Matrex Gel Red Research Articles

Characterization of hepatic l-threonine dehydrogenase of chicken

The l-threonine dehydrogenase (TDH) was purified approximately 1300-fold to a specific activity of ∼18 000 unit mg −1 from chicken ( Gallus domesticus) liver mitochondria. Purification was obtained by sequential chromatography on DEAE Cellulose, Phenyl Sepharose High Performance hydrophobic interaction, Affi-Gel Blue affinity and Matrex Gel Red A columns. The molecular weight of the subunit was estimated to be 36 kDa by sodium dodecyl-polyacrylamide gel electrophoresis. An apparent molecular mass of native protein between 62 and 74 kDa was obtained by gel filtration chromatography, suggesting a dimeric structure of TDH. The isoelectric point of TDH was determined by isoelectric focusing to be 5.3. Partial amino-terminal sequence analyses, carried out on two purified preparations of TDH, revealed a high degree of homology to the reported sequence of porcine TDH. The Michaelis constants for l-threonine and NAD for partially purified chicken hepatic TDH are 5.38 and 0.19 mM, respectively.

The Effect of Cysteine-43 Mutation on Thermostability and Kinetic Properties of Citrate Synthase fromThermoplasma acidophilum

In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic ArchaeonThermoplasma acidophilum,for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutantThermoplasmacitrate synthase is very similar to wild-type citrate synthase in its substrate and co-factor specificities, pH profile and thermal stability. The mutation, however, has decreased the enzyme activity. The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity.

Purification and Characterization of an NADPH-Cytochrome P450 (Cytochrome c) Reductase from Spearmint (Mentha spicata) Glandular Trichomes

Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2′,5′-adenosine diphosphate agarose. SDS–PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. TheKmvalues for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 μM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (−)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.

Overexpression and Purification of the Trimeric Aspartate Transcarbamoylase from Bacillus subtilis

A procedure has been developed for the overexpression and purification of milligram quantities of the Bacillus subtilis aspartate transcarbamoylase. The plasmid pEK171, carrying the B. subtilis pyrB structural gene under the control of the Escherichia coli pyrBI promoter, was transformed into the E. coli strain EK1104 and the enzyme overexpressed to approximately 50% of total soluble protein under extreme derepression of the pyrimidine pathway. The enzyme was subsequently purified by means of ammonium sulfate fractionation, anionic exchange chromatography using Q-Sepharose Fast Flow resin, negative chromatography on Matrex Gel Red A agarose, and hydrophobic interaction chromatography using Matrex Phenyl Cellufine, The purification yields approximately 60 mg of pure enzyme per liter of bacterial culture. Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties very similar to those of the enzyme purified from B. subtilis cells.

Purification and properties of glutamine synthetase from Douglas fir roots

Glutamine synthetase (GS. EC 6.3.1.2) was purified to apparent electrophoretic homogeneity from roots of Pseudotsuga menziesii (Mirb) Franco by a three‐step procedure involving diethylaminoethyl (DEAE)‐Trisacryl chromatography, affinity chromatography on Matrex Gel Red A. and preparative polyacrylamide gel electrophoresis. The enzyme was purified 40‐fold with a 16% recovery. The native enzyme had a molecular mass of 460 ± 5 kDa as estimated by gel filtration, interpolation of the Ferguson plots and non‐denaturing gradient‐PAGE. It was composed of two different subunits of 54 and 64 kDa. Affinity constants for glutamate (Glu), glutamine (Gln), ATP and ADP were 2.6, 10.5, 0.5 and 0.083 mM. respectively. The enzyme exhibited a negative cooperativity for ammonium (Hill number of 0.7) with two Km values which were 11 and 75 μM in the presence of ammonium concentrations lower and higher than 1.3 mM, respectively. Glycine and ADP appeared as potential inhibitors of the GS activity. The optimum pH values were 7.2 and 7.6 for the transferase and the biosynthetic assays, respectively. The enzyme lost 30% of its activity within 25 days of storage at 4°C. The optimum temperatures of activity were 40°C and 45°C for the transferase and bio‐synthetic activities, respectively.

