Cysteine: Depolarization-Induced Release from Rat Brain In Vitro

Compounds released on depolarization in a Ca2+-dependent manner from rat brain slices were screened to identify candidates for neuroactive substances. Lyophilized superfusates were analyzed by reversed-phase HPLC after derivatization with 9-fluorenyl N-succinimidyl carbonate. One of the compounds that showed an increase of concentration in superfusates in the presence of iodoacetamide was identified as the cysteine (Cys) derivative, S-carboxamidomethylcysteine, by fast atom bombardment mass spectrometry and other methods. This stable Cys derivative originates from endogenous, extracellular Cys. The finding led to a method for quantification of Cys in superfusates by immediate cooling of the superfusates to 0 degrees C and reaction of Cys with N-ethylmaleimide. Depolarization-induced Ca2+-dependent release of Cys was most prominent in the neocortex, followed by the mesodiencephalon, striatum, and cerebellum. This suggests that Cys is released from a neuronal compartment and might be involved in neurotransmission.     

Analysis by high-resolution two-dimensional electrophoresis of differentiation-dependent alterations in cytosolic protein pattern of HL-60 leukemic cells.

HL-60 leukemic cells were differentiated along the neutrophilic pathway with retinoic acid (RA) or along the monocytic pathway with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Using a high-resolution two-dimensional electrophoresis technique and subsequent silver staining, differentiation-dependent changes in cytosolic protein pattern of HL-60 cells were analysed and were compared with the cytosolic protein pattern of human neutrophils. The amount of 64 and 50 out of a total of 632 proteins studied was increased or decreased in RA- and 1,25(OH)2D3-differentiated HL-60 cells, respectively, in comparison to undifferentiated HL-60 cells. Thirty-three of these proteins were similarly altered in RA- and 1,25(OH)2D3-differentiated HL-60 cells. Twenty-two and 25 of the proteins altered in amount in RA- or 1,25(OH)2D3-differentiated HL-60 cells versus undifferentiated HL-60 cells were similarly altered in human neutrophils in comparison to undifferentiated HL-60 cells. Seven and 10 of the proteins altered in amount in RA- or 1,25(OH)2D3-differentiated HL-60 cells had specific equivalents in neutrophil cytosol. Our results show (i) that neutrophilic and monocytic differentiation is associated with decreases and increases in amount of cytosolic proteins; (ii) that both differentiation processes share a common set of alterations; and (iii) are associated with specific alterations in protein amount.     

Expression of the E. coli nadB gene and characterization of the gene product L-aspartate oxidase

Quinolinic acid is synthesized in E. coli by the enzymes L-aspartate oxidase and quinolinate synthase A, the genes of which are named nadB and nadA. In our previous work we cloned and characterized the two genes (Flachmann, R., Kunz, N., Seifert, J., Gütlich, M., Wientjes, F.J., L?ufer, A. & Gassen, H.G. (1988) Eur. J. Biochem. 175, 221-228). Here we report on the expression of the nadB gene under control of the inducible left promoter of the bacteriophage lambda. The yield of the active gene product L-aspartate oxidase was enhanced up to 20% of the soluble cell protein. The enzyme was purified to homogeneity in a three-step procedure and the reading frame of the L-aspartate oxidase gene was confirmed by Edman degradation of five cyanogen bromide peptides. L-Aspartate oxidase shows no classical Michaelis-Menten behaviour but is subject to a substrate inactivation. The apparent Km values were different for substrate concentrations below and above 1mM and were determined to 0.5 mM and 4.1mM, respectively. The active form of the enzyme is a monomer of 60,284 Da and contains one molecule of FAD and nine cysteine residues, four of which built up two disulfide bonds. The isoelectric point of the protein was determined to be at pH 5.6. Chemical modifications of the enzyme showed that at least one tyrosine and one histidine residue are essential for enzyme activity. The coenzyme-binding domain is located in the amino-terminal part of the polypeptide chain as revealed by a sequence comparison to other dinucleotide binding enzymes. Furthermore, there is evidence for a relationship to fumarate reductase and succinate dehydrogenase of E. coli.     

d-Ribulokinase from Klebsiella pneumoniae for continuous production of d-(−)-ribulose-5-phosphate

Summary The production of d-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to d-ribulose was catalysed by d-ribulokinase isolated from Klebsiella pneumoniae. For production of d-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for d-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of d-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of d-ribulose-5-phosphate 1^–1 per day were reached.Offprint requests to: D. Gygax     

Regulatory elements controlling expression of the Drosophila homeotic gene fork head.

The region-specific homeotic gene fork head (fkh) is expressed and required in a variety of tissues of the developing Drosophila embryo. In order to identify the cis regulatory elements directing the complex spatio-temporal expression pattern of fkh, we have studied the subpatterns directed by defined fragments of fkh genomic DNA. These experiments enabled us to distinguish separate regulatory elements specific for the different expression domains of fkh. In addition, our analysis revealed several unexpected features such as the redundancy of regulatory elements and the overlap of regulatory elements with the transcribed regions of other genes. Moreover, the separation of normally contiguous elements effecting expression in the posterior terminal fkh domain appears to lead to novel expression domains which do not correspond to known developmental units in the embryo.     

Effects of abscisic acid on somatic embryo maturation of hybrid larch

Somatic embryos of hybrid larch (Larixleptoeuropaea) whichhad been matured for 4 weeks on maturation medium, developednormally on medium supplemented with 60 µM ABA, but abnormallyon medium with no ABA. A comparative structural and histochemicalinvestigation was carried out on these two types of mature embryos.At the light microscope level, differences between both treatmentswere visible only after 2–3 weeks of maturation. At aroundthis time, abnormal development becomes evident macroscopically:ABA-minus embryos remain rather stubby as opposed to the morecylindrically shaped ABA-plus embryos. Whereas somatic embryosmatured with ABA consist of densely cytoplasmic cells showinga high rate of cell division, ABA-minus embryos are largelymade up of expanded and highly vacuolate cells, indicating thatgrowth in the latter is mainly due to cell expansion and notdivision. After 4 weeks of maturation, ABA-minus embryos beginto elongate in the hypocotyl region, and precocious germinationwas observed frequently. Again, these morphogenetic events werelargely due to abnormal timing of cell expansion. Histochemically,storage proteins were found only in somatic embryos maturedfor 4 weeks with ABA. This observation is in line with resultsobtained by total protein analysis, yielding significantly lowertotal protein contents in ABA-minus embryos both on a freshweight and a per embryo basis after 4–5 weeks of maturation.Deposition of starch grains mainly in the cortex tissue of thehypocotyl region was observed within 2 weeks of maturation invarying amounts regardless of ABA supply. Polyphenols, in particularcatechins and proanthocyanidins, were present in all embryosfrom the very onset of development. They were localized preferentiallyin the proximal suspensor cells and the basal region of theembryo. However, accumulation of polyphenols was generally muchmore pronounced in embryos matured without ABA, indicating alack of biochemical regulatory competence in those embryos. Key words: Abscisic acid, embryonal development, somatic embryo, storage protein, polyphenols