Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial characterization

Pyruvate decarboxylase (EC 4.1.1.1) from the ethanol producing bacterium Zymomonas mobilis was purified to homogeneity. This enzyme is an acidic protein with an isoelectric point of 4.87 and has an apparent molecular weight of 200,000±10,000. The enzyme showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 56,500±4,000 which indicated that the enzyme consists of four probably identical subunits. The dissociation of the cofactors Mg²⁺ and thiamine pyrophosphate at pH 8.9 resulted in a total loss of enzyme activity which could be restored to 99.5% at pH 6.0 in the presence of both cofactors. For the apoenzyme the apparent K _m values for Mg²⁺ and thiamine pyrophosphate were determined to be 24 M and 1.28 M. The apparent K _m value for the substrate pyruvate was 0.4 mM. Antiserum prepared against this purified pyruvate decarboxylase failed to crossreact with cell extracts of the reportedly pyruvate decarboxylase positive bacteria Sarcina ventriculi, Erwinia amylovora, or Gluconobacter oxydans, or with cell extracts of Saccharomyces cerevisiae.Abbreviations Tris-buffer 0,01 M tris-HCl buffer, containing 1 mM MgCl₂ 0.1 mM EDTA, 1.0 mM thiamine pyrophosphate, 2 mM mercaptopropanediol, pH 7.0     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Preparation of sealed tonoplast and plasma-membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis

Highly purified tonoplast and plasmamembrane vesicles were isolated from microsomes of Catharanthus roseus (L.) G. Don. by preparative free-flow electrophoresis. The relative amounts of tonoplast and plasma-membrane vesicles in the total microsomes varied with the pH of the grinding medium. The most electronegative fractions were identified as tonoplast using nitrate-inhibited, azide-resistant Mg²⁺-ATPase and pyrophosphatase activities as enzyme markers. The least electronegative fractions were identified as plasma membrane using glucan-synthase-II and UDPG:sterolglucosyl-transferase activities as enzyme markers. Other membrane markers, latent inosine-5-diphosphatase (Golgi), NADPH-cytochrome-c reductase (ER) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane and did not contaminate either the tonoplast or the plasma-membrane fractions. In the course of searching for a reliable marker for tonoplast, the pyrophosphatase activity was found to be essentially associated with the tonoplast fractions purified by free-flow electrophoresis from C. roseus and other plant materials. The degree of sealing of the tonoplast and plasmamembrane vesicles was probed by their ability to pump protons (measurements of quinacrine quenching) and to generate a membrane potential (absorption spectroscopy of Oxonol VI). A critical evaluation of vesicles sidedness is presented.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - Con A concanavalin A - Cyt cytochrome - LysoPC lysophosphatidylcholine - Pi orthophoshate - PP_iase pyrophosphatase - IDPase inosine-5-diphosphatase We thank Pr. Robert Dargent and André Moisan (Laboratoire de Cryptogamie, Toulouse, France) for use of their electron-microscope facilities. This work was supported by the Centre National de la Recherche Scientifique and by a grant Dynamique du fonctionnement de la vacuole from the Ministère de la Recherche et de la Technologie.     

Activation of sulphate in Anabaena cylindrica

Summary Crude cell-free extracts of Anabaena cylindrica synthesized adenosine-5-phosphosulphate (AP³⁵S) and 3-phosphoadenosine-5-phosphosulphate (PAP³⁵S) from ³⁵SO₄ ^2- in the presence of Mg²⁺, ATP and inorganic pyrophosphatase. Maximum AP³⁵S and PAP³⁵S were produced at pH 7.15 and 8.05, respectively. APS kinase was detected in the supernatant of crude cell-free extracts by a spectrophotometric procedure. ATP-Sulphurylase had an absolute requirement for Mg²⁺ and less than 30% AP³⁵S was formed when Mg²⁺ was replaced by either Mn²⁺ or Co²⁺. Nucleotide triphosphates other than ATP and 2-deoxyATP were ineffective in this reaction. Maximum enzyme activity was observed at equimolar concentrations of Mg²⁺ and ATP and excess of either of these was inhibitory. Other nucleotide triphosphates, like GTP, UTP, CTP, TTP, ITP, or 2-deoxyATP also inhibited the enzyme activity. Inhibition by GTP was competitive with respect to ATP. ATP-sulphurylase activity was not affected by cysteine, methionine or glutathione.Abbreviations APS adenosine-5-phosphosulphate - PAPS 3-phosphoadenosine-5-phosphosulphate     