Purification and Structural Characterization of Porcine L-Threonine Dehydrogenase

L-Threonine dehydrogenase was purified 10,000-fold to a specific activity ∼300 μmol · min−1 · mg−1 protein from porcine liver mitochondria. Purification to apparent homogeneity was achieved by sequential chromatography on DEAE Sepharose FF, Affi-Gel Blue, Sephacryl S-200, Matrex Gel Red A, and Matrex Gel Green A. The subunit molecular mass was estimated as 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while an apparent native molecular mass of 73 kDa was shown by gel filtration chromatography, suggesting a dimeric structure. The purified enzyme was subjected to proteolytic degradation and the resulting peptides were isolated exclusively by reverse-phase high-performance liquid chromatography. Approximately 70% of the total sequence was obtained and the N-terminal amino acid sequence of the intact polypeptide chain was thus tentatively extended to residue 40.

Purification and mechanism of delta 3,delta 5-t-2,t-4-dienoyl-CoA isomerase from rat liver.

A new enzyme, i.e., delta 3,delta 5-t-2,t-4-dienoyl-CoA isomerase, required in the NADPH-dependent metabolic pathway of odd-numbered double bond, unsaturated fatty acids, was isolated and purified to apparent homogeneity from rat liver. In the oxidation of odd-numbered double bond, unsaturated fatty acids, stepwise beta-oxidation leads to cis-5-enoyl-CoA, which is then dehydrogenated and isomerized to delta 3,delta 5-dienoyl-CoA. delta 3,delta 5-t-2,t-4-Dienoyl-CoA isomerase converts delta 3,delta 5-dienoyl-CoA to trans-2,trans-4-dienoyl-CoA, which is a substrate for NADPH-dependent 2,4-dienoyl-CoA reductase. This enzyme was purified through Matrex gel red A, blue Sepharose, DEAE-cellulose, CM-cellulose, hydroxylapatite, and Sepharose CL6B column chromatography of an ammonium sulfate precipitated fraction (30-80%) of rat liver homogenate. A native molecular weight of 200,000 with four subunits of 55,000 each was determined. The isoelectric point was 6.5. This enzyme was located in mitochondria and was inducible by clofibrate treatment. Using delta 3,delta 5-decadienoyl-CoA, delta 3,delta 5-dodecadienoyl-CoA, and delta 3,delta 5-tetradecadienoyl-CoA as substrates, the Vmax ratio was 1:0.5:0.4 and the Km's were 10.9, 5.9, and 1.4 microM, respectively. The specific activity of purified enzyme was 7 units/mg using delta 3,delta 5-decadienoyl-CoA as substrate. The mechanism of isomerization was studied by deuterium labeling. Consistent with the deuterium labeling pattern of the products, the isomerization from trans-2,cis-5-dienoyl-CoA to trans-2,trans-4-dienoyl-CoA was a two-step process through an intermediate delta 3,delta 5-dienoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)

Citrate synthases from the Archaea: Development of a bio-specific, affinity chromatography purification procedure

Citrate synthases from both thermophilic and halophilic Archaea have been purified to homogeneity using affinity chromatography on Matrex Gel Red A and elution with a combination of substrate (oxaloacetate) and product (coenzyme A). In a number of cases, purification from cell-extract to protein suitable for N-terminal sequencing can be achieved by this single-step procedure. The method is particularly useful in the rapid purification of a thermophilic archaeal citrate synthase from a cloned gene expressed in a mesophilic host.

Citrate synthases from the Archaea: Development of a bio-specific, affinity chromatography purification procedure

Citrate synthases from both thermophilic and halophilic Archaea have been purified to homogeneity using affinity chromatography on Matrex Gel Red A and elution with a combination of substrate (oxaloacetate) and product (coenzyme A). In a number of cases, purification from cell-extract to protein suitable for N-terminal sequencing can be achieved by this single-step procedure. The method is particularly useful in the rapid purification of a thermophilic archaeal citrate synthase from a cloned gene expressed in a mesophilic host.

