Mol Min Research Articles

Chiral Recognition Of Alcohol Enantiomers In Acyl Transfer Reactions Catalysed By Candida Antarctica Lipase B

A description of the substrate-enzyme interactions involved in the discrimination of see-alcohol enantiomers in acyl transfer reactions catalysed by the highly enantioselective Candida antarctica lipase B is presented. Experimentally found activities and enantioselectivities from kinetic resolutions of a series of secondary alcohol substrates were used together with molecular modelling for the elucidation of the stereoselective substrate-enzyme interactions. Matching experimental and calculated results allowed conclusions regarding the orientation of the tetra-hedral intermediates in the active site. The finding, valid for substrates of a specific activity above 1 mol min-1 mg-1 protein, describes the origin of enantioselectivity as a combination of a binding site of limited size, the ”stereospecificity pocket“, and principally different productive orientations of the two enantiomers.

Production of ? -galactosidase from thermophilic fungus Rhizomucor sp

A thermostable β-galactosidase was produced extracellularly by a thermophilic Rhizomucor sp, with maximum enzyme activity (0.21 U mg−1) after 4 days under submerged fermentation condition (SmF). Solid state fermentation (SSF) resulted in a nine-fold increase in enzyme activity (2.04 U mg−1). The temperature range for production of the enzyme was 38–55°C with maximum activity at 45°C. The optimum pH and temperature for the partially purified enzyme was 4.5 and 60°C, respectively. The enzyme retained its original activity on incubation at 60°C up to 1 h. Divalent cations like Co2+, Mn2+, Fe2+ and Zn2+ had strong inhibitory effects on the enzyme activity. The K m and V max for p-nitrophenyl-β- D-galactopyranoside and o-nitrophenyl-β - D-galactopyranoside were 0.39 mM, 0.785 mM and 232.1 mmol min−1 mg−1 respectively. The K m and V max for the natural substrate lactose were 66.66 μM and 0.20 μ mol min−1 mg−1.

Distribution of Folate Derivatives and Enzymes for Synthesis of 10-Formyltetrahydrofolate in Cytosolic and Mitochondrial Fractions of Pea Leaves.

Leaf extracts of 14-d-old pea (Pisum sativum L. cv Homesteader) seedlings were examined for folate derivatives and for 10-formyltetrahydrofolate synthetase (SYN), 5,10-methenyltetrahydrofolate cyclohydrolase (CYC), and 5,10-methylenetetrahydrofolate dehydrogenase (DHY) activities. Microbiological and enzyme assays showed that leaf folates SYN, CYC, and DHY were predominantly cytosolic. Extracts of Percoll gradient-purified mitochondria contained less than 1% of total leaf folate and less that 1% of each enzyme activity. Fractionation of whole-leaf homogenates resulted in the copurification of DHY and CYC (subunit 38 kD) and the isolation of a SYN protein (subunit 66 kD). Polyclonal antibodies were raised against purified cytosolic DHY-CYC (DHY-CYC-Ab) and cytosolic SYN (SYN-Ab), respectively. Immunoblots showed that DHY-CYC-Ab cross-reacted with a mitochondrial protein band (38 kD). Two mitochondrial protein bands (subunit Mr = 40,000 and 44,000) cross-reacted with SYN-Ab. Immunoaffinity chromatography (DHY-CYC-Ab as the immobile ligand) indicated that the bulk of mitochondrial SYN activity was not associated with mitochondrial DHY or CYC. When 9-d-old etiolated pea seedlings were exposed to light for up to 3 d, the specific enzyme activities of DHY-CYC in whole-leaf extracts rose 2-fold and more DHY-CYC-Ab cross-reacting protein was detected. In contrast, the specific activity of SYN fell from 5 to 1 [mu]mol min-1 mg-1 protein and less SYN-Ab cross-reacting protein was detected. The data suggest that in pea leaves, the bulk of one-carbon-substituted tetrahydrofolates and enzymes for the generation of 10-formyltetrahydrofolate are extra-mitochondrial.