Regulation of adenosine 5′-phosphosulfate sulfotransferase activity by H2S and cyst(e)ine in primary leaves of Phaseolus vulgaris L.

During chloroplast development in the primary leaves of Phaseolus vulgaris, the extractable activity of adenosine 5-phosphosulfate sulfotransferase increased ten-fold. When chloroplast development took place in air enriched with 3.5 l H₂S·l^-1 there was a decrease in adenosine 5-phosphosulfate sulfotransferase activity. Cyst(e)ine in concentrations up to 1 mM (in the external medium) did not affect the increase in adenosine 5-phosphosulfate sulfotransferase activity in intact plants. In plants with excised roots, 0.75 mM cyst(e)ine inhibited this increase. In green primary leaves, H₂S or cyst(e)ine treatment resulted in a decrease of extractable adenosine 5-phosphosulfate sulfotransferase activity. In intact plants, this effect of cyst(e)ine was observed at a concentration of 1 mM, and in plants with excised roots, 0.25 mM had a comparable effect.In developing plants, the extractable activities of O-acetyl-L-serine sulfhydrylase (EC 4.2.99.9) and ribulosebisphosphate carboxylase (EC 4.1.1.39.) were not affected by H₂S or cyst(e)ine.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5phosphosulfate sulfotransferase - BSA bovine serum albumin - DTE dithioerythritol - EDTA ethylenediaminetetra-acetic acid - OASSase O-acetyl-L-serine sulfhydrylase - PAPS adenosine 3-phosphate 5-phosphosulfate - POPOP 1,4 Di 2-(5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazol - RubP ribulose-bisphosphate - RubPCase ribulosebiphosphate carboxylase This is no. 8 in the series Regulation of Sulfate Assimilation in Plants. The term cysteine is used when it is clear that cystine is not involved; cyst(e)ine is used for an undefined mixture of cysteine and cystine. The concentrations are expressed in all cases relative to cysteine     

Sulfate activation in Desulfotomaculum

Enzyme activities conceivably involved in the activation of sulfate were studied with Desulfotomaculum ruminis, D. acetoxidans, D. nigrificans, D. orientis, and Desulfovibrio vulgaris. Cell lysates of these species revealed activities of at least 8 nkat/mg protein (i.e., 480 nmol per min and mg protein) of ATP sulfurylase, acetate kinase, phosphotransacetylase and adenylate kinase. ADP sulfurylase was not detected. Pyrophosphatase activity was high (73 to 97 nkat/mg protein) in Desulfotomaculum orientis and Desulfovibrio vulgaris. In these strains pyrophosphatase was activated by addition of a reductant (dithionite). In Desulfotomaculum ruminis, D. acetoxidans, and D. nigrificans, only low pyrophosphatase activity (2.5 to 6.3 nkat/mg protein) was measured, which was not reductant-activated. Some hints indicated a membrane association of the pyrophosphatase in D. ruminis, and possibly also in D. acetoxidans and D. nigrificans. Activities of a pyrophosphate-dependent acetate kinase (PP_i:acetate kinase), a PP_i:AMP kinase or a polyphosphate:AMP kinase were not detected or negligible. The results are not in favour of the assumption that pyrophosphate formed by ATP sulfurylase during sulfate activation might be utilized to form acetyl phosphate in Desulfotomaculum species. Contrary results of other authors were shown to be artefacts caused by chemical hydrolysis of acetyl phosphate in the molybdate-sulfuric acid reagent used for phosphate determination.Abbreviations P_i orthophosphate - PP_i pyrophosphate - APS adenosine phosphosulfate - AP₅A, P¹ P⁵-di(adenosine-5-)pentaphosphate - CTAB cetyltrimethylammonium bromide - MOPS 3-(N-morpholino)propanesulfonic acid - HEPES N(-2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid     