Detection of a mammalian dna methylase protein family

Purification of a DNA methylase system from nuclei of rat liver cells was performed employing affinity and ion-exchange chromatographies. Through affinity chromatography, the DNA methylase activity was condensed in a single peak(DMA- cellulose) or distributed in two peaks(Matrex Gel Red A,p- hydroxy- mercury- benzoate- agtirose). Through ion-exchange chromatography, the DNA methylase activity was detected in two peaks when usingDEAE- Sepharose eluted with a continuous 0-500 mm NaCl gradient and in four peaks when usingDEAE- Sepharose eluted with 5 mMBuffalyte 4-8{™}(chromatofocusing). The DNA methylase activity in these peaks was due to proteins distinguished by their individual isoelectric points. In SDS-denaturing conditions, the gel electrophoresis of each of these proteins always revealed a band with a molecular weight of approximately 45 KD.

Purification and Characterization of Two Types of Fumarase from Escherichia coli1

Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties.

Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae

Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns. The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a M r of 40,000. The M r of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits. This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate. Feedback inhibition by l-methionine and l-threonine was observed using the purified enzyme. The enzyme was markedly stabilized against heat treatment at high salt concentrations. Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum. The enzyme denatured in 3 m guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity. Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression. Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S. cerevisiae, Escherichia coli, and Bacillus subtilis.

Dye-ligand and immobilized metal ion interaction chromatography for the purification of enzymes of prenyl pyrophosphate metabolism

Dye-ligand and immobilized metal ion interaction chromatography were shown to be efficient techniques for the rapid batchwise fractionation, from crude plant extracts, of a series of enzymes of prenyl pyrophosphate metabolism. Isopentenyl pyrophosphate isomerase, two prenyltransferases, and a number of terpene cyclases (synthases) were readily adsorbed to Matrex Gel Red A (a dimeric triazine dye coupled to crosslinked agarose beads), and desorbed in good yield with relatively high concentrations of KCI and increasing pH. Although all of these enzymes exhibit the common feature of employing a pyrophosphorylated substrate, selective elution could not be achieved with substrate or substrate analogues bearing a pyrophosphate function. Nor could the strong binding of these enzymes to triazine dyes be attributed solely to metal ion interactions or to hydrophobic effects. In a similar way, the isomerase, the prenyltransferases, and all of the terpene cyclases bound to a column of iminodiacetate-immobilized Ni(II) and were desorbed in relatively high fold purity with 15 mM imidazole. Although all of these enzymes bear accessible histidine residues, the interactions with the chelated metal ion were not sufficiently different to permit selective enzyme desorbtion by imidazole gradient elution. However, the use of columns charged with Zn(II) or Co(II) did allow some separation of the different cyclase and transferase types. While empirical in nature, these techniques offer simple, effective, and high-capacity methods for the preliminary concentration and purification of a group of enzymes that utilize prenyl pyrophosphate intermediates of isoprenoid biosynthesis.

Purification of the benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 of Pseudomonas putida MT53 and their preliminary comparison with benzyl alcohol dehydrogenase and benzaldehyde dehydrogenases I and II from Acinetobacter calcoaceticus

Summary: A procedure was developed for purifying both the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 from a single batch of Pseudomonas putida MT53. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernate, followed by chromatography on DEAE-Sephacel, Matrex Gel Red A and Blue Sepharose CL-6B which separated the two enzymes, Phenyl Sepharose CL-4B and Matrex Gel Green A. The final preparations gave single bands on electrophoresis under denaturing and non-denaturing conditions. The subunit Mr values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are 43000 and 56300 respectively. Cross-linking studies with dimethylsuberimidate indicate that both enzymes are probably tetramers, although they run anomalously through gel-filtration columns. The benzyl alcohol dehydrogenase was fairly specific for NAD+ as cofactor but the benzaldehyde dehydrogenase had appreciable activity with NADP+ as well as with NAD+. The optimum pH values are 9.4 and 9.3 for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase respectively. Benzaldehyde dehydrogenase appears to require a monovalent cation for maximum activity. The apparent Km and maximum velocity values of the two plasmid-encoded dehydrogenases and of the chromosomally encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenases I and II from Acinetobacter calcoaceticus were determined for NAD+ and for the unsubstituted substrates and for the monomethyl ring-substituted analogues. The corresponding apparent specificity constants were then calculated. All three benzaldehyde dehydrogenases had very similar substrate profiles, as did the two benzyl alcohol dehydrogenases. The plasmid-encoded benzaldehyde dehydrogenase resembles benzaldehyde dehydrogenase I from A. calcoaceticus (which also requires monovalent cations for activity) in being much more heat-stable than benzaldehyde dehydrogenase II. Overall, the plasmid-encoded benzyl alcohol dehydrogenase from P. putida appears to be remarkably similar to the chromosomally encoded benzyl alcohol dehydrogenase from A. calcoaceticus, and the plasmid-encoded benzaldehyde dehydrogenase is very similar to the two chromosomally encoded benzaldehyde dehydrogenases, particularly benzaldehyde dehydrogenase I.

