Application of a three-dimensional drawing procedure to the evaluation of series of protein samples after analysis by gel electrophoresis and other methods

Membrane-Bound F1 ATPase from Micrococcus Sp. ATCC 398E. Purification and Characterization by Affinity Chromatography

A chemically reactive ATP analogue, 6-[(3-carboxy-4-nitrophenyl)thio]-9-β-D-ribofuranosylpurine 5′-triphosphate (Nbs6ITP) has been synthesized. It has the ability to form stable thioether bonds between the 6-position of the purine ring and aliphatic mercapto groups. The nucleotide moiety of the reagent has been covalently bound to agarose, via iminobispropylamine and N-acetyl-homocysteine as spacer with the purpose of producing an affinity chromatography material. The affinity matrix binds solubilized F1 ATPase from a crude extract of Micrococcus sp. membranes. Afterwards the enzyme can be selectively eluted from the column at a defined ATP concentration. This method is superior to the conv…

Rapid detergent exchange in solutions of the membrane protein bacteriorhodopsin by preparative high-performance liquid chromatography (HPLC)

Quantitative Bestimmung von Sterigmatocystin in verschimmelten pflanzlichen Nahrungsmitteln

Ein Verfahren fur die quantitative Bestimmung von Sterigmatocystin in pflanzlichen Nahrungsmitteln durch zweidimensionale DC-Trennung eines Extraktes und Fluorescenzintensitatsmessung der mit AlCl3 behandelten DC-Platte wird beschrieben. Durch die hier vorgeschlagene Auftrageweise auf der DC-Platte ist ein rasches Identifizieren und Auswerten von Probe- und Eichflecken moglich. Als Extraktionsmittel hat sich Acetonitril-KCl-Losung am besten bewahrt. Versuche mit zugesetztem Toxin zeigten Verluste von weniger als 5 %.

Quantitative Bestimmung von Trichothecenen HT-2 Toxin in verschimmelten pflanzlichen Nahrungsmitteln

Es wird ein Verfahren zur quantitativen Bestimmung von HT-2 Toxin in verschimmeltem Reis, Mais, Weizen, Hafer, Roggen bzw. Erbsen beschrieben. Extrahiert wurde mit einer Mischung von 9 Teilen (Volumen) Acetonitril und einem Teil wasriger 4%iger KCl-Losung. Die Extrakte musten teilweise chromatographisch vorgereinigt werden. Die terminale Bestimmung erfolgte nach zweidimensionaler dunnschichtchromatographischer Trennung durch Fluorescenzintensitatsmessung der mit H2SO4 behandelten DC-Platten. Versuche mit zugesetztem HT-2 Toxin ergaben eine Wiederfindung von 88% (Variationskoeffizient 7).

Nucleinsäuren und Proteinbiosynthese

Nucleinsauren, besonders die DNA (deoxyribonucleic acid; im deutschen Schrifttum auch Desoxynucleinsauren bzw. DNS), sind als Trager der genetischen Information von grundsatzlicher Bedeutung. In Form der mRNA (messenger ribonucleic acid), tRNA (transfer ribonucleic acid) und rRNA (ribosomal ribonucleic acid) sind sie wesentlich an der Biosynthese der Proteine beteiligt. Abbildung 11.1 fast wichtige Wechselbeziehungen zwischen Nucleinsauren und Proteinen zusammen.

ERA-experiment “space biochemistry”

Abstract The general goal of the experiment was to study the response of anhydrobiotic (metabolically dormant) microorganisms (spores of Bacillus subtilis, cells of Deinococcus radiodurans, conidia of Aspergillus species) and cellular constituents (plasmid DNA, proteins, purple membranes, amino acids, urea) to the extremely dehydrating conditions of open space, in some cases in combination with irradiation by solar UV-light. Methods of investigation included viability tests, analysis of DNA damages (strand breaks, DNA-protein cross-links) and analysis of chemical effects by spectroscopic, electrophoretic and chromatographic methods. The decrease in viability of the microorganisms was as exp…

Quantitative Bestimmung der Penicillins�ure in pflanzlichen Lebensmitteln

Es wird ein Verfahren zur quantitativen Bestimmung der Penicillinsaure in pflanzlichen Lebensmitteln (Erbsen, Reis, Haferflocken und Kokosraspeln) angegeben. Als Extraktionsmittel wird ein Gemisch von Dichlormethan/Methanol (1 + 1) benutzt. Die terminale Abtrennung der Penicillinsaure erfolgt durch zweidimensionale Dunnschichtchromatographie. Durch Umsetzung mit Diphenylborsaure-2-ethanolamin in einem Tauchverfahren wird die Penicillinsaure auf der Platte in ein stark fluorescierendes Derivat verwandelt (maximale Emission bei 440–445 nm nach Anregung bei 365–370 nm).

Analysis of T-2 toxin by HPLC and GC in samples of corn and oats

HPLC is the only physico-chemical method for the analysis of trichothecenes for which no derivatization is necessary. Hence a combination of different methods can be performed. For exclusion of any faulty interpretation of data and in order to decrease the detection limit HPLC should be followed by GC.

