Characterization of the S'-subsite specificity of V8 proteinase via acyl transfer to added nucleophiles

The S'-subsite specificity of endoproteinase Glu-C (V8 proteinase) was studied by acyl transfer reactions using Z-Glu-OMe as acyl donor and a series of amino acid- and peptide-derived nucleophiles. The partition constant, which characterizes specificity, was determined by a method based on the integrated rate equation. V8 proteinase prefers amino acid residues with hydrophobic side chains in the P'1 position. Di- and tripeptide amides are more efficient nucleophilic amino components than amino acid amides.     

Evaluation of experimental combined toxicity by use of dose-frequency curves: comparison with theoretical additivity as well as independence

Dose-frequency curves of toxic effects of a substance A were evaluated in the absence and in the presence of a fixed dose of a second substance B. Data were fitted by the curve-fitting program ALLFIT. Observed combined frequencies of A + B were compared statistically with the expected frequencies of additivity and (or) independence by the phi 2-square goodness-of-fit test. The theoretical dose-frequency curves expected for an additive response were obtained by a solely graphical procedure and the theoretical curves for independent effects were calculated from the effects of B and A at certain doses. In rotarod tests with trained mice, the combined deteriorating effect of ethanol and benzodiazepines were significantly over-additive. However, their lethal interaction appeared underadditive in mice. The lethal underadditive interaction of ethanol and phencyclidine (PCP) can be ascribed largely to independent actions of these compounds. Loss of righting reflex was additively enhanced by PCP, whereas PCP overadditively enhanced the effect of ethanol. The insecticidal action of the cholinesterase inhibitors malathion and parathion appeared additive and significantly different from independent interaction. A comparison of results from dose-response curves with isoboles showed good agreement. The method appears as an attractive alternative or as a complementary procedure to the isobolographic analysis. Combination experiments as described can be carried out and evaluated rather simply, with a minimum of expenditure and a maximum of information.     

A bacteriophage P1-encoded modulator protein affects the P1 c1 repression system

Bacteriophage P1 encodes a tripartite immunity system composed of the immC, immI, and immT region. Their basic genetic elements are the c1 repressor of lytic functions, the c4 repressor which negatively regulates antirepressor synthesis, and the bof gene, respectively. The function of the latter will be described here. We have cloned and sequenced the bof gene from P1 wild type and a P1 bof amber mutant. Based on the position of a TAG codon of the bof amber mutant the bof wild type gene was localized. It starts with a TTG codon, comprises 82 codons, and is preceded by a promoter structure. The bof protein (Mr = 7500) was overproduced in Escherichia coli from a bof recombinant plasmid and was purified to near homogeneity. The N-terminal amino acids predicted from the DNA sequence of the bof gene were confirmed by sequence analysis of the bof protein. Using a DNA mobility shift assay, we show that bof protein enhances the binding of c1 repressor to the operator of the c1 gene. In accordance with this result, in transformants of Escherichia coli, containing both a bof- and a c1-encoding plasmid, c1 expression is down-regulated. We conclude that bof acts as a modulator protein in the repression of a multitude of c1-controlled operators in the P1 genome.     

Synthesis of phosphatidylglycerol by chloroplasts from leaves of Spinacia oleracea L. (spinach)

Purified chloroplasts from leaves of Spinacia oleracea L. (spinach) incorporated glycerol 3-phosphate into diacylglycerol, monoacylglycerol, phosphatidylglycerol, phosphatidic acid, and lysophosphatidic acid. The omission of ATP or CTP, CoA or illumination decreased the incorporation markedly. The fraction of incorporated glycerol 3-phosphate found in phosphatidylglycerol was greatly reduced by the omission of bicarbonate, acetate, and ATP, or in darkness, low-osmolarity medium, or high magnesium ion concentration (10 mM). Incorporation of glycerol 3-phosphate into lipid and specifically into phosphatidylglycerol was optimal at a $\text{Mg}{}^{\mathrm{2+}}\text{CTP}$ ratio of 1, whereas the optimal ratio for $\text{Mg}{}^{\mathrm{2+}}\text{ATP}$ was closer to 2. The $\text{Mg}{}^{\mathrm{2+}}\text{CTP}$ gave lower total incorporation but a higher fraction of incorporation in phosphatidylglycerol. Triton X-100 inhibited incorporation of glycerol 3-phosphate into lipid, especially into phosphatidylglycerol.     

