Carbonic anhydrase of the alkaliphilic cyanobacterium Microcoleus chthonoplastes

The activity of carbonic anhydrase (CA) was studied in different cell fractions of the alkaliphilic cyanobacterium Microcoleus chthonoplastes. The activity of this enzyme was found in the soluble and membrane protein fractions, as well as in intact cells and in a thick glycocalyx layer enclosing the cyanobacterium cells. The localization of CA in glycocalyx of M. chthonoplastes was shown by the western blot analysis and by immunoelectron microscopy studies with antibodies to the thylakoid CA from Chlamydomonas reinhardtii (Cah3). At least one of the CA forms occurring in M. chthonoplastes CA was shown to be an alpha-type enzyme. A possible mechanism of the involvement of the glycocalyx CA in calcification of cyanobacteria is discussed.     

Carbonic Anhydrase Activity of Alkalophilic Cyanobacteria from Soda Lakes

The activity and intracellular partition of carbonic anhydrase (CA) were studied in alkalophilic cyanobacteria, an inhabitant of soda lakes at pH 9–10. In the homogenates of Rhabdoderma lineare, Rhabdoderma sp., and Microcoleus chthonoplastes, high activity of CA was found, similar to that in eukaryotic microalgae. The activity of CA calculated on the basis of chlorophyll and protein was higher for the soluble (sCA) than for membrane (mCA) protein fraction. Intact cells of all cyanobacteria under investigation also showed CA activity that implies the presence of extracellular form(s). The extracellular CA in benthic M. chthonoplastes was localized, at least partly, in a vast glycocalix (gCA) as shown by Western blotting and the measurement of enzyme activity in the isolated glycocalix preparations. Probing gCA from M. chthonoplastes with the antibodies against thylakoid CA from Chlamydomonas reinhardtii (Cah3) demonstrated that gCA belongs to the -type of enzyme and has the structure identical to that of Cah3. The extracellular CA of M. chthonoplastes manifested the maximum activity at pH 7 and 10, but not at pH 6 and 9. An increase in medium pH from 7.2 to 9.6 resulted only in slight alkalization of the cytoplasm in R. lineare, from 7.1 to 7.5. It follows that true alkalophils can maintain the pH inside the cell at the near-neutral level in spite of high pH (10.2) level in the cultural medium.     

Glycocalyx of Agmenellum quadruplicatum

The marine cyanobacterium Agmenellum quadruplicatum was shown to possess an extracellular glycocalyx similar in structure to those surrounding other bacteria from a variety of natural environments. Thin sections of cells stained with ruthenium red and frozen-etched preparations of unfixed cells indicated the glycocalyx was a network of small fibrils. The glycocalyx was present during all phases of growth, and was not degraded during nutrient limitation.     

Association of a new type of gliding,filamentous, purple phototrophic bacterium inside bundles of Microcoleus chthonoplastes in hypersaline cyanobacterial mats

An unidentified filamentous purple bacterium, probably belonging to a new genus or even a new family, is found in close association with the filamentous, mat-forming cyanobacterium Microcoleus chthonoplastes in a hypersaline pond at Guerrero Negro, Baja California Sur, Mexico, and in Solar Lake, Sinai, Egypt. This organism is a gliding, segmented trichome, 0.8–0.9 m wide. It contains intracytoplasmic stacked lamellae which are perpendicular and obliquely oriented to the cell wall, similar to those described for the purple sulfur bacteria Ectothiorhodospira. These bacteria are found inside the cyanobacterial bundle, enclosed by the cyanobacterial sheath. Detailed transmission electron microscopical analyses carried out in horizontal sections of the upper 1.5 mm of the cyanobacterial mat show this cyanobacterial-purple bacterial association at depths of 300–1200 m, corresponding to the zone below that of maximal oxygenic photosynthesis. Sharp gradients of oxygen and sulfide are established during the day at this microzone in the two cyanobacterial mats studied. The close association, the distribution pattern of this association and preliminary physiological experiments suggest a co-metabolism of sulfur by the two-membered community. This probable new genus of purple bacteria may also grow photoheterotrophically using organic carbon excreted by the cyanobacterium. Since the chemical gradients in the entire photic zone fluctuate widely in a diurnal cycle, both types of metabolism probably take place. During the morning and afternoon, sulfide migrates up to the photic zone allowing photoautotrophic metabolism with sulfide as the electron donor. During the day the photic zone is highly oxygenated and the purple bacteria may either use oxidized species of sulfur such as elemental sulfur and thiosulfate in the photoautotrophic mode or grow photoheterotrophically using organic carbon excreted by M. chthonoplastes. The new type of filamentous purple sulfur bacteria is not available yet in pure culture, and its taxonomical position cannot be fully established. This organism is suggested to be a new type of gliding, filamentous, purple phototroph.     