Photoaffinity cross-linking of F1ATPase from the thermophilic bacterium PS3 by 3′-arylazido-β-alanyl-2-azido ATP

AbstractThe photoactivatable bifunctional 3′-arylazido-β-alanyl-2-azido ATP (2,3′-DiN3ATP) has been applied to study the localization of the nucleotide-binding sites of coupling factor 1 (F1ATPase, TF1) from the thermophilic bacterium PS3 by photoaffinity cross-linking. UV irradiation of TF1 in the presence of 2,3′-DiN3ATP results in the nucleotide-dependent formation of various higher molecular mass cross-links formed by two, three or even four α- and/or β-subunits. The differences observed upon photoaffinity cross-linking by the bifunctional 2-azido ATP or 8-azido ATP analog are discussed. They are probably due to the varied maximal distance between both azido groups, or to the different …

Production and characterization of a monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol.

A monoclonal antibody was obtained by the fusion of mouse myeloma cells with splenocytes isolated from Balb/c mice, which had been immunized with diacetoxyscirpenol-hemiglutarate (DAS-hemiglutarate) and verrucarol-hemiglutarates covalently bound to ethylenediamine-modified bovine serum albumin. The anti-DAS-antibody that could be induced was of the IgM type with kappa-chains. The titer of the monoclonal anti-DAS-antibody in ascites fluid obtained from mice injected the selected cell line was much higher than those of conventional antisera. An enzyme-linked immunosorbent assay based on the competitive binding principle in which the antibody was applied had a sensitivity of 1 ng DAS per assay…

Oxidativer Endabbau und ATP-Synthese

Eukaryoten und aerobe Prokaryoten setzen Pyruvat (Zwischen- oder Endprodukt vieler Abbauwege) zu einem wesentlichen Teil unter Oxidation in „aktive Essigsaure“ um. Diese Reaktion wird durch den Multienzymkomplex Pyruvat-De-hydrogenase katalysiert, der bei Eukaryoten im inneren Kompartiment der Mito-chondrien lokalisiert ist. Auser durch die Pyruvat-Dehydrogenase wird Acetyl-CoA vor allem auch durch das Enzymsystem der s-Oxidation der Fettsauren gebildet. Dieses Enzymsystem befindet sich gleichfalls im inneren Mitochondrien-kompartiment. Der Acetyl-Rest von Acetyl-CoA wird schlieslich im Citrat-Cyclus, dessen Enzyme im Matrixraum und in der inneren Membran der Mitochondrien lokalisiert sind,…

Response ofBacillus subtilis spores to dehydration and UV irradiation at extremely low temperatures

Spores of Bacillus subtilis have been exposed to the conditions of extreme dehydration (argon/silica gel; simulated space vacuum) for up to 12 weeks at 298 K and 80 K in the dark. The inactivation has been correlated with the production of DNA-double strand-breaks. The temperature-dependence of the rate constants for inactivation or production of DNA-double strand-breaks is surprisingly low. Controls kept in the frozen state at 250 K for the same period of time showed no sign of deterioration. In another series of experiments the spores have been UV irradiated (253.7 nm) at 298 K, 200 K and 80 K after exposure to dehydrating conditions for 3 days. Fluence-effect relationships for inactivati…

High-performance liquid chromatography of trichothecenes

Kohlenhydrate und ihr Stoffwechsel

Kohlenhydrate sind neben den Proteinen unsere wichtigsten Nahrungsmittel. Die sturmische Entwicklung der Biochemie im ersten Drittel unseres Jahrhunderts ist mit der Aufklarung der Einzelschritte des anaeroben Abbaus von Glucose und anderen Kohlenhydraten untrennbar verbunden. Daher soll hier der Kohlenhydrat-Stoffwechsel als erster Stoffwechselprozes besprochen werden.

Isolation and partial characterization of a cytochrome-o complex from chromatophores of the photosynthetic bacterium Rhodospirillum rubrum FR1.

A cytochrome-o complex was isolated from chromatophores of photoheterotrophically grown Rhodospirillum rubrum FR1. The enzyme was extracted with the non-denaturating detergent taurodeoxycholate and subsequently purified by sucrose-density-gradient centrifugation and gel-permeation HPLC. The complex contains two types of cytochromes, one of them cytochrome o, and two copper atoms. It catalyzes the reduction of molecular oxygen, when N,N,N',N'-tetramethyl-p-phenylenediamine or ubiquinol 10 are offered as electron donors. The oxidase activity is inhibited by cyanide, carbon monoxide and 2-heptyl-2-hydroxyquinoline N-oxide. The molecular mass of the protein is 136 +/- 15 kDa. The subunit analys…

Coenzyme und Vitamine

Die Funktion der Coenzyme besteht vor allem in der Unterstutzung des Enzyms bei der Substratbindung und der Vorbereitung (Orientierung, Polarisierung) des Substrats auf die Umsetzung, sowie auch in der Bindung der Intermediarprodukte.

Topologie der Zelle

Die einzelnen Reaktionen des Zellstoffwechsels vollziehen sich in der Regel in entsprechend spezialisierten Bereichen (Kompartimenten) der Zelle. Die Kompartimente sind bei hoheren Zellen haufig durch Proteolipidmembranen (weniger als 10 nm stark) voneinander getrennt. Die durch Membranen innerhalb der Zelle abgegrenzten Strukturen nennt man auch Zellorganellen. Die Spezialisierung einzelner Kompartimente auf bestimmte Stoffwechselfunktionen und der dadurch erforderliche Stofftransport von Kompartiment zu Kompartiment bringt der Zelle u.a. den Vorteil, Stoffwechselreaktionen nicht nur uber die katalytische Aktivitat der einzelnen Enzyme, sondern auch uber den Stofftransport durch Membranen …

Evidence for essential primary amino groups in a bacterial coupling factor F1ATPase.

