The leaf peroxisomal form (MFP IV) of multifunctional protein functioning in fatty-acid β-oxidation

We have purified for the first time from green leaves a multifunctional protein (MFP) involved in fatty acid -oxidation. The protein, designated MFP IV, was extracted from green leaves of three-week-old cucumber (Cucumis sativus L.) plants. Chromatography on cation exchanger, separation on hydroxylapatite, and fast-protein liquid chromatography on Phenylsuperose led to a more than 7000-fold purification and to the isolation of an apparently homogeneous 80-kDa monomeric protein. This protein is immunologically related to the glyoxysomal MFP II, as evidenced by immunodecoration with antiserum raised against MFP II. Comparison of molecular masses of all MFPs presently known revealed that the MFP prepared from green leaves (MFP IV) is distinct from MFP II (76.5 kDa) and MFP I (74 kDa) from dark-grown cotyledons. By including other properties in this comparison, we demonstrated that MFP IV can also be distinguished from the glyoxysomal MFP III (81 kDa) and the bacterially expressed MFP-a (80 kDa). Moreover, MFP IV is a constituent of leaf peroxisomes and contains the activities of 2-enoyl-CoA hydratase (EC 4.2.1.17),l-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-hydroxyacyl-CoA epimerase.Abbreviation MFP multifunctional protein This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.     

Enhanced stability in vivo of a thermodynamically stable mutant form of yeast iso-1-cytochrome c

Previous work has established that the N57I amino acid replacement in iso-1-cytochrome c from the yeast Saccharomyces cerevisiae causes an unprecedented increase in thermodynamic stability of the protein in vitro, whereas the N57G replacement diminishes stability. Spectrophotometric measurements of intact cells revealed that the N57I iso-l-cytochrome c is present at higher than normal levels in vivo. Although iso-1-cytochrome c turnover is negligible during aerobic growth, transfer of fully derepressed, aerobically grown cells to anaerobic growth conditions leads to reduction in the levels of all of the cytochromes. Pulsechase experiments carried out under these anaerobic conditions demonstrated that the N57I iso-l-cytochrome c has a longer half-life than the normal protein. This is the first report of enhanced stability in vivo of a mutant form of a protein that has an enhanced thermodynamic stability in vitro. Although the N57I protein concentration is higher than the normal level, reduced growth in lactate medium indicated that the specific activity of this iso-l-cytochrome c in vivo is diminished relative to wild-type. On the other hand, the level of the thermodynamically labile N57G iso-1-cytochrome c was below normal. The in vivo levels of the N57I and N57G iso-l-cytochrome c suggest that proteins in the mitochondrial intermembrane space can be subjected to degradation, and that this degradation may play a role in controlling their normal levels.     

Purification and characterization of a 22-kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica . The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22-kDa protein was isolated from thylakoid membranes of spores and the amino-terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS-PAGE and by the disappearance of the band during spore germination. Protease activity against the 22-kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.     

Circadian expression of the light-harvesting protein of Photosystem II in etiolated bean leaves following a single red light pulse: Coordination with the capacity of the plant to form chlorophyll and the thylakoid-bound protease

The appearance of the light harvesting II (LHC II) protein in etiolated bean leaves, as monitored by immunodetection in LDS-solubilized leaf protein extracts, is under phytochrome control. A single red light pulse induces accumulation of the protein, in leaves kept in the dark thereafter, which follows circadian oscillations similar to those earlier found for Lhcb mRNA (Tavladoraki et al. (1989) Plant Physiol 90: 665–672). These oscillations are closely followed by oscillations in the capacity of the leaf to form Chlorophyll (Chl) in the light, suggesting that the synthesis of the LHC II protein and its chromophore are in close coordination. Experiments with levulinic acid showed that PChl(ide) resynthesis does not affect the LHC II level nor its oscillations, but new Chl a synthesis affects LHC II stabilization in thylakoids, implicating a proteolytic mechanism. A proteolytic activity against exogenously added LHC II was detected in thylakoids of etiolated bean leaves, which was enhanced by the light pulse. The activity, also under phytochrome control, was found to follow circadian oscillations in verse to those in the stabilization of LHC II protein in thylakoids. Such a proteolytic mechanism therefore, may account for the circadian changes observed in LHC II protein level, being implicated in pigment-protein complex assembly/stabilization during thylakoid biogenesis.Abbreviations Chl chlorophyll - CL continuous light - D dark - FR far-red light - LA levulinic acid - LHC II light-harvesting complex serving Photosystem II - PChl(ide) protochlorophyllide - PCR protochlorophyllide oxidoreductase - R red light     