Valine inhibition of β-isopropylmalate dehydrogenase takes part in the regulation of leucine biosynthesis inCandida maltosa

The -isopropylmalate (IPM) dehydrogenase (EC 1.1.1.85) ofCandida maltosa, the third pathway-specific enzyme of leucine biosynthesis, was purified, some properties of the enzyme were studied and a novel regulatory pattern was found. The K_m values of the enzyme were estimated to be 0.42 mM for -IPM and 0.34 mM for NAD⁺. It is demonstrated that the enzyme can be regulated by L-valine. The inhibition was competitive with respect to -IPM (K_i=1.84 mM) and non-competitive with respect to NAD⁺ (K_i=5.67 mM). Exogenous addition of L-valine toC. maltosa cells increased the intracellular pool of some intermediates of leucine biosynthesis (-ketoisovalerate, -IPM, -IPM), but has hardly influence on the leucine pool.     

The prime plasmalemma ATPase of the halophilic alga Dunaliella bioculata: purification and characterization

The prime plasmalemma ATPase of the halophilic green alga Dunaliella bioculata has been solubilized by Triton X-100 from a plasmalemma-rich membrane fraction and purified by anion-exchange chromatography. Vanadate-sensitive ATPase activity was totally enriched about 230-fold to a specific activity of approx. 250 nkat·mg protein^–1. The presence of Mg²⁺ or Mn²⁺ is essential for ATP hydrolysis by the enzyme. In addition to an equimolar requirement (11 Mg²⁺: ATP), there is further stimulation by Mg²⁺ (up to 20 mM) and by (100 mM) monovalent cations (K⁺ NH ₄ ⁺ >Rb⁺ -Na⁺ >Cs⁺ >Li⁺-choline⁺). Most anions have no or little effect. With a molecular mass of about 105 kDa for the single subunit, sensitivity to vanadate and N,N-dicyclohexylcarbodiimide (50% inhibition at about 1 M and 0.3 mM, respectively), strict ATP-specificity, and an acidic pH optimum, this enzyme shows the typical characteristics of the common type of H⁺-ATPase in the plasmalemma of higher plants and fungi. These results undermine the hypothesis of a wider distribution of a special (high salt) type of plasmalemma ATPase as found in the marine alga Acetabularia.Abbreviations BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - Mega-9 nonanoyl-N-methyl-glucamide - Mes N-morpholinoethanesulfonic acid - Mops N-morpholinopropanesulfonic acid - PAGE polyacrylamide-gel electrophoresis - PM plasmalemma-enriched membrane fraction - SDS sodium dodecyl sulfate This work was supported by the Deutsche Forschungsgemeinschaft; we thank Drs. M. Ikeda and D. Oesterhelt (MPI für Biochemie, Martinsried, FRG) for generous and valuable information about their work prior to publication.     

The biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase from Fusobacterium nucleatum (subsp. nucleatum)

Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na⁺ (K _m=3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent K _m for Na⁺ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, (m=65 kDa), (33 kDa), (19 kDa), and (16 kDa) with the functions of a carboxy transferase (), a carboxy lyase ( and probably ) and a biotin carrier (). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the - and the biotin containing subunits of the three decarboxylases and that from Peptostreptoccus asaccharolyticus could be detected on Western blots.     