Abstract We have found that the binding of pyridoxal-5′-phosphate to 6 primary amino groups leads to the inactivation of the enzyme. A preferential reaction of pyridoxal-5′-phosphate with the α-subunits of this enzyme can be demonstrated. The reactivity of the amino groups is influenced by various effectors. In the presence of ATP the inhibition of the ATPase activity is noncompetitive.

Conversion of the Ca2+-ATPase from Rhodospirillum rubrum into a Mg2+-dependent enzyme by 1,N6-etheno ATP

Nucleoside triphosphate hydrolysis of R.rubrum ATPase complexes can be changed from Ca2+-dependence to Mg2+-dependence by replacing ATP with 1,N6-etheno ATP. Four ATPase complexes which have been prepared by different procedures hydrolyze ATP and 1,N6-etheno ATP at different rates in dependence on the added metal ions. These differences allow an easy distinction of the various enzyme forms.

Incorporation of ATP synthetase into long-term stable liposomes of a polymerizable synthetic sulfolipid

Purification of a cytochrome aa3 terminal oxidase from protoplast membrane vesicles of Micrococcus luteus

Abstract A cytochrome aa 3 terminal oxidase was isolated from protoplast membrane vesicles of Micrococcus luteus grown under aerobic conditions. The purified complex showed similarities to cytochrome c oxidase (EC 1.9.3.1) of the electron transport chain of mitochondria and many prokaryotes. The enzyme was solubilized by subsequent treatment with the detergents CHAPS and n- dodecyl -β- d - maltoside and purified by ion-exchange chromatography using poly- l -lysine agarose and TMAE-fractogel-650 (S) columns, followed by hydroxyapatite chromatography. The purified complex is composed of two major subunits with apparent molecular masses of 54 and 32 kDa. After purification the isolated enzyme …

Prebiotic polypeptides and the origin of biological information.

Recent data on the origin of biological information are reviewed. These data corroborate the view that polyamino acids were the first informational polymers. The source of information is seen in the chemical reactivity of amino acids, their prebiotic abundance and the prebiotic environment. Evidence is presented in favor of Matsuno's protohypercycle that may have preceded Eigen's hypercycle, but that involves a translation of information from polypeptides into that of polynucleotides.

Photobiology in space: An experiment on Spacelab I

The joint European/US Spacelab Mission I, scheduled for October 1983 for a 9 day lasting Earth-orbiting flight, provides a laboratory system for various disciplines of science, including exobiology. On the pallet, in the experiment ES 029 "Microorganisms and Biomolecules in Space Hard Environment" 316 dry samples of Bacillus subtilis spores will be exposed to space vacuum and/or selected wavelenghs of solar UV radiation. After recovery action spectra of inactivation, mutation induction, reparability and photochemical damage in DNA and protein will be determined. The results will contribute to the understanding of the mechanism of the increased UV sensitivity of bacterial spores in vacuo and…

Purification, subunit structure, and kinetics of the chloroform-released F1ATPase complex from Rhodospirillum rubrum and its comparison with F1ATPase forms isolated by other methods.

Abstract A stable and homogeneous adenosine-5ʹ-triphosphatase (ATPase, EC 3.6.1.3) has been solubilized from Rhodospirillum rubrum (R . rubrum) chromatophores by chloroform extraction. Purification of the Ca2+-dependent ATPase activity was 200-fold. Ca2+ can be replaced by Mg2+, Cd2+, and Mn2+ .The Km for Ca-ATP (0.17 mᴍ) is increased about 5-fold during solubilization of the enzyme, whereas the Km values for Mg-ATP (0.029 mᴍ) and Cd-ATP (0.014 mᴍ) are not affected. The chloroform-released ATPase has a molecular weight of 400,000 ± 30,000 and consists of the following subunits (molecular weights in parenthesis): α (58,000), β (53,500), γ (39,000), δ (18,500), and ε (14,000). The amino acid …

Exobiological experiments on the first EURECA mission

Preparative separation of phospholipids by flash-HPLC

Struktur und Eigenschaften der Proteine

Proteine sind fur fast samtliche Funktionen der Zelle von grundsatzlicher Bedeutung: Als Enzyme sind sie die Trager aller biokatalytischen Funktionen, als Transportproteine in den Zellmembranen und Korperflussigkeiten sind sie fur den selektiven Stofftransport verantwortlich, als kontraktile Proteine vermitteln sie die Umwandlung chemischer Energie in mechanische Arbeit, als Skieroproteine haben sie Stutz- und Gerustfunktionen, um nur einige wesentliche Funktionen zu nennen.

Eigenschaften der Aminosäuren und Peptide

Weit uber die Halfte der Trockensubstanz der meisten Zellen besteht aus Proteinen. Die Bausteine der Proteine sind Aminosauren. Sie sollen daher zunachst behandelt werden.

3'-Arylazido-beta-alanyl-2-azido ATP, a cross-linking photoaffinity label for F1ATPases.

