Matrex Gel Blue Research Articles

Partial purification and characteristics of membrane-associated NAD +-dependent isocitrate dehydrogenase activity from etiolated pea mitochondria

NAD +-dependent isocitrate dehydrogenase from pea mitochondrial matrix has been isolated, partially purified, and characterized [1]. Of the total NAD-ICDH activity found in pea mitochondria, 35–50% is found tightly associated with the membrane. Isolation and partial purification of membrane-associated ICDH showed that this activity has physical properties that differ from those of the matrix form. Membrane-ICDH was not removed from the membrane by washing with weak detergent, conditions which result in solubilization of over 90% of the malate dehydrogenase activity. Both forms of the enzyme bind to Matrex Gel Blue A in the absence of detergent, however 4× higher salt levels are required to elute the membrane form; membrane-ICDH also binds to Blue A in the presence of detergent while the matrix form does not. Membrane-ICDH is also more anionic requiring higher salt for elution from Mono Q, and in the absence of detergent is unrecoverable from this column. Characterization of ca. 40-fold purified membrane-ICDH revealed that the membrane and matrix form of the enzyme have similar kinetic properties. The possible nature of physical differences between these activities as well as potential physiological roles is discussed.

Purification and Characterization of the Tricarboxylate Carrier from Eel Liver Mitochondria

The tricarboxylate carrier from eel (Anguilla anguilla) liver mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and Matrex Gel Blue B. On SDS-polyacrylamide gel electrophoresis, the purified fraction showed a single polypeptide band with an apparent molecular mass of 30.4 kDa. When reconstituted into liposomes, the tricarboxylate transport protein catalyzed a very active 1,2,3-benzenetricarboxylate-sensitive citrate/citrate exchange. It was purified 641-fold with a recovery of 13.3% and a protein yield of 0.02% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., requirement for a counteranion, substrate specificity and inhibitor sensitivity, were similar to those of the tricarboxylate carrier purified from rat liver mitochondria. These studies provide the first information on the mitochondrial tricarboxylate transport protein of a fish.

Purification and properties of malate dehydrogenase from Paracoccus denitrificans.

Affinity chromatography on immobilized Cibacron Blue (Matrex Gel Blue A) gel permeation chromatography on UltroPac TSK G 3000 SWG column and ion-exchange chromatography on "Mono Q" column were used to purify the malate dehydrogenase (MDH) from P. denitrificans to electrophoretic homogeneity. The last two purification steps were performed in FPLC system. The enzyme having a specific activity of about 2300 nkat/mg protein was obtained with an approximate 70% yield. MDH is a dimer with a molecular mass of 80,000 +/- 10,000 and an isoelectric point of 4.85 +/- 0.05. Absorption, fluorescence and CD-spectra were also measured and basic kinetic parameters were obtained for the homogeneous enzyme. The present paper also suggests the possibility of using the prepared enzyme for the determination of aspartate transferase (AST) in blood serum.

Purification and characterization of the reconstitutively active tricarboxylate transporter from rat liver mitochondria.

The tricarboxylate transporter has been purified in reconstitutively active form from rat liver mitochondria. The transporter was extracted from mitoplasts with Triton X-114 in the presence of cardiolipin and citrate and was then purified by sequential chromatography on hydroxylapatite, Matrex Gel Orange A, Matrex Gel Blue B, and Affi-Gel 501. Analysis of the purified material via sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of one main protein band with an apparent molecular mass of 32.5 kDa. Upon incorporation into phospholipid vesicles, the purified transporter catalyzed a 1,2,3-benzenetricarboxylate-sensitive citrate/citrate exchange with a specific transport activity of 3240 nmol/4 min/mg of protein. This value was enhanced 831-fold with respect to the starting material. Substrate competition studies indicated that the reconstituted transport could be substantially inhibited by isocitrate, malate, and phosphoenolpyruvate, but not by alpha-ketoglutarate, succinate, malonate, pyruvate, or inorganic phosphate. Moreover, in addition to 1,2,3-benzenetricarboxylate, the reconstituted exchange was sensitive to the anion transport inhibitor n-butylmalonate but was insensitive to phenylsuccinate, alpha-cyano-4-hydroxycinnamate, and carboxyatractyloside. Finally, studies with covalent modifying agents indicated the purified transporter was inhibited by sulfhydryl reagents and by diethyl pyrocarbonate, 2,3-butanedione, phenylglyoxal, and pyridoxal 5-phosphate. In conclusion, these studies describe the first procedure to yield a highly purified tricarboxylate transport protein that both displays a high specific transport activity and can be obtained in quantities that readily enable further structural as well as functional studies. Based on its substrate specificity and inhibitor sensitivity, the purified 32.5-kDa protein appears to represent the complete tricarboxylate transport system found in rat liver mitochondria. Finally, new information is presented concerning the effect of covalent modifying reagents on the function of this transporter.

Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae

A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diastereomeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C5-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (Mr) of the enzyme was estimated as 140,000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of Mr 35,000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.

Isolation and Characterization of Ceramide Glycanase from the Leech, Macrobdella decora

We have devised a simple method for achieving 890-fold purification of ceramide glycanase with 17% recovery from a North American leech, Macrobdella decora. The method includes water extraction, ammonium sulfate fractionation, and chromatography on octyl-Sepharose, Matrex gel blue A, and Bio-Gel A-0.5m columns. The final preparation showed one major protein band at 54 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By using Bio-Gel A-0.5m filtration, the native enzyme was found to have a molecular mass of 330 kDa. With GM1 as substrate, the optimum pH of this enzyme was determined to be 5.0; the enzyme was stable between pH 4.5 and 8.5. Zn2+ at 5 mM and Cu2+, Ag+, and Hg2+ at 1 mM strongly inhibited the hydrolysis of GM1 by ceramide glycanase. The ceramide glycanase released the intact glycan chain from various glycosphingolipids in which the glycan chain is linked to the ceramide through a beta-glucosyl linkage. This enzyme also cleaved lyso-glycosphingolipids such as lyso-GM1 and lyso-LacCer and synthetic alkyl beta-lactosides. Among seven alkyl beta-lactosides tested, the enzyme only hydrolyzed the ones with an alkyl chain length of four or more carbons. The enzyme also hydrolyzed 2-(octadecylthio)ethyl O-beta-lactoside and 2-(2-carbomethoxyethylthio)ethyl O-beta-lactoside. p-Nitrophenyl, benzyl, and phytyl beta-lactosides, on the other hand, were not hydrolyzed. These results suggest that the enzyme can recognize the hydrophobic portion of glycolipid substrates. The fact that 2-(2-carbomethoxyethylthio)ethyl O-beta-N-acetyllactosaminide and DiGalCer were refractory to the enzyme indicated that in the substrate the first sugar attached to the hydrophobic chain cannot be N-acetylglucosamine and galactose. Furthermore, dodecyl maltoside, Gal alpha 1----6Glc beta Cer, and the LacCer in which the --CH2OH of the galactose was converted into --CHO were also resistant to the enzyme, and Man beta 1----4 Glc beta Cer was hydrolyzed at a much slower rate than LacCer. These results indicate that the nature and the linkage of the sugar attached to the glucose have a profound effect on the action of this enzyme. The hydrolysis of glycosphingolipids by ceramide glycanase is stimulated by bile salts. Among various bile salts tested, sodium cholate at a concentration of 1 microgram/microliter was found to be most effective in stimulating the hydrolysis of various glycosphingolipids with the exception of LacCer. For LacCer, sodium taurodeoxycholate at a concentration of 2-3 micrograms/microliters was most effective. Tween 20, Nonidet P-40, and Triton X-100 did not stimulate the hydrolysis of GM1.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and characterization of an activator protein for the hydrolysis of ganglioside GM2 from the roe of striped mullet (Mugil cephalus)

After the revelation of the presence of ganglioside GM2 as the major ganglioside in the roe of striped mullet, Mugil cephalus [Li, Hirabayashi, DeGasperi, Yu, Ariga, Koerner & Li (1984) J. Biol. Chem. 259, 8980-8985], we have continued to investigate the catabolism of GM2 in this tissue. We have found that mullet roe contains a specific activator protein which stimulates the hydrolysis of GM2 carried out by the beta-hexosaminidase isolated from the same tissue. This activator has been purified by using conventional procedures including ammonium sulphate fractionation and chromatography on Sepharose 6B, DEAE-Sephadex A-50, octyl-Sepharose and Matrex Gel Blue A columns. This activator protein is also able to stimulate the hydrolysis of GM2 carried out by human beta-hexosaminidase A. Unlike human GM2-activator, the roe activator protein does not stimulate the hydrolysis of GgOse3Cer or GbOse4Cer. The molecular mass (18 kDa) of the roe activator protein was found to be similar to that of human GM2-activator; however, the pI (pH 4.1) was found to be lower than that of human GM2-activator. This is the first report on the presence of a GM2-activator protein in a source other than mammalian tissues.

