Solubilization and characterization of a galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan

Polygalacturonate 4-α-galacturonosyltransferase (PGA-GalAT), the glycosyltransferase that synthesizes the plant cell wall pectic polysaccharide homogalacturonan, has previously been identified and partially characterized in tobacco membranes. Membrane bound PGA-GalAT catalyzes the transfer of galacturonic acid from UDP-galacturonic acid (UDP-GalA) onto an endogenous acceptor to produce polymeric homogalacturonan ( Doong et al. (1995) Plant Physiol. 109, 141 –152). It is shown here that a galacturonosyltransferase is solubilized from tobacco membranes with a HEPES buffer, pH 6.8, containing 40 mM CHAPS and 2 mM EDTA. The solubilized galacturonosyltransferase was identified as putative PGA-GalAT because it transfered [¹⁴C]GalA from UDP-[¹⁴C]GalA onto exogenous homogalacturonan acceptors with degrees of polymerization (DP) of ≥ 10. Maximal solubilized PGA-GalAT activity in the presence of 0.9 μM UDP-[¹⁴C]GalA required approximately 125 μM exogenous homogalacturonan acceptor [e.g. oligogalacturonide (OGA) of DP 15]. Solubilized PGA-GalAT was active over a broad pH range of 6.3–7.8, and had an apparent K_m for UDP-GalA of 37 μM and a V_max of 290 pmol min^–1 mg^–1 protein. Approximately 44% of the PGA-GalAT activity in detergent-dispersed membranes, corresponding to 21% of the PGA-GalAT activity in intact membranes, was solubilized. PGA-GalAT solubilized with 40 mM CHAPS was shown, by exopolygalacturonase treatment in combination with size exclusion and high performance anion exchange chromatographies, to add a single α-1,4-linked galacturonic acid residue onto an OGA exogenous acceptor of DP 15 to yield an OGA product of DP 16. The significance of the apparent lack of processivity of the solubilized PGA-GalAT is discussed.     

Solid-supported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry

Solid-phase biosynthetic reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF), was used to gain insight into the biosynthesis of pectin oligomers. Sepharose supports bearing long pectic oligogalacturonides (OGAs) anchored through a disulfide-containing cleavable linker, were prepared. The OGAs (degrees of polymerization of 13 and 14) were efficiently immobilized through the reducing end via formation of an oxime linkage. These OGA-derivatized matrices were subsequently employed in novel solid-phase enzymatic reactions, with the pectin biosynthetic enzyme, alpha-1,4-galacturonosyltransferase, GalAT (solubilized from Arabidopsis thaliana) and the glycosyl donor, uridine diphosphate-galacturonic acid (UDP-GalA). Solid-supported biosynthesis was followed by cleavage of the immobilized OGAs and direct analysis of the products released into the liquid phases by MALDI-TOF mass spectrometry. In time course studies conducted with an immobilized (alpha-D-GalA)14 and limiting amounts of the glycosyl donor, the predominant product was an OGA extended by one GalA residue at the non-reducing end (i.e., (GalA)15). When UDP-GalA was added in approximately excess compared to immobilized (GalA)13, OGAs up to the 16-mer were synthesized, confirming the non-processivity of the GalAT in vitro.     

A facile enzymatic synthesis of uridine diphospho-[14C]galacturonic acid

Galacturonic acid (GalA) is a major component of plant cell-wall-derived pectins. It can be also found in the cell-surface polysaccharides of different microorganisms, including several symbiotic and pathogenic bacteria. Uridine diphosphogalacturonic acid (UDP-GalA) is a likely donor for GalA during the biosynthesis of these polysaccharides. A highly efficient, yet simple, method is presented for generating and purifying UDP-[14C]GalA. Commercially available UDP-[14C]-galactose was quantitatively oxidized (>95% conversion) to UDP-[14C]GalA in the presence of high levels of galactose oxidase and catalase, at prolonged incubation times. Following this one-step enzymatic oxidation, UDP-[14C]GalA was purified using a polyethyleneimine cellulose column with a single-step 1 M NaCl elution. The authenticity of the purified UDP-[14C]GalA was verified by its relative mobility on thin-layer chromatograms, analysis of its chemical hydrolysis products, and 1H NMR spectroscopy. Our yield of >90% is much higher than by previously described methods. The method may serve as a prototype for the preparation of other radiolabeled uronic acids and their nucleotide derivatives.     

