Partial purification of diphosphatidylglycerol synthetase from liver mitochondrial membranes

1980 ◽  
Vol 58 (10) ◽  
pp. 771-776 ◽  
Author(s):  
W. C. McMurray ◽  
E. C. Jarvis
Keyword(s):  
Divalent Cations ◽  
Fatty Acyl ◽  
Partial Purification ◽  
Salt Solutions ◽  
Mitochondrial Membranes ◽  
Absolute Dependence ◽  
Solubilized Enzyme ◽  
Cytidine Diphosphate ◽  
Zwitterionic Detergent ◽  
Nonionic Detergents

The enzyme responsible for the conversion of phosphatidylglycerol to diphosphatidylglycerol (cardiolipin) in the presence of cytidine diphosphate diacylglycerol is firmly associated with mitochondrial membranes and is not extracted with hypotonic or hypertonic media or with nonionic detergents. Some solubilization was obtained with bile salt solutions, but the zwitterionic detergent, Miranol H2M, was most effective in extracting the enzyme. The Miranol extracts were fractionated by column chromatography on Bio-Gel A-1.5 m. The solubilized enzyme is considerably more active in converting unsaturated than saturated phosphatidylglycerols, but shows little preference for the cytidine diphosphate diacylglycerols with different fatty acyl substituents. There is an absolute dependence upon divalent cations with the order of effectiveness: [Formula: see text]. In the presence of optimal levels of Co2+ other divalent cations are inhibitory with the order of inhibition: Cd2+ > Zn2+ > Ca2+ > Ba2+ > Cu2+ > Hg2+ > Ni2+. The solubilized enzyme exhibited no requirement for added phospholipids and several phospholipids inhibited the reaction in the order: diphosphatidylglycerol > phosphatidylethanolamine > phosphatidylserine > phosphatidylinositol.

Purification and properties of phosphatidylgkycerophosphate synthetase from mammalian liver mitochondria

1978 ◽  
Vol 56 (6) ◽  
pp. 414-419 ◽  
Author(s):  
W. C. McMurray ◽  
E. C. Jarvis
Keyword(s):  
Divalent Cations ◽  
Maximal Activity ◽  
Liver Mitochondria ◽  
Mitochondrial Membranes ◽  
Purification And Properties ◽  
Standard Assay ◽  
Mammalian Liver ◽  
Triton X ◽  
Cytidine Diphosphate ◽  
Additional Stimulation

The enzyme which catalyzes the synthesis of phosphatidylglycerophosphate from sn-glycerol-3-phosphate and cytidine diphosphate diacylglycerol was released from rat or pig liver mitochondrial membranes by extraction with Triton X-100 or Nonidet P-40. The detergent-extracted enzyme, like the activity of intact mitochondria, did not require added cations or lipids.The Triton extracts were fractionated by column chromatography on Bio-Gel A-1.5. The fractions obtained from the columns exhibited little activity in the standard assay system unless divalent cations were included. Additional stimulation (about twofold) was observed in the presence of added phospholipids.The cation requirement of the purified enzyme was relatively nonspecific with Mg2+, Ba2+, or Ca2+ providing maximal activity in the 10 mM range. Either Mn2+ or Co2+ were stimulatory at somewhat lower concentrations but higher concentrations were inhibitory. Other cations such as Cd2+, Zn2+, Hg2+, or Cu2+ were ineffective as cofactors, and in the presence of Mg2+ inhibited the reaction at concentrations greater than 0.5 mM.The phospholipid stimulation was obtained specifically with phosphatidylethanolamines from natural or synthetic sources. Other diacylglycerophosphatides or lysophosphatides including lysophosphatidylethanolamine were ineffective.

Solubilization and partial purification of particulate catechol-O-methyltransferase from rat liver

1977 ◽  
Vol 55 (10) ◽  
pp. 1108-1113 ◽  
Author(s):  
J. H. Tong ◽  
A. D'Iorio
Keyword(s):  
Rat Liver ◽  
Partial Purification ◽  
Catecholamine Metabolism ◽  
Ph Profile ◽  
Significant Difference ◽  
Solubilized Enzyme ◽  
Relationship Of ◽  
The Relationship ◽  
Substrate Affinities

Particulate catechol-O-methyltransferase (COMT) from rat liver has been solubilized by acetone treatment and partially purified. Results from the present study demonstrate that the solubilized, partially purified enzyme is similar to the cytosol COMT with respect to molecular weight, pH profile, sensitivity toward inhibitors, Mg2+ requirement, and substrate affinities. However, a comparison of the crude particulate COMT and the solubilized enzyme shows that there is a significant difference in their affinity for catechol substrates. This finding suggests that membrane protein and (or) lipid components may play an important role in catecholamine metabolism. The relationship of particulate COMT to [3H]norepinephrine binding was investigated. No correlation between the COMT and [3H]norepinephrine binding activities was observed in vitro.

