Electron-cytochemical localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31) in fungal cells

Summary New cytochemical method, based on biochemical experiments, was elaborated for the ultrastructural localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). The procedure was used to study the saprophytic submerged mycelium of the ascomycetous fungusClaviceps purpurea Tul. producing clavine alkaloids. The pelleted mycelium was fixed in ice cold 3% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 and washed repeatedly in the same cold buffer. The reaction mixture contained 100 mM Tris-HCl buffer pH 9.0, 10 mM phospho(enol)pyruvate, 30 mM sodium potassium tartrate, 3 mM Pb(NO₃)₂, 60 mM MgCl₂ and 30 mM NaHCO₃. Enzyme activity was localized in vacuoles, particularly inside lipid globules (spherosomes) and less frequently in membranous vesicles. Acetyl-CoA activated PEP-carboxylase both in cell free extracts and in the cytochemical staining. Aspartate inhibited the enzyme in the biochemical assay with coupled malate dehydrogenase system; the cytochemical reaction was not influenced, probably due to the interference of asparagine synthase (EC 6.3.1.1).     

Beitrag zur Kenntnis der chemischen Zusammensetzung derPhragmiti-Magnocaricetea und derAgropyro-Rumicion-Arten

Zwanzig Pflanzen der KlassePhragmiti-Magnocaricetea (Kennarten der einzelnen pflanzen-soziologischen Einheiten) und des VerbandesAgropyro-Rumicion crispi wurden den chemischen Analysen unterzogen. Die Pflanzenproben wurden auf den Wiesen der Trockengebiete S-Mährens und der SW-Slowakei gesammelt, u. zw. aus den pflanzensoziologisch, definierbaren Einheiten (Assoziationen der VerbändePhragmition, Caricion gracilis, Caricion rostratae undAgropyro-Rumicion). Die Resultate wurden mit der chemischen Zusammensetzung der in anderen Gebieten vorkommenden Pflanzen verglichen. Es zeigten sich Zusammenhänge zwischen der genetisch bedingten chemischen Zusammensetzung bestimmter Artengruppen und den Eigenschaften des Substrats. In diesem Sinne gibt es allgemeine Unterschiede zwischen den VerbändenCaricion gracilis undCaricion rostratae. Die Arten desAgropyro-Rumicion zeigen im Durchschnitt engere Bindung an die Gesellschaften desCnidion-Verbandes als an dasCaricion gracilis.     

Inhibition of phospholipid methyltransferase during zymosan induced secretion of platelet-activating factor in human polymorphonuclear leukocytes

Phagocytosis of zymosan particles coated with complement induces a time and dose dependent inhibition of the enzyme phospholipid methyltransferase in human polymorphonuclear cells. The extent of phospholipid methyltransferase inhibition induced by various concentrations of zymosan strongly correlates with the secretory process: liberation of platelet-activating factor (PAF) and β-glucuronidase. Zymosan also decreases the incorporation of ³H-methyl group into phospholipids in cells pre-labeled with (³H-methyl)-methionine. Finally, preincubation of cells with 3-deaza-adenosine and homocysteine thiolactone, inhibitors of phospholipid methyltransferase, decrease the incorporation of ³H-methyl group into phospholipids in cells pre-labeled with (³H-methyl)-methionine and modulate the release of PAF. These results suggest that phospholipid methylation plays an important role during the transduction of the secretory signal triggered by zymosan in human polymorphonuclear cells.     

Isolation of a mitochondrial factor from rat liver which potentiates the inactivation of glutamate dehydrogenase by lysosomes

Rat liver glutamate dehydrogenase (L-glutamate: NAD(P) oxidoreductase, deaminating) E.C. 1.4.1.3.) is inactivated by the mitochondrial matrix in combination with lysosomal preparations. Neither lysosomal or mitochondrial matrix extracts per se inactivate the enzyme appreciably under the conditions used. Fractionation of the matrix indicates that a low molecular weight factor is responsible for the potentiation of inactivation of glutamate dehydrogenase by lysosomes. Its absorption spectrum and chromatographic behaviour suggest that this factor is NADP.     

The effect of metals on isolated pea pyruvate decarboxylase

Pyruvate decarboxylase (PDC) was prepared from four-day-old pea seedlings by a procedure which involves extraction of plant material with a phosphate buffer, fractionation of the extract with ammonium sulphate, desalting by dialysis or gel filtration on Sephadex G-25 column, chromatography on DEAE cellulose and filtration on Sepharose 4B. The PDC preparation activity 10 000 U g^-1 protein was about 600 fold higher than that of the sodium phosphate buffer extract. According to the enzyme behaviour during the gel filtration on different carriers the molecular mass of pea PDS was estimated at about 10⁶. Magnesium ions and thiamine pyrophosphate were found to be coenzymes of PDC. Cofactors can be removed by 48 h dialysis followed by chromatography on DEAE cellulose. Apoenzym is activated optimally with the concentration of cofactors of 0.002 M. Magnesium ions can be replaced in their activation function by ions of Fe²⁺, Ni²⁺, Co²⁺, Zn²⁺ and Mn²⁺. Another ions,i.e. Ba²⁺, Ca²⁺, Cd²⁺, Hg²⁺ and Cu²⁺ are inactive. Assumption about the relation between the ion diameter and degree of activation is formulated.