A sensitive and rapid bioassay of homogalacturonan synthase using 2-aminobenzamide-labeled oligogalacturonides

Polygalacturonate 4-alpha-galacturonosyltransferase (GalA T) activity was detected in the microsomal fraction isolated from pumpkin (Cucurbia moschata Duchesne, cv. Tokyou-Kabocha) seedlings using UDP-GalA and 2-aminobenzamide (2AB)-labeled oligogalacturonides. A 2AB-labeled undecagalacturonide was elongated by the attachment of galacturonic acid (GalA) residues to give 2AB-labeled oligogalacturonides with a degree of polymerization (DP) between 12 and 17. Exogenous 2AB-labeled oligogalacturonide acceptors with a DP >3 are effective acceptor molecules for pumpkin GalA T.     

A second galacturonic acid transferase is required for core lipopolysaccharide biosynthesis and complete capsule association with the cell surface in Klebsiella pneumoniae

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae contains two galacturonic acid (GalA) residues, but only one GalA transferase (WabG) has been identified. Data from chemical and structural analysis of LPS isolated from a wabO mutant show the absence of the inner core beta-GalA residue linked to L-glycero-D-manno-heptose III (L,D-Hep III). An in vitro assay demonstrates that the purified WabO is able to catalyze the transfer of GalA from UDP-GalA to the acceptor LPS isolated from the wabO mutant, but not to LPS isolated from waaQ mutant (deficient in l,d-Hep III). The absence of this inner core beta-GalA residue results in a decrease in virulence in a capsule-dependent experimental mouse pneumonia model. In addition, this mutation leads to a strong reduction in cell-bound capsule. Interestingly, a K66 Klebsiella strain (natural isolate) without a functional wabO gene shows reduced levels of cell-bound capsule in comparison to those of other K66 strains. Thus, the WabO enzyme plays an important role in core LPS biosynthesis and determines the level of cell-bound capsule in Klebsiella pneumoniae.     

A protein-sialyl polymer complex involved in colominic acid biosynthesis. Effect of tunicamycin.

A protein-NeuAc complex involved in colominic acid biosynthesis has been identified in membrane preparations of Escherichia coli K-235. This compound had an Mr (estimated by SDS/polyacrylamide-gel electrophoresis and autoradiography) of about 100,000 and played the role of an 'initiator' or 'primer' (endogenous acceptor) in the synthesis of the whole polymer. Incubations of E. coli membranes with CMP-[14C]NeuAc (CMP-N-[14C]acetylneuraminic acid) pointed to the existence of a protein fraction (primer acceptor) that linked residues of sialic acid (N-acetylneuraminic acid, NeuAc) up to a maximal size, later releasing them as low-Mr sialyl polymers (LMrS, Mr less than 10,000). In the presence of colominic acid (final acceptor) the radioactivity linked to the protein quickly decreased, appearing stoichiometrically bound to the whole polysaccharide. When membrane preparations were previously digested with Streptomyces proteinase or de-activated by heating (80 degrees C, 10 min), no incorporation of labelled NeuAc into trichloroacetic acid-insoluble material was detected. These results suggested that colominic acid molecules are synthesized while they are bound to a proteinaceous acceptor that is subsequently excised in the presence of colominic acid, generating the native protein. The antibiotic tunicamycin inhibited the biosynthesis of colominic acid, affecting the synthesis of this protein-(NeuAc)n intermediate. All these results are described here for the first time.     