Hexose transport and membrane depolarization in Riccia fluitans

In the aquatic liverwort Riccia fluitans, the uptake of ¹⁴C-labeled 3-O-methyl glucose (3-OMG) and membrane depolarization ( _m ) caused by different hexoses has been studied as a function of time and concentration of hexose, K⁺ and H⁺, respectively. The rate of uptake of the non-metabolized 3-OMG shows two components: (A)A pH-dependent saturable uptake with a k_m value around 0.1 mM which saturates at 2.1 and 7.2 mol G _DW ^-1 h^-1 at pH 6.8 and 5.0, respectively; and (B) a pH-insensitive uptake component which increases linearly with the external 3-OMG concentration and does not saturate 4 mM. Hexoses rapidly depolarize the plasmalemma of the thallus cell and increase its electrical conductance. The maximal _m was 60±2 mV, the concentrations (mM) for half-maximal _m were 0.24 glucose, 0.32 galactose, 0.37 2-deoxy glucose, 0.38 3-OMG, 0.57 mannose, and 34 fructose. In terms of a hexose carrier model and an equivalent circuit for the hexose-induced depolarized state of the membrane, it is proposed that a hexose carrier operates either electrogenically in its protonated, pH-and voltage-sensitive state, or by transmembrane diffusion of its uncharged state.Symbols and Abbreviations _m membrane potential (mV) - g _m membrane (slope) conductance (Sm^-2) - 3-OMG 3-O-methyl glucose     

Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate

The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate+CoA+ATP phenylacetyl-CoA+AMP+PP_i and requires Mg²⁺. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 mol min^-1 x mg^-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 mol min^-1 mg^-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 ±2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn²⁺ can substitute for Mg²⁺. The apparent K _m values for phenylacetate, CoA, and ATP are 60, 150, and 290 M, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzye was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.     

Is light-dependent formation of inorganic pyrophosphate in Anacystis a photosynthetic process?

The cyanobacterium Anacystis nidulans contained levels of inorganic pyrophosphate (PP) which were about 50% of those of ATP in dark and light. Steady-state levels of PP were not decreased by the inhibitor of non-cyclic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethyl urea]. During transition from dark to light levels of PP increased rapidly. The rate of increase corresponded to a rate of synthesis of about 150 mol x mg chl^-1 x h^-1. PP formation was affected by DCMU in a similar manner to ATP synthesis.The question whether the light-dependent formation of PP is a photosynthetic process or is linked to reactions releasing PP has been studied using a newly developed cell-free system from Anacystis. Rates of ATP synthesis by phenazine metosulfate-catalyzed cyclic photophosphorylation in this system were about 170 mol x mg chl^-1 x h^-1. Formation of PP could only be observed in presence of a trapping system which converted PP to ATP, otherwise PP was split by a particle-bound inorganic pyrophosphatase. In absence of ADP neither ATP nor PP was formed.It is concluded that the light-dependent formation of PP in Anacystis is not a photosynthetic process and that the PP is derived from ATP.Abbreviations AMS adenosine 5-monosulfate - APS adenosine 5-phosphosulfate - APSase adenosine 5-triphosphate sulfurylase - chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino)ethanesulfonic acid - PCA perchloric acid - PMS phenazine metosulfate - PPase inorganic pyrophosphatase     

Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate

The effect of pyridoxal 5-phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by pyridoxal 5-phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC₅₀ of 0.5 mM, competed with the substrate for binding to the oxoglutaratecarrier (K _i = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal5-phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [³H]pyridoxal 5-phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by pyridoxal 5-phosphate. These results indicate that pyridoxal 5-phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.     

Fusicoccin action in cell-suspension cultures of Corydalis sempervirens Pers.

