scholarly journals Properties of Lubrol-extracted uridine diphosphate glucuronyltransferase

1971 ◽  
Vol 125 (4) ◽  
pp. 991-997 ◽  
Author(s):  
R. D. Howland ◽  
A. Burkhalter ◽  
A. J. Trevor ◽  
S. Hegeman ◽  
D. Y. Shirachi
Keyword(s):  
Rat Liver ◽  
Uridine Diphosphate ◽  
Soluble Enzyme ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Ionic Detergent ◽  
Michaelis Constants

1. A partially purified UDP-glucuronyltransferase was obtained by extracting rat liver microsomal preparations with Lubrol, a non-ionic detergent. 2. The soluble enzyme catalysed conjugation of both o-aminophenol and p-nitrophenol and was extremely stable when compared with untreated microsomal preparations. 3. The characteristics of the conjugation of the two phenols were found to differ with respect to pH optimum, bivalent cation requirement and Michaelis constants, suggesting that more than one enzyme is involved in the conjugation reaction.

scholarly journals The kinetic properties of citrate synthase from rat liver mitochondria

1969 ◽  
Vol 114 (3) ◽  
pp. 597-610 ◽  
Author(s):  
D. Shepherd ◽  
P. B. Garland
Keyword(s):  
Rat Liver ◽  
Citrate Synthase ◽  
Adenine Nucleotide ◽  
Sedimentation Coefficient ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Michaelis Constants

1. Citrate synthase (EC 4.1.3.7) was purified 750-fold from rat liver. 2. Measurements of the Michaelis constants for the substrates of citrate synthase gave values of 16μm for acetyl-CoA and 2μm for oxaloacetate. Each value is independent of the concentration of the other substrate. 3. The inhibition of citrate synthase by ATP, ADP and AMP is competitive with respect to acetyl-CoA. With respect to oxaloacetate the inhibition by AMP is competitive, but the inhibition by ADP and ATP is mixed, being partially competitive. 4. At low concentrations of both substrates the inhibition by ATP is sigmoidal and a Hill plot exhibits a slope of 2·5. 5. The pH optimum of the enzyme is 8·7, and is not significantly affected by ATP. 6. Mg2+ inhibits citrate synthase slightly, but relieves the inhibition caused by ATP in a complex manner. 7. At constant total adenine nucleotide concentration made up of various proportions of ATP, ADP and AMP, the activity of citrate synthase is governed by the concentration of the sum of the energy-rich phosphate bonds of ADP and ATP. 8. The sedimentation coefficient of the enzyme, as measured by activity sedimentation, is 6·3s, equivalent to molecular weight 95000.

scholarly journals Phosphatidylinositol phosphodiesterase in higher plants

1980 ◽  
Vol 192 (1) ◽  
pp. 279-283 ◽  
Author(s):  
R F Irvine ◽  
A J Letcher ◽  
R M C Dawson
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Higher Plants ◽  
Water Soluble ◽  
Apium Graveolens ◽  
Soluble Enzyme ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Detectable Activity ◽  
Ph Range

1. The lower regions of the stem of celery (Apium graveolens L.) contain a soluble enzyme that hydrolyses phosphatidylinositol. 2. The lipoidal product of hydrolysis is diacylglycerol, and the water-soluble products are 1:2-cyclic phosphoinositol and phosphoinositol in the approximate proportions of 60% and 40% respectively: this indicates that a phosphodiesterase (phospholipase C-like) activity is cleaving the phosphatidylinositol. 3. The enzyme requires a bivalent cation, Ca2+ being the most effective activator. 4. The enzyme has a pH optimum, depending on conditions of assay, of pH 5.9-6.6 and in this pH range shows no detectable activity against phosphatidylcholine or phosphatidylethanolamine. 5. The activity is stimulated by phosphatidic acid and slightly inhibited (30% at concentrations equimolar with phosphatidylinositol) by phosphatidylcholine. 6. The phosphodiesterase was also detected (but not quantified) in the tips of the flowers in cauliflowers, in outer leaves of onion and in the elongating stem of daffodils. 7. The enzyme's properties are compared with equivalent mammalian enzymes, and its possible role in the catabolism of phosphatidylinositol in higher plants is discussed.

scholarly journals Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases

1970 ◽  
Vol 120 (4) ◽  
pp. 809-814 ◽  
Author(s):  
F. J. Ballard
Keyword(s):  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Mitochondrial Enzymes ◽  
High Concentration ◽  
Michaelis Constant ◽  
Bivalent Cation ◽  
Sheep Liver ◽  
Michaelis Constants

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg2+ as bivalent cation, the Michaelis constant was approx. 2.5×10−5m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn2+ replaced Mg2+ in the reaction a lower Michaelis constant of 9×10−6m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn2+ was the added cation. With Mg2+ the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.

scholarly journals SYNTHESIS OF ERYTHRULOSE PHOSPHATE BY A SOLUBLE ENZYME FROM RAT LIVER