scholarly journals THE SOLUBILITY AND PROPERTIES OF A PURIFIED ICHTHYOCOL IN SALT SOLUTIONS OF NEUTRAL pH

1957 ◽  
Vol 3 (4) ◽  
pp. 545-557 ◽  
Author(s):  
Paul M. Gallop ◽  
Sam Seifter ◽  
Edward Meilman
Keyword(s):  
Calcium Chloride ◽  
Magnesium Chloride ◽  
Divalent Cations ◽  
Optical Rotation ◽  
Sodium Thiosulfate ◽  
Salt Solutions ◽  
Bacterial Collagenase ◽  
Neutral Ph ◽  
Calcium Magnesium ◽  
Intermediate Concentration

1. Purified citrate-extracted ichthyocol obtained from carp swim bladders has been further characterized with respect to its content of certain amino acids and carbohydrate substances. 2. The degree of solubilization or dispersion of ichthyocol by solutions of certain salts maintained in the range of neutral pH and at a temperature of 0–2°C. has been determined. 3. While a number of salts of monovalent cations had no significant solubilizing effects on ichthyocol, ammonium chloride in a concentration of 1 M did cause solution of the protein. 4. Sodium thiosulfate in a range of concentrations caused the solubilization of ichthyocol but was most effective in an intermediate concentration of 0.25 M. 5. Several salts of divalent cations, in particular the chlorides of calcium, magnesium, and barium, and magnesium thiosulfate in concentrations ranging from 0.3 to 1 M caused the immediate and complete solubilization of the ichthyocol. 6. Solutions of ichthyocol in calcium chloride, magnesium chloride, and sodium thiosulfate buffered or adjusted to pH 7.0, were studied with respect to intrinsic viscosity of the protein, optical rotation, ultracentrifugal sedimentation, and reconstitution into fibers. It was found in each case that the original characteristics of the collagen, as determined previously in acid solution, were maintained when the protein was dissolved in salt solutions of neutral pH. No evidence of denaturation or gelatinization could be found when ichthyocol was solubilized under the stated conditions. 7. Collagen in neutral solution with sodium thiosulfate, calcium chloride, or magnesium chloride was not attacked by trypsin as determined viscometrically at 20.0°C., but was rapidly degraded by a purified bacterial collagenase.

Substrate dependency for HCl secretion by isolated piglet gastric mucosa

1980 ◽  
Vol 238 (4) ◽  
pp. G353-G357 ◽  
Author(s):  
J. G. Forte ◽  
J. A. Black ◽  
J. G. Forte
Keyword(s):  
Fatty Acids ◽  
Gastric Mucosa ◽  
Short Chain Fatty Acids ◽  
Bathing Solution ◽  
Salt Solutions ◽  
Newborn Piglets ◽  
Exogenous Substrate ◽  
Saturation Kinetics ◽  
Absolute Dependence ◽  
Hcl Secretion

Gastric mucosa was isolated from newborn piglets and bathed with balanced salt solutions. In the presence of glucose (ca. 0.01 M), this gastric preparation has been shown to be responsive to histamine by relatively prompt and vigorous H+ secretory rates. Secretion is dependent on glucose concentration in the serosal bathing solution, showing saturation kinetics with an apparent Km of about 2 mM glucose. Acetate and pyruvate were about as effective as glucose in sustaining H+ secretory rates. Short-chain fatty acids supported secretory rates that were significantly lower than rates measured with glucose. The order of effectiveness was butyrate greater than valerate greater than hexanoate greater than propionate. The results show absolute dependence of H+ secretion by piglet gastric mucosa on exogenous substrate and the preferential utilization of carbohydrate sources as substrates for secretion. They suggest that it is unlikely for any specialized and essential involvement of fatty acids in the primary H+ secretory mechanism as had been previously proposed.

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.

Biosynthesis of phosphatidic acid by glycerophosphate acyltransferases in rat liver mitochondria and microsomes

1990 ◽  
Vol 68 (12) ◽  
pp. 1380-1392 ◽  
Author(s):  
Amy Y. P. Mok ◽  
William C. McMurray
Keyword(s):  
Phosphatidic Acid ◽  
Rat Liver ◽  
Neutral Lipids ◽  
Divalent Cations ◽  
Liver Mitochondria ◽  
Fatty Acyl ◽  
Subcellular Fractions ◽  
Rat Liver Mitochondria ◽  
Acyl Donor ◽  
Palmitoyl Carnitine

The acyltransferases that catalyze the synthesis of phosphatidic acid from labelled sn-[14C]glycero-3-phosphate and fatty acyl carnitine or coenzyme A derivatives have been shown to be present in both isolated mitochondria and microsomes from rat liver. The major reaction product was phosphatidic acid in both subcellular fractions. A small quantity of lysophosphatidic acid and neutral lipids were produced as by-products. Divalent cations had significant effects on both mitochondrial and microsomal fractions in stimulating acylation using palmitoyl CoA, but not when palmitoyl carnitine was used as the acyl donor. Palmitoyl CoA and palmitoyl carnitine could be used for acylation by both mitochondria and microsomes. Mitochondria were more permeable to palmitoyl carnitine and readily used it as the substrate for acylation. On the other hand, microsomes yielded a better rate with palmitoyl CoA and the rate of acylation from palmitoyl carnitine in microsomes was correlated with the degree of mitochondrial contamination. The enzymes were partially purified from Triton X-100 extracts of subcellular fractions. Based on the differences of substrate utilization, products formed, divalent cation effects, molecular weights, and polarity, the mitochondrial and microsomal acyltransferases appeared to be different enzymes.Key words: glycerophosphate, acyltransferase, mitochondria, microsomes, phosphatidic acid.

Partial Purification and Some Properties of 1-Sinapoylglucose: Choline Sinapoyltransferase ("Sinapine Synthase") from Seeds of Raphanus sativus L. and Sinapis alba L.

1986 ◽  
Vol 41 (1-2) ◽  
pp. 28-33 ◽  
Author(s):  
Walter Gräwe ◽  
Dieter Strack
Keyword(s):  
Raphanus Sativus ◽  
Divalent Cations ◽  
Sinapis Alba ◽  
Partial Purification ◽  
Acyl Donor ◽  
Sulfhydryl Reagents ◽  
Molecular Weights ◽  
Sinapis Alba L ◽  
Raphanus Sativus L

Abstract Hydroxycinnamoyltransferases which catalyze the formation of O-sinapoylcholine (sinapine) using 1-0-sinapoyl-β-ᴅ-glucose as acyl donor have been isolated from seeds of radish (Raphanus sativus L. var. sativus) and mustard (Sinapis alba L.) and purified 420-and 293-fold, respectively. The enzymes (“sinapine synthase”) had apparent molecular weights of about 60,000 daltons and showed highest activities at pH 7.2 and 7.6, respectively, at 45 °C with apparent energies of activation at 53 kJ mol-1. There were no requirements for divalent cations or sulfhydryl reagents. The apparent Km’s of the radish and mustard enzymes were 0.48 and 0.71 mᴍ for 1-sinapoylglucose and 5.3 and 6.5 mᴍ for choline, respectively. The ratios of the Vmax/Km values for 1-sinapoyl-1-feruloyl- and 1-p-coumaroylflucose were found to be 100:19:19 (radish) and 100:20:29 (mustard). 6-0-Sinapoylglucose, 3-0-sinapoylfructose, 1-0-benzoyl- and 1-0-galloylglucose were not accepted as donors.

scholarly journals Direct desaturation of intact galactolipids by a desaturase solubilized from spinach (Spinacia oleracea) chloroplast envelopes

1993 ◽  
Vol 289 (3) ◽  
pp. 777-782 ◽  
Author(s):  
H Schmidt ◽  
E Heinz
Keyword(s):  
Double Bond ◽  
Spinacia Oleracea ◽  
Membrane Lipids ◽  
Fatty Acyl ◽  
Acyl Residue ◽  
Solubilized Enzyme ◽  
Membrane Bound ◽  
Triton X ◽  
Envelope Membranes

In plants, polyenoic fatty acids are synthesized by desaturase enzymes which use acyl groups of membrane lipids as substrates. To provide direct ‘in vitro’ evidence for this reaction, we solubilized envelope membranes from spinach (Spinacia oleracea) chloroplasts with Triton X-100 to release a membrane-bound n-6 desaturase. In the presence of oxygen and reduced ferredoxin, the solubilized enzyme desaturated a variety of substrates, such as free oleic acid, free erucic acid, 1-oleoyl-sn-glycerol 3-phosphate and the three galactolipids 1-oleoyl-2-(7′-cis-hexadecenoyl)-3-beta-D-galactopyranosyl-sn-glycerol, 1,2-dioleoyl-3-beta-D-galactopyranosyl-sn-glycerol and the ether analogue 1,2-di-(9′-cis-octadecenyl)-3-beta-D-galactopyranosyl-sn- glycerol. The in vitro desaturation of these exogenously added complex lipids with ester- and ether-linked substrate chains is unambiguous evidence for lipid-linked desaturation. The enzyme measures the insertion of the new double bond from the methyl end and the existing (n-9)-cis-double bond of an appropriate acyl or alkyl chain. The distal part of the substrate group, normally the carboxy end of a fatty acyl residue, is of less importance and, in particular, its activation in thioester form is not required.

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