Abstract The synthesis of the 3′-arylazido-2-azido ATP derivative 3′-O-{3-[N-(4-azido-2-nitrophenyl)-amino]propionyl}2-azido-adenosine 5′-triphosphate (2,3′-DiN3ATP) is described. The bifunc­ tional photoreactive ATP analog is characterized spectroscopically. Photoaffinity labeling of F, ATPase from Micrococcus luteus by this analog results in the inactivation of the enzyme and in the formation of higher molecular weight cross-links,

8-Azido-adenosine 5'-triphosphate as a Photoaffinity Label for Bacterial F1 ATPase

1. 8-Azido-adenosine 5'-triphosphate (n83ATP) is a suitable photoaffinity label for F1 ATPase from Micrococcus luteus. The nucleotide is a substrate in the presence of bivalent cations and inhibits the enzyme irreversibly upon irradiation with ultraviolet light above 300 nm. 2. More than 80% of the label is covalently bound to the beta subunits in the presence of bivalent cations. Labeling and inactivation is decreased by protection with ADP, ATP or adenyl-5'-yl imidodiphosphate. To a much smaller degree the alpha subunits also become labeled. 3. n83AMP does not specifically bind to the beta subunits upon irradiation. Like n83ATP and n83ADP, it also labels the alpha subunits to a small exte…

Properties of the F0F1 ATPase Complex from Rhodospirillum rubrum Chromatophores, Solubilized by Triton X-100

1. A cold-stable oligomycin-sensitive F0F1 ATPase complex from chromatophores of Rhodospirillum rubrum FR 1 was solubilized by Triton X-100 and purified by gel filtration. 2. The F0F1 complex is resolved by sodium dodecyl sulfate electrophoresis into 14 polypeptides with approximate molecular weights in the range of 58000--6800; five of these polypeptides are derived from the F1 moiety of the complex which carries the catalytic centers of the enzyme. 3. The purified F0F1 complex is homogeneous according to analytical ultracentrifugation and isoelectric focusing. 4. The molecular weight as determined by gel filtration is about 480 000 +/- 30 000. S020,w is 1.45 +/- 0.1 S and the pI is 5.4. 5…

Structural dynamics in F1ATPase during the first reaction cycle of ATP hydrolysis

Abstract The velocity of ATP hydrolysis, catalyzed by purified F 1 ATPase from Micrococcus luteus , was decelerated on decreasing the temperature. At 13′C one reaction cycle is completed after 20 s. Hydrolysis was triggered upon rapid mixing of the enzyme with ATP. During the first reaction cycle, succeeding structural alterations of the F 1 ATPase were traced by time resolved X-ray scattering. The scattering spectra obtained from consecutive intervals of 1 s, revealed the F 1 ATPase to pass a conformational state exhibiting an expanded (6%) molecular shape. The expanded state was observed between 45% and 65% of the time required to complete the reaction cycle. This pointx out a conformatio…

Coreconstitution of bacterial ATP synthase with monomeric bacteriorhodopsin into liposomes. A comparison between the efficiency of monomeric bacteriorhodopsin and purple membrane patches in coreconstitution experiments

The conditions for coreconstitution of a bacterial ATP synthase and bacteriorhodopsin into lecithin liposomes and for light driven ATP synthesis have been optimized. A rate of maximally 280 nmol ATP min-1 mg ATP synthase-1 was achieved with monomerized bacteriorhodopsin compared with a rate of up to 45 nmol ATP min-1 mg-1 found for proteoliposomes containing bacteriorhodopsin in the form of purple membrane patches. The different rates are explained by the finding that monomeric bacteriorhodopsin is more homogeneously distributed among the liposomes than the purple membrane patches. The final activities depended on both the purification method for the two proteins and the coreconstitution pr…

Gesetzmäßigkeit biochemischer Systeme und Determination ihrer Evolution

Beim Studium der Struktur und Funktion lebender Systeme und ihrer Bausteine drangen sich immer wieder grundsatzliche Fragen auf, fur die wir bislang keine befriedigende Antworten kennen: Lebende Systeme bestehen aus leblosen Molekulen. Es gelten fur sie dieselben Naturgesetze wie fur ihre leblose Umwelt; was also ist ein lebendes System? Die Bausteine der lebenden Systeme unserer Erde sind neben dem Wasser ganz uberwiegend Kohlenwasserstoff-Verbindungen. Gibt es auf anderen Himmelskorpern eine Alternative im Sinne von anderen, „exotischen“ Biochemien? Die Grundsatze der Funktion und der Bewahrung von genetischen Informationen sowie ihrer Mutation sind fur alle lebenden Systeme gleich. Es la…

Prebiotic Evolution and the Origin of Life: Chemical and Biochemical Aspects

Evolution, as the term is used here, signifies any development or change adapting to the environment. Chemical evolution connotes changes of chemical substances, it thus signifies that changes occur fundamentally in the molecules. Frequently “chemical evolution” is used synonymously for “abiotic” or “prebiotic formation” of organic molecules in a cosmic system, usually on the prebiotic (or primitive) Earth. It is then assumed that the organic molecules were formed from the constituents of the primitive atmosphere, hydrosphere, and — in part — lithospere.