Ferric reductases of Legionella pneumophila

Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (K _m = 7.0 m) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (K _m = 15.3 m). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells.     

Purification and characterisation of an intracellular carbonic anhydrase from the unicellular green alga Coccomyxa

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified and characterised from the unicellular green alga Coccomyxa sp. Initial studies showed that cultured Coccomyxa cells contain an intracellular CA activity around 100 times higher than that measured in high-CO₂-grown cells of Chlamydomonas reinhardtii CW 92. Purification of a protein extract containing the CA activity was carried out using ammonium-sulphate precipitation followed by anion-exchange chromatography. Proteins were then separated by native (non-dissociating) polyacrylamide gel electrophoresis, with each individual protein band excised and assayed for CA activity. Measurements revealed CA activity associated with two discrete protein bands with similar molecular masses of 80 +5 kDa. Dissociation by denaturing polyacrylamide gel electrophoresis showed that both proteins contained a single polypeptide of 26 kDa, suggesting that each 80-kDa native protein was a homogeneous trimer. Isoelectric focusing of the 80-kDa proteins also produced a single protein band at a pH of 6.5. Inhibition studies on the purified CA extract showed that 50% inhibition of CA activity was obtained using 1 M azetazolamide. Polyclonal antibodies against the 26-kDa CA were produced and shown to have a high specific binding to a single polypeptide in soluble protein extracts from Coccomyxa cells. The same antiserum, however, failed to cross-react with soluble proteins isolated from two different species of green algae, Chlamydomonas reinhardtii and Chlorella vulgaris. Correspondingly, antisera directed against pea chloroplastic CA, extracellular CA from C. reinhardtii and human CAII, showed no cross-hybridisation to the 26-kDa polypeptide in Coccomyxa. The 26-kDa protein was confirmed as being a CA by N-terminal sequencing of two internal polypeptide fragments and alignment of these sequences with that of previously identified CA proteins from several different species.Abbreviations CA carbonic anhydrase - CCM CO₂-concentrating mechanism - IEF isoelectric focusing - Rubisco ribulose-l,5-bisphosphate carboxylase/oxygenase We would like to thank Drs. Cecilia Forsman, Inga-Maj Johansson and Nalle Jonsson for their valuable advice concerning the isolation of CA. This work was supported by the Swedish Natural Research Council and Seth M. Kempes Memorial foundation.     

Characterization of dihydropyrimidinase from Pseudomonas stutzeri

Dihydropyrimidinase from Pseudomonas stutzeri ATCC 17588 was purified 100-fold and characterized. It was found that dihydrouracil, dihydrothymine and hydantoin could serve as substrates for the partially purified enzyme. The K _m values for dihydrouracil, dihydrothymine and hydantoin were determined to be 19.6 M, 21.3 M and 36.4 M, respectively, while their respective V _max values were 0.836 mol/min, 0.666 mol/min and 2.21 mol/min. Between pH 7.5 and 9.0, enzyme activity was shown to be maximal. The optimum temperature for enzyme activity was 45 °C. Using gel filtration, the molecular weight of the enzyme was calculated to be approximately 115000 Da. Metal ions were found to influence the level of enzyme activity. Dihydropyrimidinase activity was stimulated by magnesium ions and inhibited by either zinc or copper ions.     

Glutathione reductase activity in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum

Glutathione reductase activity was detected and characterized in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum. The activity of the enzyme varied between 50 and 150 nmol reduced glutathione· min^-1·mg protein^-1, and the apparent K_m for NADPH was 0.125 and 0.200 mM for heterocysts and vegetative cells, respectively. The enzyme was found to be sensitive to Zn⁺² ions, however, preincubation with oxidized glutathione rendered its resistance to Zn⁺² inhibition. Nostoc muscorum filaments were found to contain 0.6–0.7mM glutathione, and it is suggested that glutathione reductase can regenerate reduced glutathione in both cell types. The combined activity of glutathione reductase and isocitrate dehydrogenase in heterocysts was as high as 18 nmol reduced glutathione·min^-1·mg protein^-1. A relatively high superoxide dismutase activity was found in the two cell types; 34.2 and 64.3 enzyme units·min^-1·mg protein^-1 in heterocysts and vegetative cells, respectively.We suggest that glutathione reductase plays a role in the protection mechanism which removes oxygen radicals in the N₂-fixing cyanobacterium Nostoc muscorum.Abbreviations DTNB 5-5-dithiobis-(2-nitrobenzoic acid) - EDTA ethylenediaminetetra-acetic acid - GR glutathione reductase (EC1.6.4.2) - GSH reduced glutathione - GSSG oxidized glutathione - OPT O-phtaldialdehyde - SOD superoxide dismutase (EC 1.15.1.1)     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Regulation of pyrimidine biosynthesis in Pseudomonas cepacia

Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the lelel of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5-triphosphate and uridine 5-phosphate.     

Properties of an enzymatic complex active in sulfite and thiosulfate oxidation by Rhodotorula sp.

An enzymatic complex from Rhodotorula was characterized and it was indicated that it possessed thiosulfate-oxidizing activity, forming tetrathionate as well as sulfite oxidase activity. Both activities coupled with ferricyanide and native cytochrome c but no with mammalian cytochrome c. Activities of these enzymes were inhibited by thiol inhibitors. Chelating agents did not affect thiosulfate oxidizing activity and only moderately inhibited sulfite oxidase. Both activities disappeared after treatment with proteolytic enzymes or sodium deoxycholate which indicates an essential role played not only by protein but also by phospholipids in the enzymatic activity of the complex. Thiosulfate oxidizing enzyme had a K _m for thiosulfate of 0.16 mM with ferricyanide as electron acceptor and of 14 M with native cytochrome c and of 0.34 mM for ferricyanide. Optimum pH for this activity was 7.8. Other properties of this enzyme were similar to those of thiobacilli and heterotrophic bacteria. The activity of sulfite oxidase was inhibited by 50% with 10 M AMP. The K _m values of this enzyme were 1 mM with ferricyanide as electron acceptor and 60 M with native cytochrome c for sulfite and 0.42 mM for ferricyanide. The enzyme did not show a specific optimum pH value with ferricyanide as electron acceptor. However, with native cytochrome c optimum pH was 7.8 for its activity. In many properties the sulfite oxidase from Rhodotorula was similar to the enzyme from Thiobacillus ferrooxidans, T. concretivorus, T. thioparus and T. novellus.Abbreviations CSH reduced glutathion - APS reductase, adenosine-S-phosphosulfate reductase - pHMB p-hydroxymercuribenzoate - NEM N-ethylmalcimide - TCA trichloroacetic acid - PPO 2,5-diphenyloxazole - POPOP 2,2-p-phenylen-bis 5-phenyloxazol     

A thioredoxin-activated fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus 6301

Fructose-1,6-bisphosphatase was isolated from the cyanobacterium Synechococcus 6301 by acid precipitation, ammonium-sulfate fractionation, and Sephadex gel chromatography. The purified enzyme needed thiols and MgCl₂ for activity. The following K_m-values were obtained: a) for fructose-1,6-bisphosphate: 1.7 mM; b) for MgCl₂: 12.5 mM; c) for dithiocrythritol: 0,56 mM; d) for glutathione: 14 mM; e) for mercaptoethanol: 22 mM; f) for cysteine: 50 mM. Thioredoxin B isolated from this organism will activate this fructose-1,6-bisphosphatase. The K_m of thioredoxin B for this fructose-1,6-bisphosphatase was determined to be 1.7 M, endicotiy that thioredoxin might activate the fructose-1,6-bisphosphatase in Synechococcus in vivo.     