Purification and properties of two oxidoreductases catalyzing the enantioselective reduction of diacetyl and other diketones from baker's yeast

The NADPH-linked diacetyl reductase system from the cytosolic fraction of Saccharomyces cerevisiae has been resolved into two oxidoreductases catalyzing irreversibly the enantioselective reduction of diacetyl (2,3-butanedione) to (S)- and (R)-acetoin (3-hydroxy-2-butanone) [so-called (S)- and (R)-diacetyl reductases] (EC 1.1.1.5) which have been isolated to apparent electrophoretical purity. The clean-up procedures comprising streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B column chromatography, affinity chromatography on Matrex Gel Red A and Superose 6 prep grade filtration led to 120-fold and 368-fold purifications, respectively. The relative molecular mass of the (R)-diacetyl reductase, estimated by means of HPLC filtration on Zorbax GF 250 and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was 36,000. The (R)-enzyme was most active at pH 6.4 and accepted in addition to diacetyl C5-, C6-2,3-diketones, 1,2-cyclohexanedione, 2-oxo aldehydes and short-chain 2- and 3-oxo esters as substrates. The enzyme was characterized by high enantioselectivity and regiospecificity. The Km values for diacetyl and 2,3-pentanedione were determined as 2.0 mM. The Mr of the (S)-diacetyl reductase was determined as 75,000 by means of HPLC filtration of Zorbax GF 250. The enzyme decomposed into subunits of Mr 48,000 and 24,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The optimum pH was 6.9. The purified (S)-enzyme reduced stereospecifically a broad spectrum of substrates, comprising 2,3-, 2,4- and 2,5-diketones, 2-oxo aldehydes, 1,2-cyclohexanedione and methyl ketones as well as 3-, 4- and 5-oxo esters. The 2,3- and 2,4-diketones are transformed to the corresponding (S)-2-hydroxy ketones; 2,5-hexanedione, however, was reduced to (S,S)-2,5-hexanediol. The Km values for diacetyl and 2,3-pentanedione were estimated as 2.3 and 1.5 mM, respectively. Further characterization of the (S)-diacetyl reductase revealed that it is identical with the so-called '(S)-enzyme', involved in the enantioselective reduction of 3-, 4- and 5-oxo esters in baker's yeast.

Chiral inversion of 2-arylpropionic acid non-steroidal anti-inflammatory drugs—1: In vitro studies of ibuprofen and flurbiprofen

The mechanism of inversion of the enantiomers of 2-arylpropionic acids was investigated in vitro using tissue homogenates. Crude rat liver homogenate was shown to mediate the inversion of R to S-ibuprofen, but not inversion of the S to the R-enanriomer. Inversion required CoA and ATP as cofactors. In contrast, R-ibuprofen was not inverted by homogenates of kidney or small intestine and there was no inversion of the enantiomers of flurbiprofen by any of these tissue homogenates. Longchain acyl-CoA synthetase was partially purified from rat liver microsomes and bound to Matrex Gel Red A. R-Ibuprofen was shown to be a substrate for this enzyme while S-ibuprofen and R and Sflurbiprofen were not substrates. These data are consistent with the hypothesis that the stereospecificity of inversion is controlled by the acyl-CoA synthetase. R-Ibuprofen-CoA did not racemize in either buffer solution (pH 7.4) or human plasma consistent with the hypothesis that racemization of the CoA thioesters is mediated enzymatically.