Pectin biosynthesis: a solubilized α1,4-galacturonosyltransferase from tobacco catalyzes the transfer of galacturonic acid from UDP-galacturonic acid onto the non-reducing end of homogalacturonan

A solubilized α1,4-galacturonosyltransferase (GalAT) from tobacco transfers galacturonic acid (GalA) residues from UDP-GalA onto oligogalacturonide (OGA) exogenous acceptors with degrees of polymerization greater than nine (R.L. Doong and D. Mohnen 1998, Plant J 13: 363–374). The solubilized GalAT has been identified as putative polygalacturonate 4-α-galacturonosyltransferase (PGA-GalAT, EC 2.4.1.43) based on its α1,4-galacturonosyltransferase activity and similar K _m for UDP-GalA, pH optimum and V _max to those of membrane-bound PGA-GalAT (R.L. Doong et al., 1995, Plant Physiol 109: 141–152). The direction of elongation of homogalacturonan catalyzed by solubilized GalAT from microsomes of tobacco (Nicotiana tabacum L. cv. Samsun) cell suspensions has now been determined. Three different types of exogenous acceptor were used to study the direction of synthesis of homogalacturonan: unmodified OGAs, OGAs derivatized by biotinylation at the reducing end, and OGAs containing a 4,5-unsaturated GalA at the non-reducing end. The unmodified OGAs and the OGAs modified at the reducing end functioned equally well as acceptors in the galacturonosyltransferase reaction. In contrast, OGAs with the 4,5-unsaturated residue at the non-reducing end were not acceptors for homogalacturonan biosynthesis. These results show that homogalacturonan biosynthesis by solubilized GalAT occurs via the addition of GalA to the non-reducing end of the polymer chain. Received: 18 June 1998 / Accepted: 22 August 1998     

Acid phosphatases in the frog (Rana esculenta) skeletal muscle. Purification and some properties of the low molecular weight enzyme.

1. The presence of high-Mr and low-Mr acid phosphatases [orthophosphoric-monoester phosphohydrolase, (acid optimum), EC 3.1.3.2] in the skeletal muscle of frog Rana esculenta was reported. 2. The subcellular localization and some characteristics of both enzymes were also described. 3. The low-Mr AcPase was purified to homogeneity. The enzyme did not absorb on Concanavalin A-Sepharose 4B indicating that this was not a glycoprotein. 4. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr 13.7 +/- 0.8 kDa in the presence of sodium dodecyl sulphate. 5. The Mr of the native enzyme was 14.0 +/- 1.1 kDa as determined by gel filtration on a Sephadex G-100 column. The isoelectric point was 6.02. 6. The enzyme was strongly inhibited by 1 mM Ag+, Hg2+, Sn2+ and Cu2+ while other cations both at 10(-2) and 10(-3) M showed little or no effect. 7. The enzyme was insensitive to NaF and tartrate but was strongly deactivated by formaldehyde, PMB, Iodoacetamide and Triton X-100. Phosphate was a competitive inhibitor (k1 = 0.83 mM). 8. The best substrate for the enzyme was p-nitrophenylphosphate but phenylphosphate, flavin mononucleotide and o-P-tyrosine were also hydrolyzed, though at different rates. 9. The enzyme activity was enhanced in the presence of methanol, ethanol, acetone and glycerol indicating a phosphotransferase activity.     

Biochemical characterization of an extracellular polygalacturonase from Trichoderma harzianum

An extracellular polygalacturonase (PGII) from Trichoderma harzianum was purified to homogeneity by two chromatography steps using DEAE-Sepharose and Sephacryl S-200. The molecular weight of T. harzianum PGII was 31,000 Da by gel filtration and SDS-PAGE. PGII had isoelectric point of 4.5 and optimum pH of 5.0. PGII was very stable at the pH 5.0. The extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (DE). PGII had very low activity toward non-pectic polysaccharides. The apparent K(m) value and K(cat) value for hydrolyzing polygalacturonic acid (PGA) were 3.4 mg/ml and 592 s(-1), respectively. PGII was found to have temperature optimum at 40 degrees C and was approximately stable up to 30 degrees C for 60 min of incubation. All the examined metal cations showed inhibitory effects on the enzyme activity. A 1,10-phenanthroline, Tween 20, Tween 80, Triton X-100 and SDS had no effect on the enzyme activity. The rate of enzyme catalyzed reduction of viscosity of solutions of PGA or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. The storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to 1 year. These properties of T. harzianum PGII with appreciable activity would be potentially novel source of enzyme for food processing.     

The purification and some equilibrium properties of the nitrite reductase of the bacterium Wolinella succinogenes.