1953 ◽  
Vol 201 (1) ◽  
pp. 161-173
Author(s):  
Frixos C. Charalampous ◽  
Gerald C. Mueller
Keyword(s):  
Rat Liver ◽  
Soluble Enzyme

scholarly journals Characterization of a spleen sulphotransferase responsible for the 6-O-sulphation of the galactose residue in sialyl-N-acetyl-lactosamine sequences

1998 ◽  
Vol 331 (1) ◽  
pp. 265-271 ◽  
Author(s):  
Robert G. SPIRO ◽  
Vishnu D. BHOYROO
Keyword(s):  
Periodate Oxidation ◽  
Maximal Activity ◽  
Sialic Acid Residue ◽  
Lymphoid Tissues ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Selectin Ligands ◽  
Strict Requirement ◽  
Lectin Chromatography ◽  
Triton X

An enzyme which catalyses the transfer of sulphate from 3´-phosphoadenosine 5´-phosphosulphate (PAPS) to C-6 of galactose in the NeuAcα2-3Galβ1-4GlcNAc (3´SLN) sequence has been found in rat spleen microsomes and its specificity indicates that it is well suited to participate in the assembly of 3´-sialyl-6´-sulpho-LacNAc [NeuAcα2-3Gal(6-SO4)β1-4GlcNAc] and 3´-sialyl-6´-sulpho-LewisX [NeuAcα2-3Gal(6-SO4)β1-4(Fucα1-3)GlcNAc] saccharide groups which have been implicated as selectin ligands. This sulphotransferase has a strict requirement for oligosaccharide acceptors which are capped by an α2-3-linked sialic acid residue, although GlcNAc in 3´SLN can be substituted by Glc, and Galβ1-4GlcNAc can be replaced by Galβ1-3GlcNAc without loss of activity. The finding that 3´-sialyl LewisX was inert as an acceptor suggested that fucosylation, in contrast with sialylation, follows the addition of the sulphate group. Since fetuin glycopeptides containing the NeuAcα2-3Galβ1-4GlcNAc sequence had a similar affinity for the enzyme as the unattached 3´SLN, it would appear that the acceptor determinants reside primarily in the peripheral trisaccharide constellation. The position of the sulphate on C-6 of galactose was elucidated by Smith periodate oxidation, hydrazine/nitrous acid/NaBH4 treatment and elder (Sambucus nigra)bark lectin chromatography of the desialylated [35S]sulphate-labelled products of the enzyme. Assays carried out with 3´SLN as acceptor indicated that the sulphotransferase had a pH optimum between 6.5 and 7.0 and a dependence on a bivalent cation best met by Mn2+ (12–25 mM); Triton X-100 (0.02 to 0.35%) brought about maximal stimulation. Tentative Km values determined for this enzyme were 4.7 µM for PAPS, and 0.72 mM and 1.16 mM for 3´SLN and fetuin glycopeptides respectively. A survey of several rat organs indicated that the PAPS:3´SLN-6-O-sulphotransferase is selectively distributed with maximal activity occurring in spleen which was substantially greater than thymus or lymph nodes. In contrast, other enzymes (i.e. PAPS:Gal-3-O-and GlcNAc-6-O-sulphotransferases) involved in the sulphation of sialyl-lactosamine and lactosamine sequences, which in the sulphated form are believed to also be selectin ligands, were more evenly distributed in lymphoid tissues. Relatively high activities for all three enzymes were found in brain.

STUDIES ON SOLUBILIZED ADENYLATE KINASE FROM MITOCHONDRIA

1966 ◽  
Vol 44 (11) ◽  
pp. 1469-1475 ◽  
Author(s):  
Marjorie A. Brewster ◽  
Ezzat S. Younathan
Keyword(s):  
Activation Energy ◽  
Rat Liver ◽  
Adenylate Kinase ◽  
Divalent Cations ◽  
Metabolic Function ◽  
Kcal Mole ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Sensitive Method

Adenylate kinase from mitochondria of rat liver was made soluble by sonication. The enzyme had a pH optimum of 8.0, temperature optimum of 30°, and activation energy of 12.2 kcal/mole. It was activated by several divalent cations in the following order of efficiency: Mg++ > Co++ > Mn++ > Ca++, with an optimal Mg++: ADP ratio of 1. The apparent Km value (ADP as substrate) was found to be 1.3 mM at pH 7.4 and 30°. The activity was sensitive to phloretin and mildly activated by aurovertin. Oligomycin, 2,4-dinitrophenol, p-chloromercuribenzoate, alloxan, and phlorizin had no effect on the activity. The metabolic function and a comparison of the properties of this solubilized mitochondrial adenylate kinase with those of similar preparations from other sources are discussed in the light of these findings. During this study, a sensitive method adaptable for a large number of assays of adenylate kinase was developed, and is described in detail.

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