Purification and characterization of a novel class III peroxidase isoenzyme from tea leaves.

A novel, basic (isoelectric point > 10), heme peroxidase isoenzyme (TP; relative molecular weight = 34,660 +/- 10, mean +/- SE) that can account for a significant part of the ascorbate peroxidase activity in tea (Camellia sinensis) leaves has been purified to homogeneity. The ultraviolet/visible absorption spectrum is typical of heme-containing plant peroxidases, with a Soret peak at 406 nm (epsilon = 115 mM-1 cm-1) and an A406/A280 value of 3.4. The enzyme has a high specific activity for ascorbate oxidation (151 mumol min-1 mg-1), with a pH optimum in the range of 4.5 to 5.0. Substrate-specificity studies have revealed significant differences between TP and other class III peroxidases, as well as similarities with class I ascorbate peroxidases. TP, like ascorbate peroxidase, exhibits a preference for ascorbate over guaiacol, whereas other class III isoenzymes are characterized by 2-orders-of-magnitude higher activity for guaiacol than for ascorbate. TP also forms an unstable porphyrin pi cation radical-type compound I, which is converted to compound II within approximately 2 min in the absence of added reductant. Amino acid sequence data show TP to be the first example, to our knowledge, of a class III peroxidase with a high specificity for ascorbate as an electron donor.

Local myocardial glucose uptake is proportional to, but not dependent on blood flow.

In nonischaemic myocardium local deoxyglucose deposition is proportional to local blood flow (y = 0.77x + 0.25 for normalized deoxyglucose deposition and blood flow). The cause for this relationship was evaluated using a mathematical model of (deoxy)glucose metabolism to elucidate whether differences in local deoxyglucose deposition are dependent on local blood flow or whether they reflect differences of the local metabolic rate. The axially distributed model consists of two blood-tissue exchange regions arranged concentrically representing the capillary and the extracapillary regions, the latter being a composite of the endothelial, interstitial and parenchymal cell regions. Exchange between the two model regions is described by a permeability-surface area product, consumption in the extracapillary region is modelled by an intraregional clearance term. Twenty blood-tissue exchange units are arranged as parallel pathways to account for the effects of flow heterogeneity and in series with a single, nonexchanging, large vessel segment (0.1 ml/g). Regional volumes (capillary 0.07 ml/g, extracapillary 0.60 ml/g) and the permeability-surface area product (0.2 ml min-1 g-1) are taken from published sources, the local myocardial blood flow is that determined experimentally (between 0.1 and 2.5 ml min-1 g-1). The only free parameter in the model is the extracapillary clearance term (0.0275-0.102 ml min-1 g-1) which was used to fit the measured tissue radioactivity concentration taking the measured arterial concentration of 3H-deoxyglucose into account. The results obtained indicate that variations in local myocardial blood flow, and hence differences in deoxyglucose supply, can not explain quantitatively the differences in local deoxyglucose deposition. However, differences of the metabolic rate (0.326 versus 0.120 micro;mol min-1 g-1) assumed to occur in parallel to the flow differences (1.26 versus 0.42 ml min-1 g-1) can well explain the different mean deoxyglucose deposition in high and low flow areas. This result supports the view that blood flow heterogeneity in the heart is paralleled by a spatial heterogeneity of glucose metabolism.

HS−:O2Oxidoreductase Activity of Cu,Zn Superoxide Dismutase

Cu,Zn-Superoxide dismutase (SOD, EC 1.15.1.1) catalyzes HS−oxidation according to the reaction: HS−+ H++ O2→ S0+ H2O2. At pH 7.8, 20°C, 280 μMO2, and 100 μMHS−, the rate of HS−oxidation is 0.2 mol min−1(mol SOD)−1. The affinities of the enzyme for HS−and O2are consistent with previous measurements for these substrates in connection with other enzymic reactions. The rate of HS−oxidation by SOD is slower than that provided by other cellular mechanisms, and it is unlikely to be of physiological significance. However, using ≤1 μMSOD the rate of HS−oxidation gradually accelerates so that after 1 h it can be 600 times faster than the initial rate. This is caused by a nonenzymic oxidative chain reaction that is not self-sustaining, but requires “seeding” from the enzymic reaction.