The bacterium Wolinella succinogenes produces a nitrite reductase enzyme that can be purified to homogeneity in high yield by a combination of detergent extraction, hydroxyapatite chromatography and Mr fractionation. Nitrite reductase activity is found to be present in both a high- and a low-Mr fraction. The high-Mr fraction has been shown to consist of the low-Mr nitrite reductase enzyme associated with a hydrophobic 'binding protein'. The amino acid composition for both proteins is reported. The nitrite reductase enzyme shows spectral characteristics indicative of the presence of c-type haem groups. Measurements at 610 nm indicate the presence of some high-spin haem groups at neutral pH. This haem subgroup undergoes a pH-linked high-spin - low-spin transition at alkaline pH. Approximately two of the six haem groups present within the enzyme bind CO with low affinity (KD = 0.4 mM). The enzyme also shows a range of redox activities with various inorganic reagents. The enzyme has been shown to exhibit dithionite reductase, oxygen reductase and CO2 reductase activities.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Candida L-norleucine,leucine:2-oxoglutarate aminotransferase. Purification and properties

A new enzyme which catalyzes the transamination of L-norleucine (2-aminohexanoic acid) and L-leucine with 2-oxoglutarate was purified to homogeneity from cells of Candida guilliermondii var. membranaefaciens. The relative molecular mass determined by gel filtration was estimated to be close to 100,000. The transaminase behaved as a dimer which consists of two subunits identical in molecular mass (Mr 51,000). The enzyme has a maximum activity in the pH range of 8.0-8.5 and at 55 degrees C. 2-Oxoglutarate, and to a lesser extent pyridoxal 5'-phosphate, were effective protecting agents against increasing temperature. The enzyme exhibits absorption maximum at 330 nm and 410 nm. L-Norleucine, and L-leucine to a lesser extent, are the best amino donors with 2-oxoglutarate as amino acceptor. The Km values for L-norleucine, L-leucine and 2-oxoglutarate determined from the Lineweaver-Burk plot were 1.8 mM, 6.6 mM and 2.0 mM respectively. A ping-pong bi-bi mechanism of inhibition with alternative substrates is found when the enzyme is in the presence of both L-norleucine and L-leucine. The inhibitory effect of various amino acid analogs on the transamination reaction between L-norleucine and 2-oxoglutarate was studied and Ki values were determined.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Purification, characterization and reaction mechanism of novel arylsulfotransferase obtained from an anaerobic bacterium of human intestine

A novel type of arylsulfotransferase was purified from Eubacterium A-44, one of the predominant bacteria of human intestine. The enzyme (Mr 315 000) was composed of four identical subunits (Mr 80 000) whose N-terminal amino acids were arginine. pI and optimal pH of the enzyme were 3.9 and 8-9, respectively. The apparent Km for p-nitrophenylsulfate using tyramine as an acceptor substrate and that for tyramine using p-nitrophenylsulfate as a donor substrate were determined to be 0.104 mM and 3.5 mM, respectively. The reaction mechanism of the enzyme was proposed as follows: a donor substrate, p-nitrophenyl [35S]sulfate, combines a histidine residue of the enzyme active site with concomitant release of a phenolic compound, p-nitrophenol. The sulfate group of the histidine residue transfers to a tyrosine group, and then to an acceptor with the binding of another donor to the histidine residue.     

Glucuronic acid directly linked to galacturonic acid in the rhamnogalacturonan backbone of beet pectins.

Sugar-beet pulp was de-esterified and submitted to 72 h hydrolysis by 0.1 M HCl at 80 degrees C. Oligomers containing a single glucuronic acid (GlcA) moiety in addition to n(>/= 2) repeats of the dimer -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1--> were isolated from the hydrolysate by ion-exchange and gel-permeation. Glycosyl linkage composition analysis and 1H NMR studies indicated that the GlcA was attached to O-3 of a galacturonic acid (GalA) residue, as shown for the two pentamers beta-D-GlcpA-(1-->3)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA-(1-->2)-L-Rhap and alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-[beta-D-GlcpA-(1-->3)]-alpha-D-GalpA-(1-->2)-L-Rhap. Substitution by GlcA was estimated as occurring on one GalA residue out of 72 in the rhamnogalacturonan fraction of the backbone of beet pectins.     

