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.

Lipid requirements for the aggregation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol

1979 ◽  
Vol 57 (6) ◽  
pp. 605-612 ◽  
Author(s):  
Patrick C. Choy ◽  
S. Blake Farren ◽  
Dennis E. Vance
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Neutral Lipids ◽  
Gel Filtration ◽  
Fold Increase ◽  
High Cholesterol Diet ◽  
Liver Cytosol ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Molecular Weight Form ◽  
Rat Liver Cytosol

Two forms of CTP:phosphocholine cytidylyltransferase were identified in rat liver cytosol by gel filtration chromatography. The low molecular weight form (L form) is the major form in fresh cytosol. The enzyme associates into a high molecular weight form (H form) upon storage of the cytosol at 4 °C. Aggregation of the purified L form of cytidylyltransferase is caused by total rat liver lipids, neutral lipids, diacylglycerol, or phosphatidylglycerol. Diacylglycerol was the only lipid isolated from the rat liver that caused aggregation of the purified enzyme. Although the addition of diacylglycerol to the cytosol did not change the amount of aggregation of the enzyme, a 2.5-fold increase in H form was observed in cytosol pretreated with phospholipase C, or in cytosol from rats fed a high cholesterol diet. In both of these cytosolic preparations, the concentration of diacylglycerol was elevated twofold. Phosphatidylglycerol did not seem to affect the association of the enzyme in cytosol since it is present in very low concentrations in the rat liver cytosol, and its degradation in cytosol by a specific phospholipase did not affect the rate of aggregation. The results suggest that diacylglycerol in an appropriate form is required for association of cytidylyltransferase in rat liver cytosol.

scholarly journals Contribution of different protein phosphatases to the dephosphorylation of 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat liver

1985 ◽  
Vol 225 (3) ◽  
pp. 665-670 ◽  
Author(s):  
G Mieskes ◽  
H D Söling
Keyword(s):  
Phosphatase Activity ◽  
Rat Liver ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Liver Protein ◽  
Key Enzyme ◽  
Phosphatase 2A

The nature of rat liver protein phosphatases involved in the dephosphorylation of the glycolytic key enzyme 6-phosphofructo-1-kinase and the regulatory enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was investigated. In terms of the classification system proposed by Ingebritsen & Cohen [(1983) Eur. J. Biochem. 132, 255-261], only the type-2 protein phosphatases 2A (which can be separated into 2A1 and 2A2) and 2C act on these substrates. Fractionation of rat liver extracts by anion-exchange chromatography and gel filtration revealed that protein phosphatase 2A is responsible for most of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase phosphatase activity (activity ratio 2A/2C = 4:1). On the other hand, 6-phosphofructo-1-kinase phosphatase activity is equally distributed between protein phosphatases 2A (2A1 plus 2A2) and 2C. In addition, the possible role of low-Mr compounds for the control of purified protein phosphatase 2C was examined. At near-physiological concentrations, none of the metabolites studied significantly affected the rate of dephosphorylation of 6-phosphofructo-1-kinase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, pyruvate kinase or fructose-1,6-bisphosphatase.

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.

scholarly journals Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

2002 ◽  
Vol 68 (4) ◽  
pp. 1980-1987 ◽  
Author(s):  
Yolanda Sanz ◽  
Fidel Toldrá
Keyword(s):  
Specific Activity ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Sulfhydryl Group ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Lactobacillus Sakei ◽  
C Terminus ◽  
Arginine Aminopeptidase

ABSTRACT An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu2+, Hg2+, and Zn2+) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The Km values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.

scholarly journals Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2

1993 ◽  
Vol 295 (2) ◽  
pp. 463-469 ◽  
Author(s):  
S A Freeman ◽  
K Peek ◽  
M Prescott ◽  
R Daniel
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
High Sequence Similarity

The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.

scholarly journals Dictyostelium discoideum contains three inositol monophosphatase activities with different substrate specificities and sensitivities to lithium

1996 ◽  
Vol 314 (2) ◽  
pp. 491-495 ◽  
Author(s):  
Peter VAN DIJKEN ◽  
Jan C. T. BERGSMA ◽  
Hoebert S. HIEMSTRA ◽  
Berber DE VRIES ◽  
Jeroen VAN DER KAAY ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Dictyostelium Discoideum ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Inositol Monophosphatase