Purification and characterization of the reconstitutively active adenine nucleotide carrier from maize mitochondria.

The adenine nucleotide carrier from maize (Zea mays L. cv B 73) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. Sodium dodecyl sulfate-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 32 kD. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5'-phosphate-sensitive ATP/ATP exchange. It was purified 168-fold with a recovery of 60% and a protein yield of 0.25% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ADP, ATP, GDP, and GTP, and was inhibited by atractyloside, bongkrekate, phenylisothiocianate, pyridoxal 5'-phosphate, and mersalyl (but not N-ethylmaleimide). Maximum initial velocity of the reconstituted ATP/ATP exchange was determined to be 2.2 mumol min-1 mg-1 protein at 25 degrees C. The half-saturation constants and the corresponding inhibition constants were 17 microM for ATP, 26 microM for ADP, 59 microM for GTP, and 125 microM for GDP. The activation energy of the ATP/ATP exchange was 48 kilojoule/mol between 0 and 15 degrees C, and 22 kilojoule/mol between 15 and 35 degrees C. Partial amino acid sequences showed that the purified protein was the product of the ANT-G1 gene sequenced previously (B. Bathgate, A. Baker, C.J. Leaver [1989] Eur J Biochem 183: 303-310).

Synthesis of Na + superionic conductor in the Na 2O-Y 2O 3-P 2O 5-SiO 2 system by the gelation induced by moist air or N 2

Sodium superionic conductors in the system Na 2O-Y 2O 3-P 2O 5-SiO 2 were prepared by sol-gel method: humidity-controlled air or N 2 was passed into the reaction mixture to avoid precipitates such as nitrate and carbonate. Highly homogeneous xerogel was obtained by supplying water at a rate of about 1.8 × 10 −4 mol min −1. The sintered samples of the xerogels with the composition between x = 5.33 and 5.60 in Na x Y 0.8P 0.4Si 3.6O 12.0 showed a single crystal phase with the superionic conduction. The sample with the composition of x = 5.33 showed highly ionic conductivity of ϱ 300 = 13.9 Ω cm at 300 °C.

Envelope Membranes from Spinach Chloroplasts Are a Site of Metabolism of Fatty Acid Hydroperoxides.

Enzymes in envelope membranes from spinach (Spinacia oleracea L.) chloroplasts were found to catalyze the rapid breakdown of fatty acid hydroperoxides. In contrast, no such activities were detected in the stroma or in thylakoids. In preparations of envelope membranes, 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid, 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, or 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid were transformed at almost the same rates (1-2 [mu]mol min-1 mg-1 protein). The products formed were separated by reversed-phase high-pressure liquid chromatography and further characterized by gas chromatography-mass spectrometry. Fatty acid hydroperoxides were cleaved (a) into aldehydes and oxoacid fragments, corresponding to the functioning of a hydroperoxide lyase, (b) into ketols that were spontaneously formed from allene oxide synthesized by a hydroperoxide dehydratase, (c) into hydroxy compounds synthesized enzymatically by a system that has not yet been characterized, and (d) into oxoenes resulting from the hydroperoxidase activity of a lipoxygenase. Chloroplast envelope membranes therefore contain a whole set of enzymes that catalyze the synthesis of a variety of fatty acid derivatives, some of which may act as regulatory molecules. The results presented demonstrate a new role for the plastid envelope within the plant cell.

