Hydrophobic interaction of soluble 3′:5′ cyclic nucleotide phosphodiesterase with octyl-sepharose

Summary Soluble cyclic nucleotide 3:5 monophosphate phosphodiesterase (PDE) (EC 3.1.4.17) obtained from beef adrenal cortex as the 100,000 g/1.5 h supernatant is usually regarded as a very hydrophilic protein. However, when subjected to hydrophobic chromatography on Octyl-Sepharose CL 413 it reveals strong hydrophobic interaction with the column matrix. The chromatographic procedure leads to multiple but distinct forms of PDE which degrade cAMP beyond 5AMP to inosine, via adenosine. The same metabolic pathway was previously observed with a membrane bound multienzyme sequence. Even the soluble PDE forms separated by gel chromatography (Sephadex G 200, Sepharose S 200 and Sepharose 6B) and soluble PDE of other tissue (heart) displayed the same metabolic pattern. These findings indicate a linkage between PDE, nucleotidase and deaminase activities. The intimate association of the enzyme is additionally supported by the phenomenon of kinetic advantage clearly observed with the most hydrophobic PDE form. Its end product, inosine, is formed more rapidly from CAMP than from the intermediate 5AMP. This paradoxical phenomenon is explained by close physical proximity between the enzymes involved in the metabolic pathway. Furthermore, when the most hydrophobic PDE form was immobilized on Octyl-Sepharose, rather than loss of catalytic activity even higher enzyme activities were measured. It is suggested that the so-called multiple forms of soluble PDE-at least in part-represent more or less preserved forms of a native, membrane bound, multienzyme sequence which degrades cyclic nucleotides.     

Nuclear cytochrome-deficient mutants of Neurospora crassa: isolation, characterization, and genetic mapping.

Summary We have isolated twenty-six nuclear, singlegene cytochrome-deficient mutants of Neurospora crassa as an initial step toward the study of the structural components and regulatory mechanisms involved in the biogenesis of the mitochondrial cytochrome system. These mutants, together with two previously described mutants, cyt-1 and cyt-2, have been classified into six distinct groups on the basis of cytochrome phenotype: a) cytochrome aa ₃ deficiency (due to mutations affecting loci designated cya); b) cytochrome b deficiency (cyb-1 locus); c) cytochrome b deficiency with a partial deficiency of cytochrome aa ₃ (cyb-2 locus); d) deficiency of both cytochromes aa ₃ and b (cyt loci); e) deficiency of both cytochromes aa ₃ and c (cyt-2 locus); and f) partial deficiency of cytochromes aa ₃ and c (cyt-12 locus).Four of seven mutations affecting cya loci have been mapped and are located on linkage groups I, II, V, and VI. It is not yet known whether these genes code for structural components of cytochrome oxidase or have a regulatory function that affects synthesis or assembly of the enzyme. The cyb-1 and cyb-2 genes are located on linkage groups V and VI, respectively, and appear to code for regulatory elements that control the biogenesis of cytochromes b and aa ₃ . The positions of the cyt mutations that cause a simultaneous deficiency of cytochromes aa ₃ and b are dispersed throughout the genome, except for two gene clusters on the left arm of linkage group I. Some of these mutants may be deficient in mitochondrial protein synthesis. Two mutations, cyt-2 and cyt-12, are located on linkage groups VI and II, respectively, and appear to affect genes that code for components of a regulatory system that controls the biogenesis of cytochromes aa ₃ and c.     

Mass spectrometry of silylated flavonol O-glycosides

The electron impact mass spectra of 19 trimethyl silylated flavonol mono-, di- and -triglycosides are reported for the first time. All spectra show wel     

Ammoniteli der GattungParapatoceras aus dem Oberen Mitteljura des Süntels (östliches Wesergebirge,Niedersachsen)

The Parapatocerates from the Süntel (Weser mountains, Northwest Germany) are described being an utmost variable subspecies respectively geographical raceParapatoceras distans (?) bentzi (Potonié) with two possibly modificatorily separated form groups. They differ from the similarP. distans(?) crioconus (Buckman) (=Crioconites Buckman) of Chippenham (England) in primary quadrilobaty and further characteristics accessible only by precise analysis. To establish the speciesdistans Baugier & Sauzé and with this the genusParapatoceras, recovery of the holotype or a determination of a neotype is required. A very similar but probably quinquelobate specimen (Parapatoceras sp.) from the Macrocephalus oolite of Hanover seems to accentuate once more the taxonomically minor degree of the quadri- or quinquelobaty inParapatoceras.     