Light-driven proton transport of bacteriorhodopsin incorporated into long-term stable liposomes of a polymerizable sulfolipid

Abstract The chromoprotein bacteriorhodopsin from Halobacterium halobium has been incorporated into liposomes made of a fully synthetic, polymerizable lipid. Bacteriorhodopsin is found to be active in these polymer liposomes. The advantage in the use of such polymer systems concerning long-term stability in comparison with liposomes made of natural lipid is demonstrated.

Überleben im Weltraumvakuum — Leben aus dem All? —

Die Frage nach dem Ursprung des Lebens auf der Erde kann bisher niemand beantworten. Wir wissen nur, das alle heute bekannten Lebensformen auf einen gemeinsamen Ursprung zuruckgefuhrt werden konnen und das man diesen Ursprung etwa 3,8 Milliarden Jahre zuruckverfolgen kann [1]. Palaontologische und palaobiochemische Befunde machen wahrscheinlich, das die vermutlich einzelligen Lebewesen dieser Zeit hinsichtlich ihres Stoffwechsels schon hoch entwickelt waren. Isotopenuntersuchungen an fossilen Kohlenstoffverbindungen dieser Epoche lassen zum Beispiel vermuten, das Kohlendioxid reduktiv assimiliert wurde, moglicherweise bereits durch Photosynthese. Da andererseits vor mehr als 4 Milliarden Ja…

Lipide und ihr Stoffwechsel

Die Lipide unterteilt man meist in die eigentlichen Fette (Neutralfette, Wachse) und die Lipoide (Phospholipide, Glycolipide, Steroide und Carotinoide).

Survival under vacuum

Quantitative determination of ochratoxin A in vegetable foods

A simple method for the quantitative determination of ochratoxin A (OA) in rice and other vegetable foods (oatmeal, coconut flakes and peas) is described. This procedure implies an acetonitrile-4% KCl -6N HCl (88+10+2) extraction of the acidic OA, subsequent twodimensional thin-layer chromatography (TLC) and detection by fluorescence after exposure to ammonia fumes (excitation at 340 nm; emission at 475 nm). The quantitative detection limit for OA in rice or coconut flakes is 2.4–4 μg/kg and the recovery is 96%. For oatmeal and peas the detection limit is only 20 μg/kg because of the interference by other metabolites.

111 Bioluminescence analysis and numerical evaluation of ATP-synthesis by native and reconstituted membranes containing bacterial ATP-synthase

ATP-synthase is a large membrane protein complex, which plays a key role in the energy metabolism of most organisms. It consists of at least eight types of subunits and can be isolated and purified from several organisms, e.g. bacteria. The enzyme couples two reversible reactions: vectorial proton transport through a membrane and synthesis of the energy rich molecule ATP. Both can be investigated with vesicles from native membranes or with reconstituted liposomes from purified ATPsynthase. The analysis is complicated because ATP-synthase catalyzes ATP-synthesis as well as ATP-hydrolysis. Furthermore the ATP level of membrane samples is influenced by adenylate kinase activities of other enzy…

The Evolution of Individuality at the Molecular and Protocellular Levels

The most important bioelements (= organoelements) hydrogen, carbon, oxygen and nitrogen, are also the most abundant elements throughout the Universe besides helium, neon, and silicon (Fig, 1). In the Universe carbon is about four times as abundant as silicon. Certainly, the abundance of elements in various celestial bodies may vary greatly depending on the history of these celestial bodies.

Multianalysenmethode zur routinemäßigen Bestimmung der Aflatoxine B1, B2, G1 und G2 sowie von Citrinin, Ochratoxin A, Patulin, Penicillinsäure und Sterigmatocystin in verschimmelten Nahrungsmitteln

Eine einfache Methode zur routinemasigen Bestimmung der Aflatoxine B1, B2, G1 und G2 sowie von Citrinin, Ochratoxin A, Patulin, Penicillinsaure und Sterigmatocystin in verschimmeltem Reis, Weisbrot und anderen pflanzlichen Nahrungsmitteln wird beschrieben. Die Mycotoxine werden zunachst mit Acetonitril/4% KCl und Cyclohexan extrahiert und dann mit Dichlormethan ausgeschuttelt. Die Aflatoxine lassen sich nach einer 2-dimensionalen Dunnschicht-Chromatographie in Chloroform/Aceton=9/1 und Dichlormethan/Acetonitril=8/2 fluorometrisch quantitativ bestimmen. Die ubrigen Mycotoxine werden in Toluol/Ethylacetat/Ameisensaure=6/3/1 und Benzol/Essigsaure=8/2 getrennt; zur einwandfreien Abtrennung von …

Conservation of optical purity of amino acids: a principal problem in biochemical and proto-biochemical systems.

Dor L-amino acids, regardless of their state (peptide-bound or free, in the solid state or in aqueous solution), tend to racemize. In a living cell this racemization is usually compensated by specific degradation and replacement of the unwanted polypeptides that contain the wrong enantiomers. But a few long-lived proteins that are synthesized at or near birth are never replaced. Well investigated is the racemization of L-aspartic acid at a rate of 0.1 to 1.14 per cent per year in proteins from lenses and dentine. Increased racemization of eye lens proteins has been related to a form of human eye disease known as brunescent cataracts. Also quite well investigated is the racemization of amino…

Solubilization of an oligomycin-sensitive ATPase complex fromRhodospirillum rubrumchromatophores and its inhibition by various antibiotics

High-performance liquid chromatography of the mycotoxin sterigmatocystin and its application to the analysis of mouldy rice for sterigmatocystin.