St-PepA, a Streptococcus thermophilus aminopeptidase with high specificity for acidic residues

The proteolytic system of lactic acid bacteria has been extensively studied over the past 10 years and peptidases from lactococci are now well known. The situation is, however, different for Streptococcus thermophilus from which only a few peptidases have been purified and characterized. The present work was conducted to characterize an aminopeptidase of S. thermophilus CNRZ 302, called St-PepA, with high specificity for acidic amino acids. St-PepA was purified by a three-step procedure from a spheroblast extract of S. thermophilus CNRZ 302. Its molecular mass was estimated to be 360 kDa by gel filtration and 45 kDa by SDS-PAGE, indicating that it had an octameric structure. Its activity against aspartate-p-nitroanilide was maximal at pH 8.5 and 62 C and highly enhanced by Zn2+, Co2+ and Mg2+. St-PepA was inhibited by metal-chelating reagents and, to a lesser extent, by agents modifying sulfhydryl groups. It showed an activity towards p-nitroanilide derivatives, di-and tripeptides, and larger peptides such as fragment 43-58 of as1-casein. It had a high substrate specificity towards N-terminal acidic amino acid residues but it could also release serine and malic acid, the a-hydroxy acid homologue of aspartic acid. Kinetic studies revealed that the affinity of St-PepA was more than 18-fold higher for aspartic acid-p-nitroanilide (K m = 0.42 mM) than for glutamic acid-p-nitroanilide (K m = 7.65 mM) with a similar V max for both substrates [about 40 mol min-1 (mg enzyme)-1]. St-PepA is heat stable, with a maximal loss of activity of 15% after incubation for 120 min at 50 C and 60C. It is likely to be involved in the nitrogen metabolism of S. thermophilus and in the development of the organoleptic characteristics of cheese and yoghurt.

Interaction between primary and secondary metabolism in Streptomyces coelicolor A3(2): role of pyrroline-5-carboxylate dehydrogenase

The activity of the proline catabolic enzyme pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) was induced up to three-hundred-fold by the addition of three hundred proline to the growth medium of the Gram-positive bacterium Streptomyces coelicolor A3(2). Rifampicin, an inhibitor of RNA polymerase activity, abolished induction, implying that regulation was at the level of activation of gene transcription. The enzyme was purified and SDS-PAGE of the highly purified enzyme preparation revealed a single subunit with M(r) 68,000. A single band of protein, which also stained for enzyme activity, was observed after native gel electrophoresis. The M(r) of the enzyme was estimated to be approximately 265,000 by native gel electrophoresis and approximately 305,000 by gel filtration, which indicated that the enzyme had a tetrameric quaternary structure. The apparent Km for pyrroline-5-carboxylate was 109 +/- 7.3 microM, whilst that for NAD+ was 43.3 +/- 2.5 microM. Product inhibition by NADH (apparent Ki 0.6mM) was observed. The observed Vmax was 22.0 +/- 1 mol min-1 (mg protein)-1. Neither 1 nor 5 mM proline had any effect on enzyme activity, whilst glutamate was a very weak inhibitor.

Estimation of L-glutaminase and L-asparaginase activities in soils by the indophenol method

Abstract A simple and rapid method was developed for determining L-glutaminase and L-asparaginase activities in soils. It involved colorimetric estimation of NH4 + released by the activity of L-glutaminase and L-asparaginase when soil was incubated with 0.25 M L-glutamine or L-asparagine in 0.1 M phosphate buffer (pH 7.6) and toluene used as a germicide. The indophenol method was applied to the assay of NH4 + for soil L-glutaminase and L-asparaginase activities. Color development in the indophenol method required one hour. The color development was stable for 2 d and was not appreciably affected by the presence of L-glutamine, L-glutamic acid, L-asparagine, and L-aspartic acid. In addition, no turbidity nor precipitate appeared in the presence of a high concentration (10 μg) of NH4 +-N. The L-glutaminase activity was estimated in three soil samples differing in pH, organic-C content, and land use. The L-glutaminase activity in the three soils ranged from 28 to 1,470 mU (10-9 mol min-1 g-1 of dry soil at 3...