Mid-log-phase cell suspensions of Corydalis sempervirens Pers., when incubated in micromolar or submicromolar concentrations of fusicoccin, strongly acidified the culture medium. High-affinity fusicoccin-binding sites were found in microsomes prepared from these cells using the radioligand [³H]-9-norfusicoccin-8-alcohol. Binding was saturable with an apparent dissociation constant (K _d) of 2.8 nM, a pH optimum of 6.0, a temperature optimum of 35° C and was rapid (t_1/2 = 8 min). The site abundance was 0.76±0.17 pmol · (mg of protein)^–1. In the same membrane preparations, the K⁺, Mg²⁺-ATPase (EC 3.6.1.3) was characterized. The enzyme was highly vanadate-sensitive (IC₅₀=6.5 M) and nucleotide-specific (ATPNTP), had a pH optimum of 6.2, an apparent K _m for ATP of 0.23±0.12 mM, and V _max of 10.6±1.8 nkat (mg of protein)^–1. Fusicoccin doubled V _max and lowered, by a factor of 2, the apparent K _m for ATP of the enzyme when the cells were incubated with the toxin for 30 min prior to homogenization of the cells. The stimulation of the enzyme was also pronounced when fusicoccin was added to the homogenization medium just prior to homogenization of the cells, but was slight to zero when the toxin was added at the microsomal stage. The pronounced stimulatory effect of fusicoccin on the ATPase was seen at pH 7.1, i.e. at a pH typical for the cytoplasmic compartment, but was not detectable at pH 6.2, the pH optimum of the enzyme. The implications of these findings for an understanding of fusicoccin action are discussed.Abbreviations [³H]ABE-FC 9-nor-8-(4-azido-3,5-[³H]-benzoyl-diaminoethyl)-fusicoccin - FC fusicoccin - FCol 9-norfusicoc-cin-8-alcohol - Mes 2(N-morpholino)ethanesulfonic acid This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, FRG and the Fonds der Chemischen Industrie, Frankfurt, FRG (literature provision).     

The influence of cation and gelling agent concentrations on vitrification of apple cultivars in vitro

Shoot tips of York and Vermont Spur Delicious apples (Malus domestica Borkh.) were cultured in vitro to test the influence of K⁺, Mg⁺⁺ and gelling agent concentrations on vitrification. These concentrations were 20.05, 14.05 and 8.05 mM K⁺, 1.5 and 3.0 mM Mg⁺⁺, 7.0 g/l Difco Bacto agar and 1.0, 1.5 and 2.0 g/l Gelrite. The lowest K⁺ level produced a higher percentage of vitrified shoots, affected tissue appearance, reduced shoot number and shoot elongation and apparently altered shoot metabolic activity. Gelrite consistently produced vitrified leaves and stems, even though media gelled with 1.5 g/l Gelrite presented the same apparent gel firmness as using 7 g/l Difco Bacto agar, which did not induce vitrification. Less shoot elongation, fewer total shoots, and more usable shoots of York were obtained on Bacto-agar, while similar but less noticeable effects were obtained with Vermont Spur Delicious. The results presented here show that vitrification can be studied in a standardized system in which the only change is substitution of one gelling agent for another.     

Pyruvate: ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition,catalytic properties,and sequence alignments

Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeon. In this communication we describe the purification and properties of pyruvate: ferredoxin oxidoreductase from this organism. The catabolic enzyme was purified 250-fold to apparent homogeneity with a yield of 16%. The native enzyme had an apparent molecular mass of 120 kDa and was composed of four different subunits of apparent molecular masses of 45, 33, 25, and 13 kDa, indicating and structure. Per mol, the enzyme contained 0.8 mol thiamine pyrophosphate, 9 mol non-heme iron, and 8 mol acid-labile sulfur. FAD, FMN, lipoic acid, and copper were not found. The purified enzyme showed an apparent K _m for coenzyme A of 0.02 mM, for pyruvate of 0.3 mM, and for clostridial ferredoxin of 0.01 mM, an apparent V _max of 64 U/mg (at 65°C) with a pH optimum near 7.5 and an Arrhenius activation energy of 75 kJ/mol (between 30 and 70°C). The temperature optimum was above 90°C. At 90°C, the enzyme lost 50% activity within 60 min in the presence of 2 M KCl. The enzyme did not catalyze the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, and hydroxypyruvate. The N-terminal amino acid sequences of the four subunits were determined. The sequence of the -subunit had similarities to the N-terminal amino acid sequence of the -subunit of the heterotetrameric pyruvate: ferredoxin oxidoreductase from Pyrococcus furiosus and from Thermotoga maritima, and unexpectedly, to the N-terminal amino acid sequence of the homodimeric pyruvate: ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the -subunits of the enzyme from A. fulgidus and the heterodimeric pyruvate: ferredoxin oxidoreductase from Halobacterium halobium.Abbreviations CoASH Coenzyme A - F ₄₂₀ Coenzyme F₄₂₀     