The Origin of Life: More Questions Than Answers

AbstractMore than 30 years of experimentation on the origin of life in the fields of chemical and molecular evolution have led to a better perception of the immensity of the problem of the origin of life on Earth rather than to its solution. At present all discussions on principal theories and experiments in the field either end in stalemate or in a confession of ignorance. New lines of thinking and experimentation must be tried. The continued exploration of our solar system, especially a better knowledge of Mars and Venus, of comets and carbonaceous meteorites may also lead to a better understanding of the prebiotic environment on Earth and will thus help us to design more appropriate preb…

Survival in extreme dryness and DNA-single-strand breaks.

A wide variety of organisms (the so-called "anhydrobiotes') is able to survive long periods of time in a state of utmost dehydration and can thus survive in extremely dry environments including artificially imposed or space vacuum. Known strategies of survival include the accumulation of certain polyols, especially disaccharides, which help prevent damage to membranes and proteins. Here we report that DNA in vacuum-dried spores is damaged to a very substantial degree by processes leading to DNA strand breaks. Most of these lesions are obviously repaired during germination, but extensive damage to DNA and enzymes after long exposure times (months to years) finally diminish the chances of sur…

Chemical and catalytical properties of thermal polymers of amino acids (proteinoids)

The significance of thermal polyamino acids (proteinoids) as abiotic predecessors of proteins is reviewed on the basis of new experimental results. Most proteinoids yield only 50% to 80% amino acid upon acid hydrolysis. They contain 40% to 60% less peptide links than typical proteins, whereas their average nitrogen content is like that of proteins. The arrangement of amino acid residues is nonrandom. The degree of nonrandomness is difficult to determine because unusual crosslinks disturb most of the sequencing methods typically applied in protein chemistry. The products obtained in a polymerization experiment are heterogeneous. They can be separated into a limited number of related fraction…

Photoaffinity labeling of the coupling factor 1 from the thermophilic bacterum PS3 by 8-azido ATP

AbstractTo localize the nucleotide binding sites of the F1ATPase (TF1) from the thermophilic bacterium PS3 we have used 14C-labeled 8-azido ATP (8-N3ATP) as photoaffmity label. 8-N3ATP is hydrolyzed by the F,ATPase in the absence of ultraviolet light. Irradiation by ultraviolet light of the enzyme in the presence of 8-N3ATP results in reduction of ATPase activity and in preferential nucleotide specific labeling of the α subunits (0.8–0.9 mol 8-N3ATP/TF1,α:β = 4:1). Inactivation and labeling do not depend on the presence of Mg2+. Both effects decrease upon addition of various nucleotide di- or triphosphates.

UV-induced cross-linking of Tet repressor to DNA containing tet operator sequences and 8-azidoadenines.

The synthesis of 8-azido-2'-deoxyadenosine-5'-triphosphate is described. The photoreactive dATP analog was characterized by thin layer chromatography, proton resonance spectroscopy, infrared spectroscopy and UV spectroscopy. Its photolysis upon UV irradiation was studied. After incorporation of this dATP analog into DNA containing the tet operator sequence the investigation of the interactions between tet operator DNA and Tet repressor protein by UV photocross-linking becomes possible. Photocross-linking of protein to DNA was demonstrated by the reduced migration of the DNA in SDS polyacrylamide gel electrophoresis. Addition of the inducer tetracycline prior to UV irradiation significantly …

Quantitative Bestimmung von Cyclopiazonsäure in pflanzlichen Lebensmitteln

Es wird ein Verfahren zur quantitativen Bestimmung von Cyclopiazonsaure in pflanzlichen Lebensmitteln beschrieben. Nach einer Extraktion mit Acetonitril/ 4%iger KCl (180+20) und Abtrennung mit Dichlormethan wird der Extrakt dunnschicht-chromatographisch zweidimensional aufgetrennt. Die Dunnschichtplatten werden nach der Behandlung mit Oxalsaure mit Methanol vorchromatographiert. Damit konnten Schwanzbildungseffekte vermieden werden. Die Detektion erfolgt nach Umsetzung mit Dimethylaminobenzaldehyd durch Absorptionsmessung in Remission bei 540 nm auf der Dunnschichtplatte. Im Bereich von 50–500 ng/Fleck ist die Toxinmenge der Peakflache proportional. Die Wiederfindungsraten liegen zwischen 7…

Energy-Linked Reactions Catalyzed by the Purified ATPase Complex (F0F1) from Rhodospirillum rubrum Chromatophores

1. The isolation of the F0F1-ATPase complex from Rhodospirillum rubrum chromatophores by the use of taurodeoxycholate is described. 2. The enzyme preparation contains about 12 polypeptides; five are subunits of the F1 moiety. 3. The ATPase activity of the purified enzyme is dependent on the addition of phospholipids. 4. Km-vales for Mg2+-ATP and Ca2+-ATP are similar to the values obtained for the membrane-bound enzyme. 5. The F0F1-ATPase complex is more than 70% inhibited by oligomycin and N,N′-dicyclohexyl-carbodiimide. 6. The F0F1-ATPase complex was integrated into liposomes. The reconstituted proteoliposomes catalyzed energy transduction as shown by ATP-dependent quenching of acridine dy…