Thermochemical study of methabenzthiazuron

A calorimetric study of methabenzthiazuron (MBT) has provided evidence of chemical transformation in the solid state at room temperature. Traces of water vapour were absorbed to form a hydrate, which produces a binary eutectic system with MBT at a temperature of 380.3 K and an estimated mole fraction of MBT of x A = 0.78. The mean value of the hydration rate of MBT at room temperature is V ̄ A → B = 3.7 × 10 −5 mol% min −1 . The mean value of the thermal decomposition rate of MBT at the melting temperature T ° m = 393.5 K is V ̄ A = 0.01% mol min −1 . The highest rate of decomposition, V ̄ E = 0.14% mol min −1 , was observed at the eutectic temperature.

Structure and Function of the Golgi Complex in Rice Cells (II. Purification and Characterization of Golgi Membrane-Bound Nucleoside Diphosphatase).

Inosine diphosphatase bound to Golgi membranes was studied in rice (Oryza sativa L. cv Nipponkai) cells. The enzyme was solubilized with Triton X-100 from isolated rice Golgi membranes and was highly purified employing a series of chromatography steps in the presence of 20% glycerol and 0.1% Triton X-100. The apparent molecular mass of the enzyme was estimated by gel filtration column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 200 and 55 kD, respectively. The isoelectric point of the enzyme was determined to be 7.5. The optimal pH for the enzyme activity was around 7 and the enzyme required Mg2+ for hydrolyzing activity. IDP, UDP, and GDP were effective substrate for the purified rice Golgi membrane-bound inosine diphosphatase, whereas activity with ADP, CDP, and thymidine 5[prime]-diphosphate was 10 to 20% of IDP. The Km values for IDP, UDP, and GDP were 0.48, 0.50, and 0.67 mM, respectively, and Vmax values were 1.85, 1.54, and 1.67 [mu]mol min-1 mg-1, respectively. These results indicate that the rice Golgi enzyme is a nucleoside diphosphatase that is specific for IDP, UDP, and GDP. Furthermore, this rice Golgi nucleoside diphosphatase stimulated the activity of glucan synthase I also localized in rice Golgi membranes. The results strongly support the view that this nucleoside diphosphatase is involved in regulation of [beta]-glucan synthesis in the plant Golgi complex.

TOL plasmid-specified xylene oxygenase is a wide substrate range monooxygenase capable of olefin epoxidation

Xylene oxygenase, which is encoded on the TOL plasmid pWWO of Pseudomonas putida mt-2, is a key enzyme system in the degradation of toluene and xylenes by this organism. It was expressed in an Escherichia coli recombinant strain carrying the xylMA structural genes. This recombinant, which expressed xylene oxygenase from the heat-shock induced lambda P L promoter, was analyzed for its potential as a biocatalytic tool so as to effect the oxidation of side chains of aromatic hydrocarbons to the corresponding alcohols. Compounds that were tested as potential substrates carried different substituents on the aromatic ring at ortho, meta, and para positions, relative to the methyl moiety. Products that accumulated after administration of the aromatic hydrocarbons to concentrated suspensions of the recombinant were identified by gas chomatography and mas spectrometry. Toluene derivatives with ortho substituents could not serve as substrates for the biocatalyst, whereas a number of meta- or para-substituted analogs were efficiently oxidized to the corresponding benzylalcohols. Bioconversion of the substrates by resting cells varied from 3 μ mol min −1 g −1 cell dry weight for 1,3,5-trimethylbenzene to 18 μ mol min −1 g −1 cell dry weight for meta-xylene. Whole cells that expressed xylene oxygenase did catalyze the oxidation of the methyl substituent attached to a benzene ring, but no conversion of n-alkylbenzene derivatives with longer side chains was observed. Although the ethyl group of ethylbenzene could not be converted by the biocatalyst, cells containing xylene oxygenase were capable of oxidizing the ethylene side group of styrene to produce styrene epoxide.

