rat liver cytosol in the 30 000-40 000 Mr fractions on gel filtration when activity was assayed at pH 5.5 (pH optimum 5.5-6.0). Activity for oestradiol but not oestrone was found in the 60 000-70 000-Mr range when assayed at pH 8.0 (pH optimum biphasic, 5.5-6.0 and 7.0-8.0). Km for oestradiol (1.3 microM) was lower than published values for hydroxysteroid sulphotransferases (15-35 microM) and previously reported oestradiol sulphotransferases (71-85 microM). At above 2 microM-oestradiol phenolic sulphotransferase activity exhibited substrate inhibition. The phenolic steroid sulphotransferase activity was found to be distinct in chromatofocusing from organic-anion-binding and bile acid-binding proteins previously identified in this Mr range. Further purification on hydroxyapatite yielded a 44-fold enriched fraction that contained two monomeric bands, Mr 32 500 and 29 500.

scholarly journals Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents

1988 ◽  
Vol 250 (1) ◽  
pp. 53-58 ◽  
Author(s):  
F Flamigni ◽  
C Guarnieri ◽  
C M Caldarera
Keyword(s):  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Effective Concentration ◽  
The Other ◽  
Limited Effect ◽  
Liver Cytosol ◽  
Rat Liver Cytosol ◽  
Dependent Mechanism

Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated ‘A’, eluted near the void volume (Mr greater than or equal to 60,000), and the other designated ‘B’, eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.

Partial purification of a cortisol binding protein from rat liver cytosol

Steroids ◽  
1970 ◽  
Vol 16 ◽  
pp. 207-216 ◽  
Author(s):  
M. Beato ◽  
W. Schmid ◽  
W. Braendle ◽  
C.E. Sekeris
Keyword(s):  
Rat Liver ◽  
Binding Protein ◽  
Partial Purification ◽  
Liver Cytosol ◽  
Rat Liver Cytosol

scholarly journals Dephosphorylation of 1d-myo-inositol 1,4-bisphosphate in rat liver

1988 ◽  
Vol 254 (3) ◽  
pp. 655-660 ◽  
Author(s):  
A J Morris ◽  
D J Storey ◽  
C P Downes ◽  
R H Michell
Keyword(s):  
Phosphatase Activity ◽  
Rat Liver ◽  
Substrate Concentration ◽  
Gel Filtration ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Inositol Monophosphatase ◽  
Liver Cytosol ◽  
Rat Liver Cytosol

Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2] in rat liver is catalysed by a cytosolic phosphatase that removes the 1-phosphate group. The Km for Ins(1,4)P2 is approx. 17 microM. Li+ (100 mM) causes 50% inhibition of Ins(1,4)P2 phosphatase activity when activity is measured at the very low substrate concentration of 10 nM, but on raising the substrate concentration to 100 microM there is a greater than 10-fold increase in sensitivity to Li+, suggesting that Li+ acts mainly, but not entirely, as an uncompetitive inhibitor of Ins(1,4)P2 phosphatase. In addition, rat liver cytosol shows Li+-sensitive phosphatase activity against 1D-myo-inositol 1-,3- and 4-monophosphates. The Ins(1,4)P2 1-phosphatase and inositol monophosphatase activities all share an apparent Mr of 47 x 10(3), as determined by gel-filtration chromatography. However, the Ins(1,4)P2 1-phosphatase is more sensitive to inactivation by heat, and can be separated from inositol monophosphatase activity by anion-exchange chromatography. We conclude that rat liver cytosol contains an Ins(1,4)P2 1-phosphatase that is distinct from, but in many ways similar to, inositol monophosphatase.

scholarly journals The alkaline phospholipase A1 of rat liver cytosol

1983 ◽  
Vol 209 (3) ◽  
pp. 865-872 ◽  
Author(s):  
R M C Dawson ◽  
R F Irvine ◽  
N L Hemington ◽  
K Hirasawa
Keyword(s):  
Rat Liver ◽  
Water Soluble ◽  
Univalent Cations ◽  
Phospholipase A1 ◽  
Ph Optimum ◽  
Liver Cytosol ◽  
Tentative Conclusion ◽  
Bivalent Cations ◽  
Rat Liver Cytosol ◽  
Negative Zeta Potential

1. Rat liver cytosol contains a heat-sensitive phospholipase A1 active against phosphatidylethanolamine, 1-acylglycerophosphoethanolamine and, to a very much lesser extent, phosphatidylcholine and phosphatidylinositol. 2. Activity towards a pure phosphatidylethanolamine substrate is invoked by the presence of water-soluble cations that do not precipitate at the pH optimum of the enzyme (9.5). In this activation bivalent cations, e.g. Mg2+, Ca2+, Mn2+, Sr2+ and Ba2+, are effective at much lower concentrations (2.5-5 mM) than univalent cations K+, Na+ and NH4+ (100 mM). 3. In the absence of such cations the enzyme can be activated by cationic amphiphiles containing quaternary nitrogen or by basic proteins. 4. It is concluded that these agents activate the enzyme by reducing the negative zeta potential on the substrate at the high pH optimum (9.5) and allow interaction with the enzyme whose isoelectric point is at 7.15. 5. The activated enzyme is markedly inhibited by mixing the phosphatidylethanolamine substrate with many other phospholipids that exist in cell membranes, e.g. phosphatidylcholine, phosphatidylinositol. On the other hand, both phosphatidylcholine and phosphatidylinositol can be hydrolysed much more readily if they are mixed with an excess of phosphatidylethanolamine. 6. Such results on the inhibition and substrate specificity of the enzyme, coupled with birefringence measurements, allow the tentative conclusion that phospholipid substrates are only attacked when they exist in a hexagonal or non-bilayer structure and not in the bilayer (lamellar) form.

scholarly journals Effects of sex hormones on enzymic esterification of 2-(N-hydroxyacetamido)-fluorene by rat liver cytosol

1970 ◽  
Vol 120 (2) ◽  
pp. 409-416 ◽  
Author(s):  
Prabhakar D. Lotlikar
Keyword(s):  
Rat Liver ◽  
Adult Male ◽  
Male Rat ◽  
Female Rat ◽  
Liver Cytosol ◽  
Adult Male Rat ◽  
Derivatives Of ◽  
Sex And Age Differences ◽  
Rat Liver Cytosol

1. Enzymic esterifications of 2-(N-hydroxyacetamido)fluorene and several other hydroxamic acids by liver cytosol were studied. Determination of 2-acetamido-3-methylthiofluorene was used for the assay. 2. With rat liver cytosol, requirement for ATP, Mg2+ and SO42− suggested formation of phosphate and sulphate esters of 2-(N-hydroxyacetamido)fluorene. 3. Rats showed sex and age differences in their activity. Liver from adult male rat was at least twice as active as liver from adult female rat. No such sex differences were found in mice, hamsters and guinea pigs. 4. Administration of testosterone (300μg/day) subcutaneously for 8 days increased the activity in the female rat by 100%, whereas diethylstilboestrol (100μg/day) had no effect. In the male rat diethylstilboestrol treatment decreased the activity by 60%, whereas testosterone pretreatment was without any effect. 5. Among various endocrine ablations such as adrenalectomy, castration, adrenalectomy–castration and hypophysectomy in the adult male rat, hypophysectomy was found to be the most effective in decreasing the activity of the liver to about 50% of control values. 6. Like 2-(N-hydroxyacetamido)fluorene, various other N-hydroxy derivatives of 2-acetamido-7-fluorofluorene, 2-acetamidophenanthrene, 4-acetamidobiphenyl and 4-acetamidostilbene were also shown to be esterified to different extents by rat liver cytosol.

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