Abbau der Proteine und Stoffwechsel der Aminosäuren

Neben den Kohlenhydraten sind die Proteine die wichtigsten Nahrungsstoffe. Man hat ermittelt, das im erwachsenen Menschen taglich etwa 400 g korpereigenes Protein hydrolytisch gespalten und neu synthetisiert werden. Davon werden etwa 60 bis 80 g nach der Hydrolyse zu Aminosauren oxidativ abgebaut oder in Glucose umgewandelt. Zu etwa 50% kann dieser Anteil durch Eigensynthese aus anderen Metaboliten wieder gedeckt werden (nichtessentielle Aminosauren), etwa die Halfte der in Proteinen vorkommenden Aminosauren (essentielle Aminosauren) kann jedoch nicht im menschlichen Organismus resynthetisiert werden. Dieser Anteil mus in jedem Fall mit der Nahrung aufgenommen werden.

Alterations of Activities of Ribonucleases and Polyadenylate Polymerase in Synchronized Mouse L Cells

The activities of the three known catabolic and the one anabolic polyadenylate enzymes have been determined in synchronized L5178y cells: endoribonuclease, exoribonuclease, 5'-nucleotidase and poly(A) polymerase (Mg2+-dependent). These four enzymes were found primarily in the nuclear fraction. The activity of poly(A) polymerase remains essentially constant during the transition from G1 to S phase. However, the poly(A) catabolic enzyme activities increase parallel with DNA synthesis; the endoribonuclease activity increases 4-fold during G1 to S phase, the exoribonuclease and the nucleotidase activities increasing 30-fold and 16-fold. During the S phase the poly(A)-degrading enzymes are far m…

F1-ATPase from Micrococcus sp. ATCC 398. Purification by Ion-Exchange Chromatography and Further Characterization. (Auto)proteolysis and Dissociative Effects

The preparation of highly purified F1-ATPase from Micrococcus sp. ATCC 398 by application of DEAE-Sepharose CL-6B chromatography as final step is described. This enzyme consists of five subunits of different molecular weight: alpha (65000), beta (55000),gamma (35000), delta (20000), and epsilon (17000). Disc electrophoresis on 5% polyacrylamide gels removes the epsilon-polypeptide yielding an active ATPase complex with four different subunits: alpha, beta, gamma, delta. Additionally, by variation of the ionic strength delta can (partly) removed allowing the isolation by disc electrophoresis of an active ATPase complex which consists only of three different subunits alpha, beta, and gamma. I…

Filter paper disk techniques for assay of nucleotidase

A DE filter disk technique for assaying the activity of nucleotidase is described. This method is based on the observation that nucleotides bind to the filters at 5 mM Tris-HCl (pH 7.8) while nucleosides do not. As parameter for the nucleotidase activity the decrease of bound nucleotides is determined. In parallel experiments the amount of the product (nucleoside) formed can be measured by DEAE Sephadex column chromatography. The filter disk technique can be applied for the determination of vmax and Km of a nucleotidase by using different ribonucleosidase monophosphate substrates.

Regulation und Integration des Stoffwechsels

Wie bereits in Abschn. 2.4 geschildert wurde, ist die eukaryotische Zelle in viele durch Membranen von einander getrennte Raume (Kompartimente) geteilt. So findet der oxidative Abbau der Fettsauren oder des Pyruvats in den Mitochon-drien statt. Die Fettsaure-Synthetase, das Enzymsystem der Fettsauresynthese (s. Abschn. 9.4.1), ist dagegen im Cytosol lokalisiert. Aber auch im Cytosol liegen die einzelnen Enzyme der Glycolyse, der Glucogenese, der Glucoseoxidation, der Fettsauresynthese usw. sicher nicht statistisch verteilt vor. Hinweise auf spezielle Mikrobereiche fur die einzelnen Enzymsysteme (Mikrokompartimentierung) konnen beispielsweise in der Bindung der Glucose-6-phosphatase (ein Sch…

HPLC: a tool for the analysis of T-2 toxin and HT-2 toxin in cereals.

An analytical procedure for the determination of trichothecenes in various cereals is described. HPLC was performed with a reversed-phase (C18) column eluted with methanol:water (60:40, v/v). Compounds were detected with a refractive index detector. The elution patterns of free and contaminated samples were compared. The recovery of added T-2 toxin (2 and 5 micrograms/g) in rye and wheat was approximately 80%. The application of this method allows for combined use with other sensitive methods such as mass spectrometry and gas chromatography. The described method is operationally simple, relatively inexpensive, and requires no derivatization.

Purification and molecular weight determination of the membrane protein cytochrome o-complex from Rhodospirillum rubrum by high-performance liquid chromatography (HPLC)

Time-dependent monomerization of bacteriorhodopsin in triton X-100 solutions analyzed by high-performance liquid chromatography

Abstract Bacteriorhodopsin from Halobacterium halobium was monomerized in Triton X-100 solutions. The process of delipidation was monitored by size-exclusion high-performance liquid chromatography under conditions that preserved the native conformation of the protein. The effects on the process of monomerization of the concentration and pH of the Triton X-100 solutions were investigated. The monomeric bacteriorhodopsin separated was active in light-dependent proton translocation when incorporated into soy bean lecithin liposomes.