Carbonic anhydrase activity in acetate grown Methanosarcina barkeri

Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K _i=0.5 mM), by azide (apparent K _i=1 mM), and by cyanide (apparent K _i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H₂/CO₂ grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO₂ and CH₄. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO₂ with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein).     

Respiratory activity in the marine cyanobacterium Spirulina subsalsa and its role in salt tolerance

Intracellular ion concentration and respiratory activity in the marine cyanobacterium Spirulina subsalsa was analyzed during cell transition from saline to hypersaline medium. During salt upshock, an early phase of Na⁺ and Cl^- influx was observed, followed by an adaptation phase where both Na⁺ and Cl^- were excluded from the cell. Respiration in intact cells was enhanced during salt upshock. S. subsalsa spheroplasts exhibited a high rate of O₂ uptake, which was further enhanced in cells grown in hypersaline medium, upon addition of NaCl to the assay mixture. This effect was found to be specific to sodium ions. Plasma membrane fractions from cells grown in hypersaline medium exhibited a high rate of cytochrome oxidase activity, which was further stimulated by NaCl, and was sensitive to DCCD. Immunoblot analysis of Spirulina plasma membrane polypeptides with anti-cytochrome oxidase serum demonstrated high content of 53.4 kDa polypeptide of cytochrome oxidase, which was enriched in membranes obtained from hypersaline Spirulina cells. The enhanced respiration, and more specifically the enrichment of cytochrome oxidase activity in salt-adapted cells in situ, as well as its stimulation by NaCl in vitro and inhibition by DCCD, suggest that cytochrome oxidase is involved in the extrusion of sodium ions from cells of the salt-tolerant Spirulina subsalsa.Abbreviations DCCD dicyclohexylcarbodiimide - CCCP carbonylcyanide m-chlorophenyl hydrazone - TMPD N, N, N, N, tetramethyl p-phenylenediamine dichloride     

Site of action of the natural algicide,cyanobacterin, in the blue-green alga,Synechococcus sp.

Cyanobacterin is a secondary metabolite produced by the cyanobacterium, Scytonema hofmanni. Highly purified cyanobacterin was found to inhibit the growth of many cyanobacteria at a minimum effective dose of 2 g/ml (4.6 M). The antibiotic had no effect on eubacteria including the photosynthetic Rhodospirillum rubrum. The site of action of cyanobacterin was further investigated in the unicellular cyanobacterium, Synechococcus sp. Electron micrographs of antibiotic-treated Synechococcus cells indicated that cyanobacterin affects thylakoid membrane structure. The antibiotic also inhibited light-dependent oxygen evolution in Synechococcus cells and in spheroplasts. These data support our conclusion that cyanobacterin specifically inhibits photosynthetic electron transport. This activity is similar to herbicides such as 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU). The anhydro analog of cyanobacterin had no biological activity.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DCPIP dichlorophenolindophenol     

Electron-transport chain and coupled oxidative phosphorylation in methanol-grown Paracoccus denitrificans

Methanol dehydrogenase of Paracoccus denitrificans was shown to be very similar to the enzyme of Pseudomonas sp, M. 27. The K _m value for methanol with excess activator (ammonium ions) is 35 M. The pH optimum for enzyme activity with 2,6-dichlorophe-nolindophenol as electronacceptor was at 9.0 A CO-binding type of cytochrome c was present only in cells grown with methanol as carbon and energy source.It has been shown that methanol-oxidation involves electron-transport via cytochrome c and an a-type cytochrome to oxygen. Antimycin A did not inhibit this electron transport and 90% inhibition was obtained by 375 M potassium cyanide. Electron transport from endogenous substrates is possible via cytochrome b and possibly cytochrome o to oxygen. Potassium cyanide inhibited 90% of the electron transport via this pathway at a concentration of 1.42 mM. Measurement of respiration-driven proton translocation proved that during oxidation of methanol to formaldehyde by oxygen one mole of adenosine triphosphate is synthesized in the site 3 region of the electron transport chain. The H⁺/O value found confirmed the H⁺/site ratio of 3–4 found in heterotrophic grown cells. During electron transport from endogenous substrates to oxygen there is a possible synthesis of 3 moles of adenosine triphosphate.In heterotrophically grown cells electron transfer to oxygen follows almost only the branch of the respiratory chain containing cytochrome o. In methanol-grown cells the pathway via the a-type cytochrome seems more important.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - EPR electron paramagnetic resonance - S.D. standard deviation - ATP adenosine triphosphate     