Degradation of quinoline by immobilized Comamonas acidovorans in a three-phase airlift reactor

Quinolie degradation by Comamonas acidovorans was studied in a continuously operated three-phase airlift reactor. Porous glass beads were applied as support matrix for cell imobilization by colonization. Under steady-state conditions (S approximately 0), cell attachment was poor at low dilution rates but imporved considerably with increasing dilution rate. Conversion of quinoline was investigated below and above the washout for suspended culture (D(crit) = mu(max) = 0.42 h(-1)). With immobilized cells the reactor could be operated at D > mu(max), and complete conversion of quinoline was achieved as long as the specific quinoline feed rate D*S(0)/X did not exceed the maximum specific degradation rate (r(S, max)). The biofilm thickness was about 100 mum, and its efficiency was about 54% compared to suspended organisms. If quinoline overloads were supplied to the reactor, quinoline, as overloads were supplied to the reactor, quinoline, as well as its pathway intermediates, appeared in the reactor and conversion was low. Hence, the immobilized microorganisms remained viable and active. They could survive quinoline overloads. If the quinoline feed rate was reduced agains, complete conversion was reestablished. (c) 1995 John Wiley & Sons, Inc.     

Effects of some factors on protoplast formation of a microalga,Prototheca zopfii

The effects of experimental factors on protoplast formation of Prototheca zopfii Kru¨ger in 0.85 m NaCl using Macerozyme R-200 were studied based on a fractional factorial experimental design. The rate of protoplast formation was mainly affected by the incubation temperature and the age of algal cells. The optimal condition for the maximum protoplast yield was determined based on a response surface model. These were: mid-logarithmic phase cells and Macerozyme concentration of 4% at a temperature of 35°C.     

Localization of the enzymes involved in H2 and formate metabolism inSyntrophospora bryantii

Cell-free extracts of crotonate-grown cells of the syntrophic butyrate-oxidizing bacteriumSyntrophospora bryantii contained high hydrogenase activities (8.5–75.8 µmol · min^–1 mg^–1 protein) and relatively low formate dehydrogenase activities (0.04–0.07 µmol · min^–1 mg^–1 protein). The K _M value and threshold value of the hydrogenase for H₂ were 0.21 mM and 18 µM, respectively, whereas the K _M value and threshold value of the formate dehydrogenase for formate were 0.22 mM and 10 µM, respectively. Hydrogenase, butyryl-CoA dehydrogenase and 3-OH-butyryl-CoA dehydrogenase were detected in the cytoplasmic fraction. Formate dehydrogenase and CO₂ reductase were membrane-bound, likely located at the outer aspect of the cytoplasmic membrane. Results suggest that during syntrophic butyrate oxidation H₂ is formed intracellularly while formate is formed at the outside of the cell.     