Organic Anion Transport and Reabsorptive Fluid Flux in Diabetic Retinal Vessels: a Microperfusion Study

Microperfusion techniques were used to characterize in vitro the fluorescein transport and fluid movement across isolated diabetic rabbit retinal vessels. Unbranched segments of afterioles of alloxan-induced diabetic rabbits were isolated and cannulated on a microperfusion apparatus. Movement of 3 H-inulin, the reference tracer, and 14C-fluorescein, the test tracer, across the vascular walls was studied using microperfusion techniques adapted by us to study vascular permeability. The diabetic retinal vessels showed a fluid flux from bath to lumen of 3·6 ± 0·6 nl min-1 mm-1 with perfusion rates of 7·1 ± 0·3 nl min-1. This value was 43% (P ≤ 0·05) lower when compared with normal values obtained in previous work. Bath to lumen fluorescein fluxes were 2·1 ± 0·5 and 7·6 ± 1·6 × 10-12 mol min-1 mm,-1, for fluorescein concentrations of 0·87 × 10-4 and 1·5 × 10-4 M, respectively. These values are 23% (P ≤ 0·05) and 38·7% (P ≤ 0·05), respectively, lower when compared to values obtained from normal retinal vessels. Probenecid, a competitive inhibitor of organic anion transport, added to the bath, had no effect. Increasing bath concentrations of fluorescein caused increasing fluid flux from bath to lumen, and again, probenecid added to the bath has no effect. These results show a significant decrease of fluorescein transport and of net fluid flux across diabetic retinal vessel walls from outside into the lumen, suggesting that in experimental alloxan-induced diabetes there is an early, probably functional, involvement of the retinal vessels in the initial alteration of the blood-retinal barrier (BRB).

IMPLICATIONS OF HYPERGLYCEMIA FOR POST-EXERCISE RESYNTHESIS OF GLYCOGEN IN TROUT SKELETAL MUSCLE

Rates of whole-body glucose turnover and muscle-specific glucose utilization were determined in rainbow trout (Oncorhynchus mykiss) at rest and at intervals during recovery from burst swimming. Plasma glucose level was high in the experimental animals (range 6­38 mmol l-1), but hyperglycemia was not related specifically to exercise. Estimated glucose turnover, 19.3±2.6 (rest) and 15.8±3.9 µmol min-1 kg-1 (recovery), was also highly variable, but was linearly associated with plasma glucose concentration (turnover=0.97[glucose]+0.57, r=0.93) in both resting and recovering fish. While utilization of glucose in the whole animal was clearly responsive to plasma glucose availability, estimated total skeletal muscle disposal of glucose accounted for less than 15 % of glucose turnover, indicating that glucose was utilized largely by tissues other than locomotory muscle. Rates of glucose utilization in white muscle (range 0.5­4 nmol min-1 g-1) provide direct evidence that glucose, regardless of plasma concentration, accounted for less than 10 % of glycogen repletion during exercise recovery. In red muscle, glucose uptake was influenced by plasma glucose level below 10­12 mmol l-1 (utilization range 1­15 nmol min-1 g-1), but was independent of glucose concentration above about 12 mmol l-1 (utilization plateaued at 15­20 nmol min-1 g-1). Trout red muscle is similar to mammalian white muscle in the sense that glucose is estimated to account incompletely for glycogen restoration (25­60 %), suggesting dependence on both glycogenesis and glyconeogenesis during recovery. It is concluded that hyperglycemia may be important to the pattern of substrate incorporation into red muscle glycogen and to the rate of repletion observed, but glucose availability has, as predicted from earlier indirect studies, little relevance to white muscle glycogen restoration. The regulatory mechanisms that govern apparently very high glucose turnover rates during extreme hyperglycemia, concomitant with low disposal rates in skeletal muscle, require further investigation.