Characterization of a malate dehydrogenase in the cyanobacterium Coccochloris peniocystis

A malate dehydrogenase (MDH) was characterized from the cyanobacterium Coccochloris peniocystis. The enzyme was purified approximately 180-fold and had a molecular weight of about 90000. The enzyme had a pH optimum of pH 6.7 to 7.5; a K_m (malate) of 5.6 mM and K_ms for NAD and NADP of 24 M and 178 M, respectively, although similar V_max were obtained with either pyridine nucleotide. Enzyme activity was inhibited by ATP, citrate, oxalacetate, acetyl CoA and CoA. Enzyme assays with uniformly ¹⁴C-labelled malate caused no ¹⁴CO₂ release, indicating this MDH is not a malic enzyme. Electrophoresis and S-200 gel filtration of the partially purified enzyme indicated a single MDH was present in this preparation. A second, less abundant, MDH was present in crude extracts. The presence of MDH in this organism is consistent with the operation of a glyoxylate cycle which, in the absence of a TCA cycle, would provide organic acids required in secondary carbon metabolism. ATP inhibition of MDH may allow for light regulation of MDH activity since, in the light, oxaloacetic acid is generated by phosphoenolpyruvate carboxylase activity.Abbreviations MDH malate dehydrogenase - PEPcase phosphoenolpyruvate carboxylase - MOPS 3-[N-Morpholino] propane sulfonic acid - TRIS Tris(hydroxymethyl)-aminomethane - EDTA Disodium Ethylenadiamine Tetraacetate - MES 2[N-Morpholino]-ethane Sulfonic Acid - EPPS N-2-Hydroxyethylpiperazine Propane - MW Molecular weight - OAA Oxaloacetic acid     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

L-Phenylalanine ammonia-lyase fromPhaseolus vulgaris: Modulation of the levels of active enzyme bytrans-cinnamic acid

The extractable activity ofl-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [³⁵S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of³⁵S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.Abbreviations AOPP l--aminoxy--phenylpropionic acid - CA trans-cinnamic acid - PAGE polyacrylamide gel electrophoresis - PAL l-phenylalanine ammonia-lyase - SDS sodium dodecyl sulphate     

NADP+-dependent acetaldehyde dehydrogenase from Zymomonas mobilis

An NADP⁺-linked acetaldehyde dehydrogenase (EC 1.2.1.4) from the ethanol producing bacterium Zymomonas mobilis was purified 180-fold to homogeneity. The enzyme is a cytosolic protein with an isoelectric point of 8.0 and has an apparent molecular weight of 210000. It showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 55000, which indicates that it consists of four probably identical subunits. The apparent K _m values for the substrate acetaldehyde were 57 M and for the cosubstrate NADP⁺ 579 M. The enzyme was almost inactive with NAD⁺ as cofactor. Several other aldehydes besides acetaldehyde were accepted as a substrate but not formaldehyde or trichloroacetaldehyde. In anaerobically grown cells of Zymomonas mobilis the enzyme showed a specific activity of 0.035 U/mg protein but its specific activity could be increased up to 0.132 U/mg protein by adding acetaldehyde to the medium during the exponential growth phase or up to 0.284 U/mg protein when cells were grown under aeration. The physiological role of the enzyme is discussed.Abbreviations ALD-DH acetaldehyde dehydrogenases from Z. mobilis - DTT dithiothreitol - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - SDS sodium dodecylsulfate Dedicated to Prof. Dr. H.-G. Schlegel, Universität Göttingen, on the occasion of his 65th birthday