Dark modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast

The properties of the system which reverses light modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in pea chloroplasts were examined. A factor catalyzing dark modulation of these enzymes was found. This factor cochromatographed with thioredoxin in all systems used (Sephacryl S-200, Sephadex G-75, DEAE-cellulose). Inhibition of dithiothreitol-dependent modulation and of dark reversal by antibody against Escherichia coli thioredoxin further suggest that the dark factor is in fact thioredoxin. It appears that the reaction is the reverse of the previously described dithiothreitol-dependent thioredoxin-catalyzed modulation of enzymes. The limiting step in vitro seems to be the oxidation of thioredoxin during the dark period.     

Histochemical evidence for lysosomal storage of acid glycosaminoglycans in splenic cells of rats treated with tilorone

Summary Tilorone, an agent with antiviral and antitumor activities, has previously been reported to produce clear cytoplasmic vacuoles in many cell types of the rat. The present study on rat spleen was planned to investigate the ultrastructural and histochemical features of the tilorone-induced vacuoles occurring in sinus endothelium, trabecular smooth muscle cells, and macrophages of the red pulp. Evidence was obtained that the vacuoles represent lysosomes overloaded with acid glycosaminoglycans (aGAG). The main purpose of the present study was to overcome the technical difficulties of preserving the intralysosomal storage materials which were highly water-soluble and non-fixable by aldehyde fixatives. Preservation, at least for the light microscopical level, was achieved by freeze drying and by means of cationic dyes which served also to characterize the storage materials on the basis of their acidities. Tissue slices were used to determine the critical MgCl₂ concentration necessary to abolish Alcian blue staining; cartilage and mast cells served as references. For the storage material in sinus endothelium, the critical MgCl₂ concentration was found to be >0.7 M, as compared to >0.5 M for cartilage and >0.9 M for mast cells. The storage materials in trabecular cells and macrophages were slightly less acidic than cartilagineous matrix and more heterogeneous than that in sinus endothelium. Ultrastructurally, positive staining with high iron diamine (HID) confirmed the presence of aGAG within the tilorone-induced vacuoles.     

Die Variabilität vegetativer und generativer Flechtenstrukturen und ihre Bedeutung für die Systematik

Die Entstehung von Stützleisten in den Thalli von Cladonia chlorophaea und Cetraria islandica, sowie die Entwicklung sekundärer Soredien im Inneren der Podetien von Cladonia chlorophaea werden beschrieben. Gemeinsam mit anderen Entwicklungsvorgängen von Flechten zeigen die Beobachtungen eine sehr geringe Determination der Flechtengewebe. Die Struktur wird weitgehend durch die Funktion bestimmt, während gleichzeitig bestimmte ontogenetische Abläufe unveränderbar sind. Die Bedeutung dieser Erscheinungen für die Systematik wird diskutiert.     

Structural preferences for the binding of chromium nucleotides by beef heart mitochondrial ATPase

The mono- and bidentate forms of adenosine 5'-diphosphate, chromium (III) salt (CrADP) were separated using Sephadex G-10 column chromatography. The isomeric purity of the two forms was monitored using high voltage electrophoresis and column chromatography. The same techniques were employed to assess the purity of the mono-, bi-, and tridentate forms of adenosine 5'-triphosphate, chromium (III) salt (CrATP). Distinct differences in the interaction of beef heart mitochondrial ATPase with the various isomers of chromium nucleotides were seen in kinetic studies. Monodentate CrADP was a competitive inhibitor of the ATP hydrolysis activity of both purified ATPase and submitochondrial particles. However, when ITPase activity was examined, noncompetitive inhibition was observed. The bidentate isomer of CrADP did not affect ATPase activity. Enzymatic synthesis of the transition state analog of ATP synthesis and hydrolysis, Pi-CrADP occurred exclusively with the monodentate isomer of CrADP. It was also found that only the mono- and tridentate forms of CrATP were potent inhibitors of ATP hydrolysis by beef heart mitochondrial ATPase. These results are discussed in terms of possible ATP synthesis and hydrolysis mechanisms.