Purification and partial characterization of a glutamyl-tRNA synthetase from the unicellular green algaScenedesmus obliquus,mutant C-2A′

The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNA^Glu by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482–1484). The synthetase from the yellow pigment mutant C-2A of the unicellular green alga Scenedesmus obliquus was purified by sequential column chromatography on Sephacryl S-300, Blue Sepharose, phosphocellulose P11 and by fast protein liquid chromatography (FPLC) on Mono Q. After denaturing sodium dodecylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, proving the apparent homogeneity of the glutamyl-tRNA synthetase. A molecular mass of 105 ± 10 kDa was determined for the native protein by chromatography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The purified protein is active within a pH range from 7.0 to 9.0 with a maximum activity at pH 8.0. Kinetics for the binding of glutamate to the tRNA, performed with highly purified enzyme preparations, showed a K _m value of 2.3 M ± 0.3 for glutamate.Abbreviations ALA 5-aminolevulinic acid - FPLC fast protein liquid chromatography - Glu glutamate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SDS sodium dodecylsulfate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine This work was supported by a grant of the Deutsche Forschungsgemeinschaft. U.C. Vothknecht is grateful for a Nachwuchs-förderungsstipendium des Landes Hessen. The authors want to thank Ms. B. Böhm, J. Gade and K. Eckhardt for skillful technical assistance. The authors also want to thank Dr. C.G. Kannangara (Carlsberg Institute, Kopenhagen, Denmark) for the donation of tRNA from barley and Dr. D. Jahn (FB Biology/Microbiology, Philipps-University, Marburg, FRG) for the tRNA^Glufrom E. coli.     

Influence of enzyme solution on protoplast culture and transient gene expression in maize (Zea mays L.)

The influence of two enzyme solutions, differing only in the presence or absence of Macerozyme, on protoplast yield, colony formation and transient GUS (-glucuronidase) activity was studied. For all parameters tested the presence of Macerozyme during protoplast isolation had a negative influence. Using an enzyme solution without Macerozyme suspension aggregates gave up to 4.4 times higher protoplast yield and plating efficiencies were increased up to 10-fold. Further, protoplasts isolated without macerozyme showed a 5.2-fold higher GUS activity in transient gene expression. Apart from the presence of Macerozyme, longer incubation (3 compared with 1.5 h) of cell aggregates in the enzyme solution also had a negative effect on transient transformation efficiency. These data demonstrate that protoplast isolation conditions have a profound effect on transient gene expression and it is proposed that these factors will also influence stable transformation efficiency.Abbreviations CP cellulase pectolyase - CPM cellulase pectolyase Macerozyme - 2,4-d 2,4-dichlorophenoxyacetic acid     

Interaction between fatty-acid and starch synthesis in isolated amyloplasts from cauliflower floral buds

The interaction of fatty-acid synthesis with starch synthesis has been studied in intact amyloplasts isolated from floral buds of cauliflower (Brassica oleracea L.). These amyloplasts perform acetate-dependent fatty acid synthesis at maximum rates only at high external ATP concentrations. Neither pyruvate nor malate inhibit acetate-dependent fatty-acid synthesis. In contrast, acetate is inhibitory to the low pyruvate-dependent fatty acid synthesis. These observations indicate that neither pyruvate nor malate are used as natural precursors of fatty-acid synthesis. In contrast to fatty-acid synthesis, the rate of glucose-6-phosphate-dependent starch synthesis is already saturated in the presence of much lower ATP concentrations. Rising rates of starch synthesis influence negatively the process of acetate-dependent fatty acid synthesis. This inhibition appears to occur under both limiting and saturating concentrations of external ATP, indicating that the rate of ATP uptake is limiting when both biochemical pathways are active. The rate of starch synthesis is modulated specifically by the concentration of 3-phosphoglycerate in the incubation medium. This observation leads to the conclusion that the activity of ADP-glucose pyrophosphorylase is of primary importance for the control of both, starch and fatty-acid synthesis. Using the modified approach of Kacser and Burns (1973; Symp. Soc. Exp. Biol.27, 65–104) we have quantified the contribution of the rate of starch synthesis to the control of the metabolic flux through fatty-acid synthesis.Abbreviations ADPGlc-PPase ADPglucose pyrophosphorylase - Glc6P glucose-6-phosphate - PGA 3-phosphoglyceric acid     

Simultaneous fructose and ethanol production from sucrose,usingZymomonas mobilis 2864 co-immobilised with invertase

Summary Fructokinase negativeZymomonas mobilis UQM 2864, was co-immobilised with invertase in alginate and incubated on sucrose-based media in batch and fedbatch culture. The highest fructose concentration achieved was 138 g/l using fed-batch cultivation with sugar-cane syrup-simultaneously producing 79.9 g/l or 10.1% (v/v) ethanol in less than 24 hours. The ethanol and fructose yields were 95 and 84% respectively. Co-immobilisation resulted in faster fermentation times, particularly for the batch fermentations, and complete utilisation of substrate.     

TGGE and HIEF: a comparison of two methods in the detection of carriers of the Z mutation of the alpha-1-antitrypsin gene

Alpha-1-antitrypsin (-1-AT) deficiency can lead to juvenile liver cirrhosis and lung emphysema in adulthood. The deficiency Z allele is caused by a base transition. Temperature gradient gel electrophoresis (TGGE) and hybrid isoelectric focusing (HIEF) were used to detect carriers of the Z mutation of the -1-AT gene. The resulting data were compared. To verify carriers at the sequence level, a manual nonradioactive sequencing strategy was established. Among our sample of carriers of the Z mutation, two were not detected by HIEF that could be identified by TGGE. DNA of all TGGE identified individuals harboring the Z mutation of the -1-AT gene were sequenced nonradioactively. All carriers harbored a G to A transition at position 11.940. This mutation is described to cause the altered protein.     

Sequence analysis of cohesive ends of the actinophage RP3 genome and construction of a transducible shuttle vector

Abstract The sequence of the cohesive ends of actinophage RP3 DNA has been determined. As with all other phages of Gram-positive bacteria that have been studied sofar, RP3 DNA has 3'-protruding ends. A shuttle cosmid has been constructed containing this cos area which can be efficiently transduced by phage RP3 to host cells of Streptomyces rimosus .     

Inactivation of the streptokinase gene prevents Streptococcus equisimilis H46A from acquiring cell-associated plasmin activity in the presence of plasminogen

Abstract The effects of some physico-chemical parameters on production of extracellular α-L-arabinofuranosidase by Aspergillus nidulans were examined. Highest levels of α-L-arabinofuranosidase were generated with cultures grown on 1% (w/v) purified beet pulp arabinan at 30°C and at an initial pH of 7.0. The enzyme was shown to be very sensitive to the action of proteases. Zymogram overlay of a protein profile obtained by SDS-PAGE revealed the occurrence of a band ( M _r 36 000) exhibiting α-L-arabinofuranosidase activity. The isoelectric pH of the enzyme lay near 4.3. Temperature and pH optima for the activity of crude α-L-arabinofuranosidase preparations were 55°C and 5.5, respectively. Enzyme activity was greatly reduced by thiol reagents such as Hg²⁺ and p -hydroxymercuribenzoate and showed a K _m value of 2.7 mM on p -nitrophenyl α-L-arabinofuranoside as substrate.