Ketol-Acid Reductoisomerase from Barley (Hordeum vulgare) (Purification, Properties, and Specific Inhibition).

Ketol-acid reductoisomerase (KARI, EC 1.1.1.86) was purified to homogeneity from etiolated barley shoots (Hordeum vulgare) using anion exchange, Red-Sepharose, hydrophobic interaction, and chromatofocusing steps. Purification yielded 0.25 to 0.27 mg of pure KARI per 100 g fresh weight of starting material. The specific activity of the purified enzyme was 6 [mu]mol of NADPH oxidized min-1 mg-1 with acetohydroxybutyrate as substrate. The native enzyme had an apparent molecular weight of 115,000 as estimated by gel filtration and appeared to be a homodimer with a subunit molecular weight of 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Km values of the purified KARI for acetolactate, acetohydroxybutyrate, and NADPH (determined with acetohydroxybutyrate) were 11, 38, and 4.3 [mu]M, respectively. The Vmax obtained with acetohydroxybutyrate was 1.8 [mu]mol min-1 mg-1; the corresponding value for acetolactate was 0.16 [mu]mol min-1 mg-1. The enzyme showed optimum activity at pH 7.5. When either acetolactate or acetohydroxybutyrate was used as substrate, the experimental herbicidal compound 2-dimethyl-phosphinoyl-2-hydroxyacetic acid inhibited the purified KARI in a time-dependent and reversible manner. The initial inhibition was strictly competitive. The inhibition constant values were 0.46 (using acetolactate as substrate) and 0.19 [mu]M (acetohydroxybutyrate), respectively.

Kinetic study of Bi 1.8Pb 0.4Ca 2Sr 2Cu 3O y superconductor in water

The reaction of Bi 1.8Pb 0.4Ca 2Sr 2Cu 3O y powder in water was studied quantitatively. It was found that the [H 3O +] ion would act as a catalyst in this reaction and the initial rate equation was R 0 = - d[A] 0 dt = k[A] 0[ H 3 O +] 0.2 0 , where [ A] represented the surface area of the superconducting powder. The rate constant, k, obtained at 10, 25 and 40°C was 3.98, 8.8 and 19.6 × 10 -4 mol min -1 cm -2 M 0.8, respectively. The activation energy and pre-exponential factor calculated from the Arrhenius equation were respectively 39.1 kJ mol -1 and 6.4 × 10 3 mol min -1 cm -2 M 0.8.

Phosphatidate Kinase, a Novel Enzyme in Phospholipid Metabolism (Purification, Subcellular Localization, and Occurrence in the Plant Kingdom).

Microsomal membranes from suspension-cultured Catharanthus roseus cells possess an enzymic activity that catalyzes the ATP-dependent phosphorylation of phosphatidic acid (PA) to form diacylglycerol pyrophosphate (H. Behrbohm, J.B. Wissing [1993] FEBS Lett 315: 95-99). This enzyme activity, PA kinase, was purified and characterized. Plasma membranes, obtained from C. roseus microsomes by aqueous two-phase partitioning, were extracted, and PA kinase was purified 3200-fold by applying different chromatographic steps that resulted in a specific activity of about 10 [mu]mol min-1 mg-1. Sodium dodecyl sulfate-gel electrophoresis of the fractions obtained from the final chromatographic step revealed a 39-kD protein that correlated with the enzyme activity; PA kinase activity could be eluted from this protein band. Subcellular localization, investigated with C. roseus cells, showed that the activity was confined to membrane fractions, and at least 80% was associated with plasma membranes. The data revealed the same distribution within the cellular membranes of PA kinase as reported for diacylglycerol kinase, which is a typical plasma membrane-located enzyme. Furthermore, PA kinase activity was detected in the calli of 16 different plant species and in the different organs of C. roseus plants and obviously occurs ubiquitously in the plant kingdom.