Metabolic pathways and energetics of the acetone-oxidizing,sulfate-reducing bacterium,Desulfobacterium cetonicum

Acetone degradation by cell suspensions of Desulfobacterium cetonicum was CO₂-dependent, indicating initiation by a carboxylation reaction. Degradation of butyrate was not CO₂-dependent, and acetate accumulated at a ratio of 1 mol acetate per mol butyrate degraded. In cultures grown on acetone, no CoA transfer apparently occurred, and no acetate accumulated in the medium. No CoA-ligase activities were detected in cell-free crude extracts. This suggested that the carboxylation of acetone to acetoacetate, and its activation to acetoacetyl-CoA may occur without the formation of free acetoacetate. Acetoacetyl-CoA was thiolytically cleaved to two acetyl-CoA, which were oxidized to CO₂ via the acetyl-CoA/carbon monoxide dehydrogenase pathway. The measured intracellular acyl-CoA ester concentrations allowed the calculation of the free energy changes involved in the conversion of acetone to acetyl-CoA. At in vivo concentrations of reactants and products, the initial steps (carboxylation and activation) must be energy-driven, either by direct coupling to ATP, or coupling to transmembrane gradients. The G of acetone conversion to two acetyl-CoA at the expense of the energetic equivalent of one ATP was calculated to lie very close to 0kJ (mol acetone)^-1. Assimilatory metabolism was by an incomplete citric acid cycle, lacking an activity oxidatively decarboxylating 2-oxoglutarate. The low specific activities of this cycle suggested its probable function in anabolic metabolism. Succinate and glyoxylate were formed from isocitrate by isocitrate lyase. Glyoxylate thus formed was condensed with acetyl-CoA to form malate, functioning as an anaplerotic sequence. A glyoxylate cycle thus operates in this strictly anaerobic bacterium. Phosphoenolpyruvate (PEP) carboxykinase formed PEP from oxaloacetate. No pyruvate kinase activity was detected. PEP presumably served as a precursor for polyglucose formation and other biosyntheses.Abbreviations MV ²⁺ Oxidized methyl viologen - PEP Phosphoenolpyruvate - PHB Poly--hydroxybutyrate     

Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria

Anaerobic degradation of alkylbenzenes with side chains longer than that of toluene was studied in freshwater mud samples in the presence of nitrate. Two new denitrifying strains, EbN1 and PbN1, were isolated on ethylbenzene and n-propylbenzene, respectively. For comparison, two further denitrifying strains, ToN1 and mXyN1, were isolated from the same mud with toluene and m-xylene, respectively. Sequencing of 16SrDNA revealed a close relationship of the new isolates to Thauera selenatis. The strains exhibited different specific capacities for degradation of alkylbenzenes. In addition to ethylbenzene, strain EbN1 utilized toluence, but not propylbenzene. In contrast, propylbenzene-degrading strain PbN1 did not grow on toluene, but was able to utilize ethylbenzene. Strain ToN1 used toluene as the only hydrocarbon substrate, whereas strain mXyN1 utilized both toluene and m-xylene. Measurement of the degradation balance demonstrated complete oxidation of ethylbenzene to CO₂ by strain EbN1. Further characteristic substrates of strains EbN1 and PbN1 were 1-phenylethanol and acetophenone. In contrast to the other isolates, strain mXyN1 did not grow on benzyl alcohol. Benzyl alcohol (also m-methylbenzyl alcohol) was even a specific inhibitor of toluene and m-xylene utilization by strain mXyN1. None of the strains was able to grow on any of the alkylbenzenes with oxygen as electron acceptor. However, polar aromatic compounds such as benzoate were utilized under both oxic and anoxic conditions. All four isolates grew anaerobically on crude oil. Gas chromatographic analysis of crude oil after growth of strain ToN1 revealed specific depletion of toluene.     

Construction and characterization of an absolute deletion mutant of Escherichia coli ribonuclease II

Abstract The degradation of mRNA plays a central role in the control of protein synthesis. In Escherichia coli , the rnb gene encodes ribonuclease II (RNase II), one of the two main exonucleases involved in mRNA decay. We have constructed strain CMA201, in which the rnb promoter region and the gene were deleted from the chromosome and replaced by a tet^tr cassette. This is the first rnb absolute deletion mutant that shows the complete absence of rnb -specific mRNA. This strain has growth characteristics similar to the wild-type, even though it has no RNase II activity, and it should be useful in studies of mRNA metabolism.