Penicillium chrysogenum extracellular acid phosphatase: purification and biochemical characterization

An extracellular acid phosphatase (EC 3.1.3.2) from crude culture filtrate of Penicillium chrysogenum was purified to homogeneity using high-performance ion-exchange chromatography and size-exclusion chromatography. SDS-PAGE of the purified enzyme exhibited a single stained band at an Mr of approx. 57,000. The mobility of the native enzyme indicated the Mr to be 50,000, implying that the active form is a monomer. The isoelectric point of the enzyme was estimated to be 6.2 by isoelectric focusing. Like acid phosphatases from several yeasts and fungi the Penicillium enzyme was a glycoprotein. Removal of carbohydrate resulted in a protein band with an Mr of 50,000 as estimated by SDS-PAGE, suggesting that 12% of the mass of the enzyme was carbohydrate. The enzyme was catalytically active at temperatures ranging from 20 degrees C to 65 degrees C with a maximum activity at 60 degrees C and the pH optimum was at 5.5. The Michaelis constant of the enzyme for p-nitrophenyl phosphate was 0.11 mM and it was inhibited competitively by inorganic phosphate (ki = 0.42 mM).     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.     

Characterization of pectin methyltransferase from soybean hypocotyls

Pectin methyltransferase (PMT) catalyzing the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the C-6 carboxyl group of galactosyluronic acid residues in pectin was found in a membrane preparation of etiolated hypocotyls from 6-d-old soybean (Glycinemax Merr.). The enzyme was maximally active at pH 6.8 and 35–40 °C, and required 0.5% (w/v) Triton X-100. The incorporation of the methyl group was significantly enhanced by addition of a pectin with a low (22%) degree of methyl-esterification (DE) as exogenous acceptor substrate. The apparent Michaelis constants for SAM and the pectin (DE22) were 0.23 mM and 66 μg · ml⁻¹, respectively. Attachment of the methyl group to the carboxyl group of the pectin via ester linkage was confirmed by analyzing radiolabeled product from incubation of the enzyme with [¹⁴C]methyl SAM and the acceptor pectin. Size-exclusion chromatography showed that both enzymatic hydrolysis with a pectin methylesterase and a mild alkali treatment (saponification) led to the release of radioactive methanol from the product. Enzymatic hydrolysis of the product with an endopolygalacturonase degraded it into small pectic fragments with low relative molecular mass, which also supports the idea that the methyl group is incorporated into the pectin. The soybean hypocotyls were fractionated into their cell wall components by successive extraction with water, EDTA, and alkali treatment. Among the resulting polysaccharide fractions, high PMT activity was observed when a de-esterified polysaccharide derived from the EDTA-soluble fraction (the pectic fraction) was added as an alternative acceptor substrate, indicating that the enzyme may be responsible for producing methyl-esterified pectin in vivo. Received: 10 September 1999 / Accepted: 11 October 1999     

Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species.

Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate. The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.     

Characterization of aspartate aminotransferase from the cyanobacterium Phormidium lapideum

Aspartate aminotransferase (AspAT) was purified to homogeneity from cell extracts of the non-N2-fixing cyanobacterium Phormidium lapideum. The NH2-terminal sequence of 25 amino acid residues was different from the sequences of the subfamily Ialpha of AspATs from eukaryotes and Escherichia coli, but it was similar to sequences of the subfamily Igamma of AspATs from archaebacteria and eubacteria. The enzyme was most active at 80 degrees C and was stable at up to 75 degrees C. Thermal inactivation (60-85 degrees C) of the enzyme followed first-order kinetics, with 2-oxoglutarate causing a shift of the thermal inactivation curves to higher temperatures. However, at 25 degrees C the kcat of P. lapideum AspAT was nearly equal to the values of AspATs from mesophilic organisms. The enzyme used L-aspartate and L-cysteine sulfinate as amino donors and 2-oxoglutarate as an amino acceptor. The Km values were 5.0 mM for L-aspartate, 5.7 mM for L-glutamate, 0.2 mM for 2-oxoglutarate, and 0.032 mM for oxaloacetate.