Biosynthesis of Δ-aminolevulinate in Cyanidium caldarium: Characterization of tRNAGlu, ligase, dehydrogenase and glutamate 1-semialdehyde aminotransferase

The unicellular red algae Cyanidium caldarium converts the intact carbon skeleton of glutamate to δ-aminolevulinate. The components required for this conversion are: tRNAGlu, ligase, dehydrogenase, glutamate 1-semialdehyde aminotransferase, ATP, NADPH and Mg2+. The enzymes and the tRNA involved in δ-aminolevulinate synthesis were isolated from Cyanidium caldarium and characterised. Soluble proteins from greening cells were separated by gel filtration on Sephacryl S-300 and passed sequentially through Blue Sepharose, Matrex gel Red A and chlorophyllin Sepharose. The ligase and the dehydrogenase activities were bound to Blue Sepharose as well as Matrex gel red A. Chlorophyllin Sepharose retained the tRNAGlu, while glutamate 1-semialdehyde aminotransferase passed through the affinity columns and was collected in the run-off protein fraction. The run-off fraction also contained δ-aminolevulinate dehydratase and porphobilinogen deaminase. Glutamate was converted to glutamate 1-semialdehyde in the presence of tRNAGlu, ATP, NADPH and Mg2+, either by the Blue Sepharose-bound protein fraction or the Matrex gel Red A-bound protein fraction. In the presence of the aminotransferase-containing fraction, the glutamate 1-semialdehyde produced was converted into δ-aminolevulinate. Two compounds in addition to glutamate 1-semialdehyde and glutamyl tRNAGlu were produced from glutamate when the Blue Sepharose-bound protein fraction was incubated with tRNAGlu, ATP, NADPH and Mg2+. One of these was converted into δ-aminolevulinate upon incubation with the run-off protein fraction and was assumed to be an additional intermediate in the δ-aminolevulinate biosynthesis pathway. The tRNA preparation from Cyanidium caldarium gave a minor and a major peak of glutamate acceptor activity when analysed by High Pressure Liquid Chromatography on a C18 column containing trioctyl methylammonium chloride. The ability to convert glutamate to glutamate 1-semialdehyde was associated with the major peak of glutamate acceptor activity. Glutamate 1-semialdehyde aminotransferase of Cyanidium caldarium was stimulated by low concentrations (up to 100 μm) of pyridoxal 5′ phosphate and pyridoxamine 5′ phosphate. At higher concentrations (0.5 to 5mm), pyridoxamine 5′ phosphate was further stimulatory while pyridoxal 5′ phosphate was inhibitory. Gabaculine inhibited glutamate 1-semialdehyde aminotransferase and was more effective than the gabaculine-pyridoxal 5′ phosphate adduct, m-carboxyphenyl pyridoxamine 5′ phosphate.

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Purification and preliminary characterization.

A quick, reliable, purification procedure was developed for purifying both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from a single batch of Acinetobacter calcoaceticus N.C.I.B. 8250. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernatant, followed by chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CL-6B and Matrex Gel Red A, and finally gel filtration through a Superose 12 fast-protein-liquid-chromatography column. The enzymes co-purified as far as the Blue Sepharose CL-6B step were separated on the Matrex Gel Red A column. The final preparations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II gave single bands on electrophoresis under non-denaturing conditions or on SDS/polyacrylamide-gel electrophoresis. The enzymes are tetramers, as judged by comparison of their subunit (benzyl alcohol dehydrogenase, 39,700; benzaldehyde dehydrogenase II, 55,000) and native (benzyl alcohol dehydrogenase, 155,000; benzaldehyde dehydrogenase II, 222,500) Mr values, estimated by SDS/polyacrylamide-gel electrophoresis and gel filtration respectively. The optimum pH values for the oxidation reactions were 9.2 for benzyl alcohol dehydrogenase and 9.5 for benzaldehyde dehydrogenase II. The pH optimum for the reduction reaction for benzyl alcohol dehydrogenase was 8.9. The equilibrium constant for oxidation of benzyl alcohol to benzaldehyde by benzyl alcohol dehydrogenase was determined to be 3.08 x 10(-11) M; the ready reversibility of the reaction catalysed by benzyl alcohol dehydrogenase necessitated the development of an assay procedure in which hydrazine was used to trap the benzaldehyde formed by the NAD+-dependent oxidation of benzyl alcohol. The oxidation reaction catalysed by benzaldehyde dehydrogenase II was essentially irreversible. The maximum velocities for the oxidation reactions catalysed by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were 231 and 76 mumol/min per mg of protein respectively; the maximum velocity of the reduction reaction of benzyl alcohol dehydrogenase was 366 mumol/min per mg of protein. The pI values were 5.0 for benzyl alcohol dehydrogenase and 4.6 for benzaldehyde dehydrogenase II. Neither enzyme activity was affected when assayed in the presence of a range of salts. Absorption spectra of the two enzymes showed no evidence that they contain any cofactors such as cytochrome, flavin, or pyrroloquinoline quinone. The kinetic coefficients of the purified enzymes with benzyl alcohol, benzaldehyde, NAD+ and NADH are also presented.