A heme-nonapeptide tracer for electron microscopy

Summary A heme-nonapeptide (H-9-P)¹, applicable to electron microscopic cytochemistry via peroxidase-like activity, was prepared by passing horse heart cytochrome c through a column with Sepharose and covalently attached trypsin. After purification by column chromatography (Sephadex G50 Superfine, Biogel P-2) a maximal yield of 50% and purity of >99% was achieved. A concise schedule allows for inexpensive preparation of H-9-P with standard laboratory equipment. H-9-P has the following properties: Its structure is (14) Cys-Ala-Gln-Cys-His-Thr-Val-Glu-Lys (22) with heme attached to Cys (14) and (17). MW=1630, pI=4.95, _E(max) ^{pH 7} = 397.5 nm, _{22 °C, pH 7} ^{397.5 nm} = 1.11 × 10⁵ [Liter/Mole x cm]. With the use of a diaminobenzidine-H₂O₂-medium — as applied for cytochemistry — we determined spectrophotometrically a pH_opt=12.5 and an apparent K₅ = 3.14 × 10^{– 3} [M]. Glutardialdehyde leads to considerable de-activation and, according to SDS-polyacrylamide-gel-electrophoresis, to diffuse crosslinking accompanied by a shift of the active pH-region towards neutral pH values. An attempt was made to optimize the cytochemical assay. The peroxidase-like activity of H-9-P is well comparable to that of other heme-tracers; only horseradish peroxidase has a higher turnover number. When injected to mice or added to cell suspensions, even high concentrations of H-9-P did not entail any signs of toxicity.Abbreviations AAA amino acid analysis - AHC ammoniumhydrogencarbonate - BSA bovine serum albumin - Cyt c cytochrome c - DAB 5,3-diaminobenzidine - GA glutardialdehyde - H-8-P heme-octapeptide - H-9-P heme-nonapeptide - H-11-P heme-undecapeptide - HR-POX horseradish peroxidase - MW molecular weight - PAGE polyacrylamid-gel-electrophoresis - pI isoelectric point - SDS sodiumdodecylsulphate - SG-TLC silicagel-thin-layer-chromatography This work was supported by the Österreichische Forschungsfonds     

The structure of the O-antigenic polysaccharide from lipopolysaccharide of Vibrio cholerae strain H11 (non-O1).

After acid degradation of the lipopolysaccharide (LPS) of Vibrio cholerae strain H11 (non-O1), a tetrasaccharide was obtained, the structure of which was determined by quantitative and methylation analyses, periodate oxidation, one- and two-dimensional NMR spectroscopy, and fast-atom-bombardment and four-sector tandem mass spectrometry as beta-D-GalANGro-(1-3)-beta-D-QuiNAc-(1-4)-alpha-D-GalANGr o-(1-4)-NeuAc, in which GalANGro is N-galacturonoyl-2-aminoglycerol and QuiN 2-amino-2,6-dideoxy-glucopyranose. In addition, the trisaccharide beta-D-GalANGro-(1-3)-beta-D-QuiNAc-(1-4)-D-altro-hept ulose and the disaccharide alpha-D-GalANGro-(1-4)-NeuAc were isolated from acid-degraded lipopolysaccharide; the occurrence of sedoheptulose in lipopolysaccharide has not been described before. Based on the result of methylation analysis showing that galacturonic acid was the terminal sugar of the polysaccharide chain, and on the assumption that the tri- and the disaccharide represented the reducing and the non-reducing ends of the polysaccharide, respectively, the chemical structure of the O-specific chain of V. cholerae H11 is proposed as alpha-D-GalANGro-(1-4)-alpha-NeuAc-(2-3)-beta-D-GalANGro-(1- 3)-beta-D-QuiNAc- (1-[4)-alpha-D-GalANGro-(1-4)-alpha-NeuAc-(2-3)-beta-D-GalANGro -(1-3)-beta-D- QuiNAc-(1-]n-(1-4)-D-altro-heptulose. However, other possible structures can not be ruled out since the tri- and the disaccharide could be localised at different positions.     

