scholarly journals Partial purification of a diacylglycerol lipase from bovine aorta

1994 ◽  
Vol 298 (1) ◽  
pp. 213-219 ◽  
Author(s):  
M W Lee ◽  
D L Severson
Keyword(s):  
Lipase Activity ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Short Chain ◽  
Partial Purification ◽  
Diacylglycerol Lipase ◽  
Bivalent Cations ◽  
Triton X ◽  
Hydrolysis Of ◽  
Long Chain

A diacylglycerol (DG) lipase has been purified from a soluble subcellular fraction of bovine aorta by (NH4)2SO4 precipitation in the presence of 5.0% (w/v) Triton X-100, followed by chromatography on DEAE-Sephacel, heparin-Sepharose and octyl-Sepharose in the presence of either CHAPS or Triton X-100 detergents. Under basal conditions, the hydrolysis of a short-chain [3H]dioctanoylglycerol ([3H]diC8) substrate was much greater than that of a long-chain 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) substrate. Lipase activity measured with 1-[14C]POG was markedly enhanced by Triton X-100. In the presence of 0.1% Triton X-100, specific enzyme activities in the octyl-Sepharose fraction determined with 1-[14C]POG or 1-stearoyl-2-[1-14C]-arachidonoyl-sn-glycerol as substrates were the same as that measured with [3H]diC8. MgCl2 (5mM) or CaCl2 (2 mM) also selectively stimulated lipase activity (up to 10-13-fold) measured with the long-chain (1-[14C]POG) substrate only. The increase in relative specific activity in the octyl-Sepharose fraction was 60-fold and 155-fold, based on hydrolysis of [3H]diC8 and 1-[14C]POG (+ Triton X-100), respectively. Unlabelled diC8 was a competitive inhibitor of 1-[14C]POG hydrolysis, suggesting that a single lipase hydrolyses both the short-chain and long-chain DG substrates; selective stimulatory effects of non-ionic detergents and bivalent cations on the hydrolysis of 1-[14C]POG may be due to effects on the physical properties of the substrate preparation. Monoacylglycerol lipase, DG kinase and cholesterol esterase activities could not be detected in the partially purified lipase preparation.

Monoacylglycerol lipase activity in cardiac myocytes

1988 ◽  
Vol 66 (9) ◽  
pp. 1013-1018 ◽  
Author(s):  
David L. Severson ◽  
Mariette Hee-Cheong
Keyword(s):  
Lipase Activity ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Fatty Acyl ◽  
Supernatant Fraction ◽  
Monoacylglycerol Lipase ◽  
Pellet Fraction ◽  
Rat Hearts ◽  
Triton X ◽  
Hydrolysis Of

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100 000 × g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100 000 × g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 μM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 μM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA > free fatty acids > fatty acyl carnitine.

The lipase activity of a partially purified lipolytic enzyme of Escherichia coli

1978 ◽  
Vol 56 (5) ◽  
pp. 324-328 ◽  
Author(s):  
G. Nantel ◽  
Georgia Baraff ◽  
P. Proulx
Keyword(s):  
Escherichia Coli ◽  
Lipase Activity ◽  
Slow Rate ◽  
Water Soluble ◽  
Lipolytic Enzyme ◽  
Ph Optimum ◽  
Chain Acyl ◽  
Marked Preference ◽  
Hydrolysis Of ◽  
Long Chain

Escherichia coli lipase was found to have a broad pH optimum between pH 8 and 10. Long-chain acyl triacylglycerols such as trioleoylglycerol were hydrolysed at a relatively slow rate, whereas, the shorter-chain acyl derivative tricapryloylglycerol was not. Triacylglycerols and diacylglycerols were broken down at a rate 10- to 15-fold greater than that for monoacylglycerol. Simple esters such as methyloieate and cetylpalmitate were hydrolysed at rates greater than that of triacylglycerol. Water-soluble esters such as p-nitrophenylacetate were not attacked. Hydrolysis of lipase substrates occurred more readily in the presence of an anionic detergent such as taurocholate. The enzyme had no marked preference for the 1- or 3-position of triacylglycerols but attacked these positions much more readily than position 2. The enzyme also catalyzed transacylation reactions with simple alcohols such as methanol or ethanol.

scholarly journals Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

1980 ◽  
Vol 191 (1) ◽  
pp. 117-124 ◽  
Author(s):  
R Zecher ◽  
H U Wolf
Keyword(s):  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Half Maximum ◽  
Purification And Characterization ◽  
Dimeric Molecule ◽  
Maximum Inhibition ◽  
Optimum Ph ◽  
Triton X

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

scholarly journals Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase

1979 ◽  
Vol 177 (3) ◽  
pp. 833-846 ◽  
Author(s):  
M C Scrutton ◽  
I Beis
Keyword(s):  
Rat Liver ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Detectable Effect ◽  
Formyltetrahydrofolate Dehydrogenase ◽  
Hydrolysis Of

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7–0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.