Bestimmung von T-2-Toxin in pflanzlichen Nahrungsmitteln

Ein Verfahren zur quantitativen Bestimmung von T-2-Toxin in verschimmeltem Reis bzw. Mais durch Extraktion, dunnschicht-chromatographische Trennung und Fluorescenzintensitatsmessung der mit H2SO4 behandelten DC-Platte wird vorgestellt. Fluorescenzabsorptions- und Fluorescenzemissionsspektrum werden angegeben. Die Abhangigkeit der Mycotoxinkonzentration von der Fluorescenzintensitat wird durch eine Gleichung beschrieben. Die analytische Detektion von T-2-Toxin last sich durch ein 4-(p-Nitrobenzyl)pyridin-Derivat und dessen Absorptionsspektrum bestatigen. Versuche mit zugesetztem T-2-Toxin zeigten einen Analysenfehler von weniger als 10%.

Enzyme und Biokatalyse

Enzyme (Fermente) sind biologische Katalysatoren. Die Substanz (Metabolit), deren chemische Umsetzung sie katalysieren, nennt man Substrat.

Self-instructed condensation of amino acids and the origin of biological information

In contemporary cells biological information is largely stored in nucleic acids. Therefore, a prerequisite in many theories on the origin of cellular life is the pre-existence of self-replicating polynucleotides that had to be formed by abiotic processes on the prebiotic Earth. It is usually assumed that the spontaneous synthesis of a self-replicating polynucleotide could take place readily. However, serious stereochemical obstacles exist which make such a synthesis extremely improbable. Amino acids, on the other hand, which are abundantly formed in prebiotic simulation experiments, are relatively easily polymerized to macromolecules (protoproteins) that share with modern proteins many prop…

Pump and Displacement Currents of Reconstituted ATP Synthase on Black Lipid Membranes

Purified ATP synthase (F0F1) from Rhodospirillum rubrum was reconstituted into asolectine liposomes which were then adsorbed to a planar lipid bilayer. After the addition of an inactive photolabile ATP derivative (caged ATP), ATP was released after illumination with u.v.-light, which led to a transient current in the system. The transient photocurrent indicates that the vesicles and the planar membrane are capacitatively coupled. Stationary pump currents were obtained after addition of protonophores. These currents are specifically inhibited by oligomycin and stimulated threefold by inorganic phosphate (Pi). In analogy oligomycin sensitive pump currents in the reverse direction coupled to n…

UV-induced cross-linking of proteins to plasmid pBR322 containing 8-azidoadenine 2′-deoxyribonucleotides

Abstract An efficient method of cross-linking DNA to protein is described. The method is based on the incorporation of photoactive 8-azidoadenine 2′-deoxyribonucleotides into DNA. We have found that 8-N 3 dATP is a substrate for E. coli DNA polymerase I and that 8-N 3 dATP can be incorporated into plasmid pBR322 by nick-translation. Subsequently we were able to cross-link a set of different proteins to 8-azido-2′-deoxyadenosine-containing pBR322 by UV irradiation (366 nm). No DNA-protein photocross-linking was observed under the same conditions when the non-photoactive plasmid pBR322 was used.

Quantitative determination of moniliformin in vegetable foods and feeds

A suitable and simple method for the quantitative determination of moniliformin in vegetable foods and feeds is described. The mycotoxin was extracted by Soxhlet extraction with methanol from mouldy maize, rice, rye, oats, wheat and barley samples. Moniliformin was determined by thin-layer chromatography (TLC) using N-methylbenzthiazolon-2-hydrazone (MBTH) as a new derivatization reagent for this mycotoxin. The moniliformin derivative was assayed at 518 nm. Quantification could be performed after calibration. A linear relationship between mycotoxin amount and peak area was found from 100 to 400 ng/spot. The detection limit is 75 ng/spot.

Desiccation by Exposure to Space Vacuum or Extremely Dry Deserts: Effect on Microorganisms

General Limits of Growth at Low Water Activities Dormant Life Molecular Events Induced by Desiccation Survival at Extremely Low Water Activity Survival Under Extremely Dry Desert Conditions Keywords: anhydrobiosis; desiccation; extreme environments; freeze-drying; panspermia thesis; space: survival in space; space vacuum; water activity

Eine einfache Darstellung von 8-Azidoadenosin-5′-triphosphat; ein Agens zur Photoaffinitätsmarkierung von ATP-bindenden Proteinen

Eine vereinfachte Synthese von 8-Azido-ATP (3) wird beschrieben. Die neue Methode ist kurzer und fuhrt zu wesentlich besseren Ausbeuten als die bekannte Methode. Neben 3 entstehen dabei 8-Azido-ADP und 8-Azido-AMP. Es wurden mit einem Reaktionsansatz alle drei 8-Azidonucleotide gleichzeitig erhalten. 8-Azido-ATP (3) wurde durch Elementaranalyse, Dunnschichtchromatographie, Infrarotspektroskopie und UV-Absorptionsspektroskopie charakterisiert. Die Photolyse bei verschiedenen pH-Werten wurde untersucht. Durch F1ATPase wird 3 umgesetzt. A Facile Synthesis of 8-Azidoadenosine 5′-Triphosphate; a Photoaffinity Label for ATP-Binding Proteins A simple procedure for the synthesis of 8-azido-ATP (3) …