Heteropycnosis of an underreplicating chromosome

Drosophila nasutoides has an extraordinary genome since 62% of its DNA resides in chromosome4. This element mainly consists of constitutive heterochromatin which does not polytenize. Earlier studies of heterochromatin attributed little attention to the fact that condensed chromosomes often vary in condensation. This paper reports that chromosomes of the same complement display different degrees and kinetics of condensation. InD. nasutoides, even sex specific differences can be observed. The results of a comparative microphotometric study on neuroblast metaphases in both sexes revealed the following picture. The process of chromosome condensation is not restricted to mitotic prophase but continues into the metaphase. The mean condensation is not equal for all chromosomes. In the metaphase of the female, Feulgen density increases from theX chromosome, via3 and2, to chromosome4. In the male, the order isX, 2, 3, Y, and4. During the metaphase of the male, chromosomes condense with similar kinetics. In contrast, chromosomes of the female display asynchrony as monitored by area and length determinations. TheX chromosomes of the female probably have enhanced shortening during prophase. This would explain the metaphase of the female where theX chromosomes shorten less than the autosomes, and why each of theX chromosomes is 15% shorter than theX chromosome in the metaphase of the male. Further differences were observed in the longitudinal and lateral compaction of the chromosomes in males and females. The sex chromosomes and chromosome3 condense by shortening, while chromosomes2 and4 preferentially reduce their diameter. The large amount of DNA engaged in heteropycnosis and the isochromosome nature allow the identification of chromosome4 during interphase. At this stage, a new category of extreme DNA packaging was detected. The interphase density of chromosome4 can exceed that of metaphase by a factor of up to 8. Two events account for this high degree of condensation:(1) the homologues are particularly associated due to somatic pairing and (2) the arms are further tightened as a result of pericentric folding. The features of the isochromosome suggest that the interaction of chromatids during interphase is essentially caused by specific DNA sequences. The data confirm that heteropycnosis not only interferes with gene expression but also strongly inhibits DNA synthesis in endocycles.     

Continuous production of L-alanine from fumarate in a two-stage membrane reactor

Summary L-alanine was produced continuously from fumaric acid by means of soluble aspartase and L-aspartate--decarboxylase. The two reaction steps were carried out in two membrane reactors in series at different pH and temperature. The retention of the soluble enzymes within the reactor vessels was achieved by means of ultrafiltration membranes.     

Structural reactions to polarized light of microvilli in photoreceptor cells of the moth Spodoptera

Summary Intact armyworm moths (Spodoptera exempta, Farn. Noctuidae) were illuminated by polarized monochromatic light to induce structural changes in the rhabdomeres of the compound eyes. The degree of distortion of their microvilli depends on the light energy absorbed per time unit. Under polarized light, the number of quanta absorbed varies with the position of the plane of polarization relative to the axis of the microvilli (intrinsic dichroism). Therefore, in Spodoptera, different degrees of deformations could be demonstrated in differently oriented rhabdomeres of both types of ommatidia. Moreover, in rhabdoms of the lobed type with fan-like arranged microvilli, different reactions were regularly seen in differently oriented microvilli of one rhabdomere. This indicates that microvilli may react to light individually.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 114 (Bionach)     

Sequence complexity of poly(A) RNA fromPhysarum polycephalum

Summary Poly(A) RNA from S phase, G₂ phase and starved macroplasmodia of Physarum contain mRNA sequences which when translated in vitro, yield similar patterns of polypeptides after fluorography.Reassociation of nick-translated DNA (Cot) allows the isolation of highly labeled single copy DNA which, after saturation hybridization with poly(A) RNA, gives values of 23% for growth and 17% for starvation.Homologous cDNA/poly(A) RNA hybridization reactions (Rot) indicate that 22–28% of the genome is transcribed during growth and 12% during starvation and that about half of the cDNA reacts with 0.1% of the genome and could represent 50–80 RNA species, each present in about 1,000 copies per nucleus. Up to 25,000 different RNA species, 1–5 copies each per nucleus, are estimated to be present during growth, and about 15,000 during starvation. Heterologous cDNA/poly(A) RNA hybridization reactions (Rot) indicate that the RNA sequences in S and G₂ phase of the cell cycle are similar, with RNA sequences being more abundant in G₂ phase.During starvation about 25% of the sequences present during growth cannot be detected and those sequences present during growth have become diluted during starvation.