Heparin-agarose chromatography for the purification of tetrahydrofolate utilizing enzymes: C 1-tetrahydrofolate synthase and 10-formyltetrahydrofolate synthetase

Rapid and convenient purification procedures based upon heparin-agarose chromatography for C 1-tetrahydrofolate synthase from Saccharomyces cerevisiae and 10-formyltetrahydrofolate synthetase from Clostridium acidi-urici have been developed. The purification of the yeast enzyme involves three chromatographic steps that can be done rapidly, with no intervening dialyses, and results in high yield. The first step alone, heparin-agarose chromatography, is sufficient to purify the enzyme from yeast bearing a cloned copy of the ADE3 gene that overexpresses the protein. The other steps in the purification from wild-type yeast are matrex gel red A and phenyl-Sepharose chromatography. The purification of the clostridial enzyme involves protamine sulfate fractionation and heparin-agarose chromatography. Heparin-agarose also binds two other enzymes that use tetrahydrofolate, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase. Thus, heparin-agarose should prove useful in purification of a variety of enzymes that utilize tetrahydrofolate or its derivatives as a cofactor.

Butanol-Ethanol Dehydrogenase and Butanol-Ethanol-Isopropanol Dehydrogenase: Different Alcohol Dehydrogenases in Two Strains of Clostridium beijerinckii (Clostridium butylicum)

Alcohol-producing strains of Clostridium beijerinckii (Clostridium butylicum) produce, besides acetone, either n-butanol and ethanol or n-butanol, ethanol, and isopropanol as their characteristic products. Alcohol dehydrogenase has been isolated from a strain (NRRL B593) of C. beijerinckii producing isopropanol and from a strain (NRRL B592) not producing isopropanol. Butanol-ethanol dehydrogenase activities were present in both strains, but isopropanol dehydrogenase activity was present only in the isopropanol-producing strain. The butanol-ethanol dehydrogenase of strain NRRL B592 had M(r) 66,000 and a K(m) of 6 muM for butyraldehyde. In contrast, the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593 had a M(r) 100,000 and K(m)s of 9.5 and 1.0 mM for butyraldehyde and acetone, respectively. In a purification by four different types of separatory methods (DEAE-cellulose, hydroxyapatite, Sephacryl S-300, and Matrex Gel Red A), butanol-ethanol-isopropanol dehydrogenase activities of strain NRRL B593 were purified up to 200-fold (10 to 30% yield), and these activities were not separated. Gel electrophoresis followed by activity stain also revealed distinct mobilities for the butanol-ethanol dehydrogenase of strain NRRL B592 and the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593. In cell extracts from both strains, a higher alcohol dehydrogenase activity was measured with NADP(H) than with NAD(H). The 150- to 200-fold-purified alcohol dehydrogenase from strain NRRL B593 did not show any NAD(H)-linked activities. The K(m) for NADPH was 31 muM (with butyraldehyde as cosubstrate) and 18 muM (with acetone as cosubstrate) for the alcohol dehydrogenase of strain NRRL B593. This study showed that the alcohol dehydrogenases from two strains of C. beijerinckii differed significantly.