Diagnostic Enzymology of a-L-Iduronidase with Special Reference to a Sulphated Disaccharide Derived from Heparin

1982 ◽  
Vol 62 (2) ◽  
pp. 193-201 ◽  
Author(s):  
J. J. Hopwood ◽  
Vivienne Muller
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Ph Profile ◽  
Ph Values ◽  
Substrate Structure ◽  
Cultured Skin ◽  
Bivalent Cations ◽  
Competitive Inhibitors ◽  
Specific Ion Effect ◽  
Hydrolysis Of

1. Iduronosyl anhydro[1-3H]mannitol 6-sulphate (IMs), iduronosyl anhydro[1-3H]mannitol, phenyl iduronide (PhI) and 4-methylumbelliferyl iduronide have been compared as substrates for the diagnostic estimation of α-l-iduronidase activity present in human leucocyte and cultured skin fibroblast homogenates. The pH profile of leucocyte and fibroblast iduronidase activity was dependent on substrate structure and concentration, the ionic strength and the nature of the buffer ion used in the assay mixture. 2. NaCl, KBr and Na2SO4 were shown to be parabolic competitive inhibitors of IMs activity, the K1 with fibroblast homogenates being 34, 13.4 and 0.22 mmol/l respectively. NaCl and KBr were shown to have a primary salt effect on the interaction between enzyme and substrate but Na2SO4 appeared to have a specific ion effect at a cationic binding site. 3. NaCl inhibited the hydrolysis of IMs at all pH values studied, whereas NaCl concentrations of 0.2 mol/l inhibited the hydrolysis of PhI at pH values below 3.8 but activated the enzyme at higher incubation pH values. 4. Cu2+ was shown to be a potent non-competitive inhibitor of IMs enzyme activity with an apparent Kl, of approximately 0.02 mmol/l. The enzyme activity was inhibited by Fe2+ (Kl 4 mmol/l), Hg2+ and Ag+, but has not significantly been affected by other univalent or bivalent cations. 5. The presence of solvent and salt effects on apparent Km but not the Vmax. suggest that the binding of IMs to the enzyme involved charge neutralization, and it is inferred that two cationic binding sites are present at the active site. It is postulated that one site specifically binds to the iduronic acid carboxyl group, the other to the 6-sulphate of the anhydromannitol moiety.

scholarly journals Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans

1977 ◽  
Vol 161 (2) ◽  
pp. 357-370 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Cytochrome C ◽  
Electron Acceptor ◽  
Specific Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Transport Chain ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Triton X ◽  
Sepharose 4B

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.

scholarly journals The biosynthesis of granulose by Clostridium pasteurianum

1974 ◽  
Vol 144 (3) ◽  
pp. 503-511 ◽  
Author(s):  
R L Robson ◽  
R M Robson ◽  
J G Morris
Keyword(s):  
Specific Activity ◽  
Competitive Inhibitor ◽  
Partial Purification ◽  
Clostridium Pasteurianum ◽  
Saturation Curve ◽  
Wild Type ◽  
Competitive Inhibitors ◽  
Fine Control ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

1. Mutant strains of Clostridium pasteurianum were obtained, which are unable to synthesize granulose (an intracellularly accumulated amylopectin-like α-polyglucan). 2. These mutants lacked either (a) ADP-glucose pyrophosphorylase (EC 2.7.7.27), or (b) granulose synthase (i.e. ADP-glucose–α-1,4-glucan glucosyltransferase, EC 2.4.1.21). 3. Although both of these enzymes were constitutively synthesized by the wild-type organism, massive deposition of granulose in a sporulating culture coincided with a threefold increase in the specific activity of ADP-glucose pyrophosphorylase. 4. The soluble ADP-glucose pyrophosphorylase was partially purified (33-fold). Its ATP-saturation curve was not sigmoidal and its activity was not enhanced by phosphorylated intermediates of glycolysis, pyruvate, NAD(P)H or pyridoxal 5′-phosphate. ADP at relatively high concentrations acted as a competitive inhibitor (Ki=19mm). 5. The dependence of granulose synthase on a suitable polyglucan primer was demonstrated by using enzyme obtained from a granulose-free mutant strain (lacking ADP-glucose pyrophosphorylase). 6. Partial purification of granulose synthase from wild-type strains was facilitated by its being bound to the native particles of granulose. No activator was discovered, but ADP, AMP and pyridoxal 5′-phosphate were competitive inhibitors, ADP being most effective (Ki about 0.2mm). 7. It would appear that the synthesis of granulose in Cl. pasteurianum is not subject to the positive, fine control that is a feature of glycogen biosynthesis in most bacteria.

Diacylglycerol lipase and kinase activities in rabbit aorta and coronary microvessels

1986 ◽  
Vol 64 (10) ◽  
pp. 976-983 ◽  
Author(s):  
David L. Severson ◽  
Mariette Hee-Cheong
Keyword(s):  
Fatty Acid ◽  
Lipase Activity ◽  
Kinase Activity ◽  
Rabbit Aorta ◽  
Monoacylglycerol Lipase ◽  
Ph Optimum ◽  
Diacylglycerol Lipase ◽  
Dependent Protein ◽  
Hydrolysis Of ◽  
Hydrolysis Activity

Diacylglycerol lipase and kinase activities were measured in particulate and soluble fractions from rabbit aorta (intima–media) and coronary microvessels. With rabbit aorta, the hydrolysis at the sn-1 position of 1-palmitoyl-2-oleoyl-sn-glycerol had a pH optimum of 5–6 and was greater than hydrolysis at the sn-2 position (pH optimum of 6.5). Only the 2-monoacylglycerol accumulated during incubations at pH 5 and 6.5. These results are consistent with an ordered two-step reaction sequence where the fatty acid at the sn-1 position is released first, followed by the hydrolysis of the fatty acid from the 2-monoacylglycerol by a monoacylglycerol lipase with a neutral pH optimum. Lipase activity (sn-2 hydrolysis) at pH 6.5 was greater than kinase activity at all substrate concentrations. The presence of arachidonate at the sn-2 position of the diacylglycerol increased kinase activity but had little effect on lipase activity. Kinase activity was mainly particulate, whereas 50–60% of diacylglycerol lipase and 50% of monoacylglycerol lipase activity were soluble. Diacylglycerol lipase and kinase were also present in coronary microvessel preparations. Diacylglycerol lipase (sn-2 hydrolysis) activity in coronary microvessels was not enhanced by preincubation of the enzyme preparation with cAMP-dependent protein kinase.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

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