Rat osseous plate alkaline phosphatase: Effect of neutral protease digestion on the hydrolysis of pyrophosphate and nitrohenylphospate

Collagenase treatment, commonly used to prepare alkaline phosphatase-rich matrix vesicles from epiphyseal cartilage growth plates, seems to affect the integrity of this membrane-bound enzyme. Alkaline phosphatase-rich rat osseous plates were incubated with 1000 U/mL collagenase for 3 h, at 37°C and after purification on Sepharose 4B, kinetic studies were performed using nitrophenylphosphate and pyrophosphate as substrates.The optimum apparent pH for the hydrolysis of p-nitrophenylphosphate and pyrophosphate increased from 9.4 to 10.25 and from 8.0 to 9.0, respectively, as a consequence of collagenase treatment. In the absence of Mg²⁺ ions, the enzyme hydrolyzed PNPP with K_M = 322.5 ± 15.3 M and V = 965.2 ± 45.8 U/mg, while in the presence of 2 mM Mg²⁺ ions, V increased 66%. Cobalt (K_0.5 = 5.3 ± 0.3 M) and manganese (K_0.5 = 0.72 ± 0.03 M) ions stimulated the PNPPase activity of the collagenase-treated enzyme, but with a lower apparent affinity when compared with that of not-treated enzyme. In the absence of Mg²⁺ ions pyrophosphate was hydrolyzed according to Michaelis-Menten kinetics (K_M = 105.1 ± 6.3 M and V = 64.9 ± 3.9 U/mg), but site-site interactions (n_H = 1.2) were observed in the presence of 2 mM Mg²⁺ ions (V = 110.8 ± 5.5 U/mg; K_0.5 = 42.7 ± 2.0 M).To our knowledge this is the first report showing significant alterations on phosphohydrolytic activity and metal binding properties of bone alkaline phosphatase due to associated neutral proteases in collagenase preparations often used for the isolation of matrix vesicles.     

Microsatellite markers in the hexaploid Prunus domestica species and parentage lineage of three European plum cultivars using nuclear and chloroplast simple-sequence repeats

In a previous study, cDNA microsatellite markers were described in apricot (Prunus armeniaca L.). Specific PCR primers were designed to amplify the microsatellite-containing regions from genomic DNA in different Prunus species. In the present work, cDNA microsatellite markers were developed in the hexaploid Prunus domestica L. species and polymorphism was ascertained in a segregating plum population. Co-dominant mendelian segregation of alleles was demonstrated and microsatellite polymorphism displayed up to 6 alleles per SSR locus per individual. Parentage lineage of three full-sib European plum cultivars (cv. Cacanska najbolja, Cacanska rana and Cacanska lepotica) was reconstructed by the analysis of the above nuclear SSR markers, completed by four chloroplastic microsatellite loci. The six most informative nuclear loci enabled discrimination between the three Cacak cultivars and unrelated individuals as well as the previously proposed parents, Wangenheim and Pozegaca. Data obtained support previous evidence that these cultivars originated from the Stanley cultivar. However, SSR analysis finally excluded Wangenheim as the other possible parent. Based on the results obtained with nuclear and chloroplast SSR loci, we propose the origin of those three Cacak cultivars in a cross between Stanley as the mother plant and Ruth gerstetter as the pollinator. Furthermore, we demonstrate the utility of these apricot SSR markers for genotype fingerprinting of the hexaploid plum cultivars.     