Purification and properties of short chain acyl-CoA, medium chain acyl-CoA, and isovaleryl-CoA dehydrogenases from human liver.

Short chain acyl-CoA (SCA), medium chain acyl-CoA (MCA), and isovaleryl-CoA (IV) dehydrogenases were purified to homogeneity from human liver using ammonium sulfate fractionation followed by DEAE-Sephadex A-50, hydroxyapatite, Matrex Gel Blue A, agarose-hexane-CoA, and Bio-Gel A-0.5 column chromatographies. The specific activities of the final preparations were enriched 507-, 750-, and 588-fold over those from the second ammonium sulfate fractionation step. The native molecular weights were estimated to be 168,000, 178,000, and 172,000, respectively, by gel filtration. Each of them exhibited, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single protein band with molecular weights of 41,000, 44,000, and 42,000, respectively, indicating a homotetrameric structure. UV/visual spectra, fluorescence spectra, and other evidence indicated that each contains 1 mol of FAD per subunit. They all utilized electron transfer flavoprotein (ETF) or phenazine methosulfate (PMS) as an electron acceptor. The products of SCA dehydrogenase/butyryl-CoA, MCA dehydrogenase/octanoyl-CoA, and IV dehydrogenase/isovaleryl-CoA reactions were identified as crotonyl-CoA, 2-octenoyl-CoA, and 3-methylcrotonyl-CoA, respectively, using gas chromatography. Kinetic parameters Vappmax and Kappm) of these enzymes for various acyl-CoA substrates, as well as Kappm values for ETF and PMS are presented. In general, the substrate specificities of human SCA, MCA, and IV dehydrogenases are slightly less stringent than those of their rat counterparts and resemble those of their bovine and porcine counterparts. The pattern of substrate specificity for these enzymes determined using ETF as electron acceptor significantly differed from that determined using PMS. All of them were severely inhibited by (methylenecyclopropyl)acetyl-CoA.

Monoclonal antibodies against an intracellular phospholipase A2 from rat liver and their cross-reactivity with other phospholipases A2.

The membrane-associated phospholipase A2 from rat liver mitochondria was solubilized and partially purified by AcA 54 gel filtration and Matrex gel blue A chromatography. The approximately 2500-fold purified preparation was injected into mice to prepare monoclonal antibodies against phospholipase A2 after fusion of spleen cells and mouse SP2/0 myeloma cells. Hybridoma supernatants were assayed for antibody production in enzyme-linked immunosorbent assay with partially purified phospholipase A2 as antigen. Positive clones were tested for their ability to bind phospholipase A2 in a specific immunoprecipitation assay involving protein-A--Sepharose to which rabbit anti-(mouse immunoglobulins) and monoclonal antibodies from hybridoma supernatants were complexed. Twelve clones producing antibodies that bound mitochondrial phospholipase A2 were identified. The binding of all of these antibodies to protein fractions eluted from AcA 54 and Matrex gel blue A columns coincided with the phospholipase A2 activity in these fractions. All monoclonal antibodies showed cross-reactivity with rat liver cytosolic and solubilized rat platelet phospholipase A2. Extracellular phospholipase A2 from rat and pig pancreas or Crotalus atrox were not recognized by the anti-(mitochondrial phospholipase A2) antibodies.

Some aspects of rat platelet and serum phospholipase A 2 activities

Rat platelet lysate contained appreciable phospholipase A 2 activity. In agreement with literature data this enzymatic activity eluted in the void volume of a Sephadex G-100 column. When the void volume peak was chromatographed over a Matrex gel blue A column, part of the phospholipase A 2 activity ran through, whereas the remainder was bound to the gel. The latter activity could be eluted with buffers containing a high salt concentration. In contrast, phospholipase A 2 activity solubilized from rat platelet lysates by treatment with high salt eluted from Sephadex G-100 columns with an apparent molecular weight of 10–15 kDa. This solubilized enzyme completely bound to Matrex gel blue A and, in the presence of Ca 2+ also to an alkylphosphocholine-AH Sepharose affinity column. No indications were obtained for the presence of inactive phospholipase A 2 and activator proteins in rat platelet lysates as described by Etienne, J., Grüber, A. and Polonovski, J. ((1980) Biochim. Biophys. Acta 619, 693–698; (1982) Biochemie 64, 377–380). Phospholipase A 2 activity, both the associated form in platelet lysate and the monomeric form as eluted from Sephadex G-100 was slightly inhibited by trifluoperazine but calmodulin exerted no stimulation. Likewise, phospholipase A 2 activity from rat serum eluted in the void volume of a Sephadex G-100 column. Rather than indicating the presence of high molecular weight forms of the enzyme, this is apparently caused by association with lipids or other proteins, in that chromatography in the presence of high salt revealed a molecular weight similar to that found for solubilized platelet phospholipase A 2 activity.

Purification and properties of D-3-hydroxybutyrate dehydrogenase from Paracoccus denitrificans

D-3-Hydroxybutyrate dehydrogenase from Paracoccus denitrificans has been purified to near homogeneity. The enzyme was prepared using DEAE-cellulose chromatography, affinity chromatography on immobilized Cibacron blue (Matrex Gel Blue A) and gel permeation chromatography. The pure enzyme was obtained by chromatofocusing as the final isolation step. The purification procedure yielded the enzyme with a specific activity of about 100 units/mg protein. The enzyme is specific for D-3-hydroxybutyrate and NAD and it exhibits anomalous kinetics (hysteresis) at low enzyme and coenzyme concentrations. It is relatively stable in the presence of EDTA at pH 7–8 higer salt concentrations. D-3-Hydroxybutyrate dehydrogenase is a tetramer with a molecular weight of 130 000 ± 10 000, its isoelectric point equals 5.10 ± 0.05. The enzyme is applicable to the determination of acetoacetate and D-3-hydroxybutyrate concentrations.

Purification and characterization of short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases from rat liver mitochondria. Isolation of the holo- and apoenzymes and conversion of the apoenzyme to the holoenzyme.

Short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases were purified to homogeneity from rat liver mitochondria by sequential chromatography on DEAE-Sephadex A-50, hydroxyapatite, Matrex Gel Blue A, agarose-hexane-CoA, and Bio-Gel A-0.5m. Molecular, immunological, and catalytic properties of the pure acyl-CoA dehydrogenases were investigated. The native molecular weights of these three enzymes were 160,000, 180,000, and 180,000, respectively. The subunit molecular weights of the three enzymes were estimated to be 41,000, 45,000, and 45,000, respectively, indicating that these enzymes are each composed of four subunits of equal size. The FAD content was calculated to be 1 mol/mol of subunit. While FAD binding by short-chain acyl-CoA dehydrogenase was very tight, that by medium-chain acyl-CoA and long-chain acyl-CoA dehydrogenases was less tight. The medium- and long-chain acyl-CoA dehydrogenases were also purified to homogeneity as FAD-free apoenzymes. The apoenzymes were converted to the fully active holoenzymes by incubation with FAD. The three acyl-CoA dehydrogenases were immunologically distinct from each other, i.e. the antibodies raised against the individual enzymes were monospecific and did not cross-react with any other acyl-CoA dehydrogenases. Our preparations of the three enzymes exhibited substrate specificities (as defined in Vappmax and Kappmax) significantly more specific than those of the previous preparations isolated from other sources. The substrate specificities were assessed also by measuring the activities in mitochondrial sonicates after selectively precipitating each enzyme with their individual monospecific antibodies. Butyryl-CoA was almost exclusively dehydrogenated by short-chain acyl-CoA dehydrogenase while C6-C10 acyl-CoAs were mainly dehydrogenated by medium-chain acyl-CoA dehydrogenase. C14-C22 acyl-CoAs were exclusively dehydrogenated by long-chain acyl-CoA dehydrogenase. C24 acyl-CoAs were not dehydrogenated by this enzyme. Lauroyl-CoA appeared to be jointly dehydrogenated by the latter two enzymes. Branched-chain acyl-CoAs were not dehydrogenated by short-chain acyl-CoA dehydrogenase. In the presence of electron-transfer flavoprotein or phenazine methosulfate, 2-enoyl-CoAs were identified as products from the corresponding enzyme/acyl-CoA reactions.

Studies on the poly(vinyl alcohol) degrading enzyme. Part IV. Purification and properties of secondary alcohol oxidase with an acidic isoelectric point.

An enzyme which catalyzes the oxidation of poly(vinyl alcohol) (PVA) has been purified from a fraction adsorbed to DEAE-Sephadex at pH 7.0 from PVA-degrading enzyme activities produced by a bacterial symbiotic mixed culture in a culture broth when the culture was grown in a minimal medium where PVA served as a sole source of carbon and energy. The enzyme was separated from a coexisting oxidized PVA hydrolase by dye-ligand chromatography on Matrex Gel Blue A. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoreses in the absence and presence of SDS.The enzyme is a single polypeptide with a molecular weight of about 40,000 and has an isoelectric point of 4.5. The amino acid composition of the enzyme has been determined and found to have no histidine. The N- and C-terminal amino acid residues are both alanine. The enzyme solution is pink and shows absorption maxima at 276, 364, and 469 nm. One atom of non-heme iron has been detected per molecule in the enzyme.The enzyme catalyzes ...

Purification of prostaglandin E 2-9-oxoreductase from human decidua vera

Prostaglandin E 2-9-oxoreductase (PGE 2-9-OR), the enzyme which converts prostaglandin E 2 (PGE 2) to prostaglandin F 2α (PGF 2α), has been detected in human decidua vera. A 105-fold purification was achieved when the centrifuged homogenate was fractionated sequentially by DEAE—Trisacryl, hydroxyapatite—agarose gel, ultrogel AcA 44 and Matrex gel blue A gel chromatographies. The following kinetic constants for PGE 2-9-OR have been obtained. The equilibrium constant with respect to PGE 2 is 83 μM, the Michaelis constant, K m, for PGE 2 is 80 μM, for NADPH 1.6 μM. The maximal velocity for the forward reaction is V 1 = .203 pmol/min. The enzyme was inhibited by progesterone, oestradiol-17β, cortisol and pharmaceutical drugs. An activating effect could be demonstrated with Ca 2+ and oxytocin. The occurrence of PGE 2-9-OR in the decidua vera suggests that this enzyme may be responsible for the transformation of PGE 2 to PGF 2α in these tissues. This may be an important mechanism for the initiation and maintenance of uterine contractions.

A simple procedure for purification of NADH-specific dihydropteridine reductase from mammalian liver.

A simple and convenient purification method which can yield a homogeneous preparation from even a small amount of starting material was devised for NADH-specific dihydropteridine reductase from rat liver. The procedure is essentially composed of two steps, i.e., affinity chromatography on Matrex gel blue A and hydrophobic chromatography on Phenyl-Sepharose. Prior to the Matrex gel blue A chromatography, the crude extract of rat liver was oxidized to dissociate NADH, bound in the enzyme-NADH complex, from the enzyme. Low molecular weight substances in the extract were removed by Sephadex G-25 gel filtration; then the enzyme was purified by successive chromatographies on Matrex gel blue A and Phenyl-Sepharose columns. Thus about 0.1 mg of purified enzyme was obtained from 3 g of rat liver with 40% recovery. The preparation showed a single protein band on polyacrylamide and SDS-gel electrophoresis. Using NADH and tetrahydro-6-methylpterin, the maximal velocity of the enzyme was determined to be 64.2 mumol quinonoid-dihydro-6-methylpterin reduced/min/mg. Km values of the enzyme were 0.85 microM and 3.4 microM for NADH and quinonoid-dihydro-6-methylpterin, respectively. This simple purification method was also applicable to livers from other mammalian sources such as human and bovine.

25] Isolation and properties of an NADP+-dependent PGI2-specific 15-hydroxyprostaglandin dehydrogenase from rabbit kidney

Publisher Summary This chapter presents a procedure for the isolation and assay of nicotinamide adenine dinucleotide phosphate (NADP + )-dependent prostaglandin (PG) I 2 -specific 15-hydroxyprostaglandin dehydrogenase from rabbit kidney. For purification described in the chapter, frozen rabbit kidneys were thawed and minced. Portions of 50–60 g were homogenized with four volumes of buffer in a Waring blender. A 10-ml portion of the homogenate was centrifuged at 30,000 g for 60 min, and the supernatant solution was assayed for enzyme activity. The remainder of the homogenate was centrifuged at 9000 g for 75 min, and the supernatant solution was collected. Further steps were (1) ammonium sulfate fractionation, (2) diethylaminoethyl-cellulose chromatography, (3) matrex gel blue a chromatography, (4) sephadex G-100 gel filtration, and (4) hydroxyapatite chromatography. The enzyme activity was measured spectrophotometrically in a cuvette with a l-cm light path at 25 ±0.5°. The cuvette contains 3 ml of aqueous solution consisting of 290 μmol of sodium pyrophosphate at pH 10.3, 1.25 μmol of NADP + , and 267 nmol of PGI 2 added in 0.01 ml of sodium pyrophosphate. The blank contains no PGI 2 but was identical otherwise. The reaction was started by the addition of enzyme, and NADPH production was followed by measuring the change in absorbance at 340 nm.

The rapid purification of a phosphotransferase from wheat shoots

A phosphotransferase from wheat shoots which specifically phosphorylates the 5′-position of nucleosides has been purified by a simple procedure involving chromatography on Matrex Gel Blue A (Cibacron Blue F3GA).