The small ion lithium, a very effective agent in the treatment of manic depressive patients, inhibits the mammalian enzyme inositol monophosphatase, which is proposed as the biological target for the effects of lithium. In this study we investigated Dictyostelium discoideum inositol monophosphatase activity. Partial purification of the proteins in the soluble cell fraction using anion-exchange chromatography revealed the presence of at least three enzyme activities capable of degrading inositol monophosphate isomers. The first activity was similar to the monophosphatase found in mammalian cells, as it degraded Ins(4)P, Ins(1)P and to a lesser extent Ins(3)P, was dependent on MgCl2 and inhibited by LiCl in a non-competitive manner. The second enzyme activity was specific for Ins(4)P; the enzyme activity was not dependent on MgCl2 and not inhibited by LiCl. The third monophosphatase activity degraded especially Ins(3)P, but also Ins(4)P and Ins(1)P; increasing concentrations of MgCl2 inhibited this enzyme activity, whereas LiCl had no effect. In vivo, LiCl induces a reduction of inositol levels by about 20%. In [3H]inositol-labelled cells LiCl causes a 6-fold increase in the radioactivity of [3H]Ins(1)P, a doubling of [3H]Ins(4)P and a slight decrease in the radioactivity in [3H]Ins(3)P. These data indicate that the biological effects of lithium in Dictyostelium are not due to depletion of the inositol pool by inhibition of inositol monophosphatase activity.

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.

Synthetic 1-thio-β-D-glucosiduronic acids as substrates for rat-liver β-glucuronidase

1970 ◽  
Vol 48 (7) ◽  
pp. 799-804 ◽  
Author(s):  
C. Hétu ◽  
R. Gianetto
Keyword(s):  
Rat Liver ◽  
Molecular Sieve ◽  
Enzyme Preparation ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Filtration Step ◽  
Hydrolysis Of ◽  
Menten Constant

The hydrolysis of 1-thio-β-D-glucosiduronic acids by rat liver was studied using synthetic phenyl 1-thio-β-D-glucosiduronic acid, sodium (2-benzothiazolyl 1-thio-β- D-glucosid)uronate, and sodium (p-nitrophenyl 1-thio-β-D-glucosid)uronate. It was found that rat liver preparations can hydrolyze the β-D-glucuronides of 2-benzothiazolethiol and p-nitrothiophenol but not the β-D-glucuronide of thiophenol.Partial purification of the enzyme from a lysosomal preparation using ammonium sulfate fractionation, gel filtration on a molecular sieve, and anion-exchange chromatography showed that β-glucuronidase (EC 3.2.1.31) is the enzyme responsible for the hydrolysis of these thioglucuronides. The pH optimum and Michaelis–Menten constant (Km) were determined for both substrates using an enzyme preparation obtained after the gel filtration step. The glucuronide of 2-benzothiazolethiol was found to be almost as good a substrate as that of phenolphthalein for rat-liver β-glucuronidase, while the glucuronide of p-nitrothiophenol is hydrolyzed at a much slower rate. Possible explanations of the fact that β-glucuronidase hydrolyzes only certain thioglucuronides are suggested.

scholarly journals Mechanism of action of β-oxoacyl-CoA thiolase from rat liver cytosol. Direct evidence for the order of addition of the two acetyl-CoA molecules during the formation of acetoacetyl-CoA

1983 ◽  
Vol 213 (1) ◽  
pp. 153-158 ◽  
Author(s):  
P M Jordan ◽  
P N B Gibbs
Keyword(s):  
Rat Liver ◽  
Direct Evidence ◽  
Gel Filtration ◽  
Acetyl Coa ◽  
Liver Cytosol ◽  
Enzyme Reactions ◽  
Single Turnover ◽  
The One ◽  
Enzyme Intermediate ◽  
Rat Liver Cytosol

Single-turnover enzyme reactions were employed with beta-oxoacyl-CoA thiolase purified from rat liver cytosol to determine the order of binding of the two acetyl-CoA molecules to the enzyme during the formation of acetoacetyl-CoA. Equimolar quantities of [1-14C]acetyl-CoA and enzyme were mixed initially in a rapid mixing device and the reaction was quenched by addition of an excess of unlabelled acetyl-CoA. Degradation of the resulting acetoacetyl-CoA revealed that the larger proportion of the radioactivity was in C-3. In the converse experiment, in which unlabelled acetyl-CoA was mixed with enzyme and the reaction was quenched with [1-14C]acetyl-CoA, radioactivity was incorporated preferentially into C-1. Similar results were obtained when [14C]acetyl-enzyme complex isolated by gel filtration was reacted with unlabelled acetyl-CoA, the radioactivity appearing largely in C-3. These findings lead to the conclusion that of the two molecules of acetyl-CoA that are bound by the enzyme and converted into acetoacetyl-CoA, it is the one giving rise to C-3 and −4 that is bound initially to the enzyme in the form of the acetyl-enzyme intermediate complex.

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