Some properties of 2-5A binding/nucleolytic activities in gel filtered rabbit reticulocyte lysates

Rabbit reticulocyte lysates, gel filtered on Sephadex G-25 with or without ATP (or its analogs), were preincubated at 37°C and their subsequent binding to p₃A₄,3-[³²P]pCp was studied. Lysates filtered without ATP or in the presence of 0.1 mM 8-bromo-ATP, 1,N⁶-etheno-ATP, or ITP showed a time-dependent decrease in binding activity. This decrease was completely prevented when lysates were filtered with 0.1 mM ATP, 2-deoxy-ATP, --methylene-ATP, or ATP--S. The stability of binding provided by ATP or 2-deoxy-ATP analogs corresponds to a more active 2–5A dependent endonucleolytic (RNAase L) activity based on studies using [³H] viral mRNA. Chromatography on heparin-agarose showed that ATP-supplemented gel-filtered reticulocyte lysates had a different p₃A₄,3-[³²P]pCp binding activity elution-profile than lysates gel-filtered in the absence of ATP. Covalent cross-linking of periodate-oxidized p₃A₄,3-[³²P]pC to gelfiltered lysates, preincubated at 0°C or 37°C for 30 min, showed the following results: (1) all lysates gave a major cross-linking of the radioactive ligand to an 80 000 dalton polypeptide, regardless of the temperature of preincubation, (2) Iysates gel-filtered without ATP, with 0.1 mM ITP, or --methylene-ATP, showed a significant reduction in the cross-linking of the 80 000 dalton protein, after preincubation at 37°C for 30 min. This decrease was accompanied by an increase in the labeling of two smaller polypeptides.Abbreviations used 2 5-oligoadenylates oligonucleotides consisting of 5-adenylic acid residues joined by a 2 5-phosphodiester linkage     

Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi

Our fungal culture collection was screened for fructosyl peptide oxidase, an enzyme that could be used for the determination of glycated hemoglobin in diabetic subjects with hyperglycemia. Fructosyl peptide oxidases were found in strains of eight genera: Achaetomiella, Achaetomium, Chaetomium, Coniochaeta, Eupenicillium, Gelasinospora, Microascus and Thielavia. By their substrate specificity toward N-fructosyl valyl-histidine (-keto-amine) and N-fructosyl lysine (-keto-amine), fructosyl peptide oxidases could be categorized into two groups: (1) enzymes that oxidize both -keto-amine and -keto-amine, and (2) enzymes that preferably oxidize -keto-amine. A fructosyl peptide oxidase from Achaetomiella virescens ATCC 32393, active toward both N-fructosyl valyl-histidine and N-fructosyl lysine, was purified to homogeneity and characterized. The enzyme was monomeric (M_r=50,000), was most active at 40 °C and pH 8.0, and had a covalently bound flavin as a prosthetic group. Apparent K_m values for N-fructosyl valyl-histidine and N-fructosyl lysine were 2.30 and 1.69 mM, respectively. N-fructosyl valyl-histidine was consumed and the same molar amount of valyl-histidine was produced by the fructosyl peptide oxidase reaction. This enzyme could be useful for the measurement of hemoglobin A_1C, the N-terminal valine residue of the -subunit of which is glycated.Abbreviations HbA_1C Hemoglobin A_1C - FPOX Fructosyl peptide oxidase - FAOX Fructosyl amino acid oxidase - Fru-ValHis N-fructosyl valyl-histidine - Fru-Val N-fructosyl valine - Fru-Lys N-fructosyl lysine - Fru-Gly Fructosyl glycine - TOOS N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt