Studies on Mitochondrial Proteins. II. Localization of Components in the inner membrane labelling with diazobenzenesulfonate,a non-penetrating probe

The topography of the inner membrane of rat liver mitochondria was studied using a probe, diazobenzenesulfonate, which interacts preferentially with surface components. Inner membranes were examined both in a native orientation as found in the intact mitochondrion or in an inverted state as found in isolated inner membranes prepared by sonication.Enzyme inactivation as a consequence of diazobenzenesulfonate labeling was employed to determine the localization of a number of inner membrane activities. In inner membranes labeled on the outer surface, NADH and succinate oxidation were strongly inhibited while ATPase and $\text{ascorbate}\text{-N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenylene-diamine}$ (TMPD) oxidase activities were unaffected. In inner membranes labeled on the inner surface. ATPase and succinate oxidation were inactivated while NADH oxidation and ascorbate-TMPD oxidase were unaffected. Succinate dehydrogenase was inhibited only by labeling the inner surface while NADH dehydrogenase was inhibited to a similar extent by treatment of either surface.Sodium dodecylsulfate-polypeptides (66 000 and 26 000) on the outer surface of the inner membrane and five polypeptides (80 000, 66 000, 51 000-48 000, and 26 000) on the inner surface. These results indicate a highly asymmetric localization of inner membrane components.     

Resolution and chromatographic configuration analysis of 2-hydroxy fatty acids

2-Hydroxyoctadecanoic acid was resolved into D and L isomers as salts of 1-phenylethylamine enantiomers The diastereomers of phenylethylamides of 2-hydroxy fatty acids and the corresponding derivatives with protected hydroxy group (acetyl, methyl, trifluoro-acetyl, trimethylsilyl) are well separated by thin-layer or gas-liquid chromatography. This allows a simple microanalysis of configuration and optical purity of 2-hydroxy fatty acids. With this method 2-hydroxy fatty acids from sphingomyelin of the honey-bee were shown to belong exclusively to the D series.     

Arterio-venöse Kurzschlüsse mit Klappenmechanismen im Bereich der dorsalen Haut der Rattenhinterpfote

Zusammenfassung In der dorsalen Haut der Hinterpfoten von Ratten wurden Klappen am Abgang kleinerer Gefäße von großen Arterien beobachtet. Diese Arterien verlaufen in einer subkutanen, größere Gefäße und Nerven führenden Bindegewebsschichte über den Streckersehnen.Die kleineren Gefäße haben den Wandbau einer Vene und zweigen etwa im rechten Winkel von der Arterie ab. An ihrer Abgangsstelle ist neben den Klappen manchmal ein sphinkterartiger Muskelring ausgebildet.Diese Gefäßabschnitte werden für arterio-venöse Anastomosen gehalten und auf die Bedeutung solcher an dieser Stelle bisher nicht beschriebenen Verschlußeinrichtungen für die Regulation der Kurzschlüsse wird hingewiesen.

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Arteriovenous anastomoses with valve mechanisms in the dorsal skin of the hindpaw of rats

Summary In the dorsal skin of the hindpaws of rats valves were observed at those sites where smaller vessels branched from larger arteries. These arteries run in a subcutaneous layer of connective tissue, which lies above the extensor tendons and in which the larger vessels and nerves are found.These smaller vessels, the walls of which resemble those of veins, were seen branching off at approximate right angles to their artery of origin. Besides the valves a sphincterlike muscle ring was observed in some cases at the point of branching.These vascular segments are held to be arteriovenous anastomoses. Such locking devices at these points have not been described until now and seem to be important in the regulation of arteriovenous shunts.

     

Die Ultrastruktur des Epithels des Wolffschen Ganges und des Ductus deferens beim Schafembryo

Zusammenfassung Die Epithelzellen der Wolffschen Gänge bzw. der Samenleiter von 28 Schafembryonen der Größe von 1,1–45 cm Scheitel-Steiß-Länge (SSL) wurden elektronenmikroskopisch untersucht. Der Golgiapparat von Embryonen in der Phase der aktiven Urnierensekretion zeichnet sich durch Zisternen mit kondensiertem Inhalt aus, von denen sich kleine coated vesicles abschnüren. Bei Feten mit inaktiver Urniere wurden keine Zisternen mit kondensiertem Inhalt beobachtet. Die Abschnürung kiemer coated vesicles vom Golgiapparat tritt jedoch in allen untersuchten Altersstadien auf. Eine zweite Population großer coated vesicles schnürt sich vom apikalen, basalen und interzellulären Plasmalemm ab, besonders häufig beim fast geburtsreifen Fetus. Die Vereinigung von Golgivesikeln mit einem Inhalt von mittlerer Elektronendichte mit multivesikulären Körpern kann in allen untersuchten Stadien festgestellt werden. Glykogen findet sich bereits bei 1,1 cm SSL in Lagern; ab ca. 20 cm SSL ist eine erhebliche Glykogenvermehrung zu beobachten. Stereozilien und glattes endoplasmatisches Retikulum treten erst ab 45 cm SSL auf.

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Fine structure of the epithelia of the Wolffian duct and of the vas deferens in fetal sheep

Summary The ultrastructure of the epithelium of the Wolffian duct and of the vas deferens of fetal sheep ranging from 1,1 to 45 cm CRL has been studied. The Golgi apparatus of specimens possessing an actively secreting mesonephros shows condensation of the contents of the cisternae with budding of small coated vesicles. In the fetuses possessing inactive mesonephros no condensation of the contents of Golgi cisternae is to be observed, but the budding of small coated vesicles continues. A second population of large coated vesicles originating by invagination of the plasma membrane occurs on the periphery of the epithelium cells, namely in the apical cell poles of the near term fetuses. The convergence and subsequent fusion of Golgi vesicles of medium electron density is already found in patches in the 1,1 cm CRL fetus; it increases in abundance in specimens of more than 20 cm CRL. Stereocilia and agranular endoplasmatic reticulum appear in full-term sheep fetuses.

     

Proteins of the thermophilic fungus Humicola lanuginosa. II. Some physicochemical properties of a cytochrome c

A cytochrome c from Humicola lanuginosa is unique among eukaryotic cytochromes c in having phenylalanine as Residue 74. This protein has certain properties which differ from those of other cytochromes c to which it is generally similar. The Humicola cytochrome c is as stable as horse heart cytochrome c in urea, but more stable than both horse heart and yeast cytochromes c in acidic and alkaline conditions. Spectrophotometric titration of the four tyrosyl residues of the Humicola protein was nonsigmoidal with a pK_app of 11.4. Solvent perturbation difference spectra indicate that 50% of the tyrosyl residues are exposed to solvent in the native protein, and that the single tryptophanyl and all four tyrosyl residues become exposed in 8 m urea. Certain unusual features in both the optical rotatory dispersion and circular dichroism spectra in the 290-250-nm region are tentatively attributed to the substitution of phenylalanine for tyrosine at position 74.     

The molecular weight and substrate specificity of 20 β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

The molecular weight of 20β-hydroxysteroid dehydrogenase was 111,000 when determined by agarose gel fitration and 106,000 by density gradient centrifugation. From gel electrophoresis in sodium dodecyl sulfate, after treatment with urea and 2-mercaptoethanol, the molecular weight was 27,000, consistent with the native molecule containing four subunits. After gel electrophoresis at pH 8.1, a single band was detected which stained for protein and activity with 5α-pregnan-20β-ol-3-one and 5α-androstan-3α,17β-diol. 20β-hydroxysteroid dehydrogenase was inactivated at pH 4.5 and the time course of inactivation was independent of the steroid used for activity measurements. Steroid substrates did not protect 20β-hydroxysteroid dehydrogenase against acid inactivation or affect enzyme fluorescence. It was concluded that the activity observed with the two substrates occurred at the same active center and that under the experimental conditions little steroid was bound to the enzyme in the the absence of coenzyme.     

Prophage lambda at unusual chromosomal locations. II. Mutations induced by bacteriophage lambda in Escherichia coli K12

An Escherichia coli strain deleted for the primary λ attachment site was lysogenized with λ at secondary sites. Some lysogens became mutants because of prophage insertion in the affected gene. Mutagenesis by phage λ is not random with respect to the gene affected: most mutants were pro, although certain other genes could be mutated at lower frequencies. In the case of several independent ilv and gal mutants, the sites of prophage insertion were in the same segment of the ilv region and galT gene respectively. The galT location may also be a preferred site for the insertion of DNAs other than prophage λ. Insertion of prophage λ within an operon can reduce the expression of operator-distal genes. A trpC λ insertion mutant expresses the operator-distal trpB function constitutively at a low level. This expression probably derives from a promoter located in the left arm of the prophage.     

Analysis of the transformation effect in cytochrome b559 of photosystem II in terms of the model of the heme-quinone redox interaction

Transformation of three-component redox pattern of cytochrome (Cyt) b559 in PS II membrane fragments upon various treatments is manifested in decrease of the relative content (R) of the high potential (HP) redox form of Cyt b559 and concomitant increase in the fractions of the two lower potential forms. Redox titration of Cyt b559 in different types of PS II membrane preparations was performed and revealed that (1) alteration of redox titration curve of Cyt b559 upon treatment of a sample is not specific to the type of treatment; (2) each value of R_HP defines the individual shape of the redox titration curve; (3) population of Cyt b559 may exist in several stable forms with multicomponent redox pattern: three types of three-component redox pattern and one type of two-component redox pattern as well as in the form with a single E_m; (4) transformation of Cyt b559 proceeds as successive conversion between the stable forms with multicomponent redox pattern; (5) upon harsh treatments, Cyt b559 abruptly converts into the state with a single E_m which value is intermediate between the E_m values of the two lower potential forms. Analysis of the data using the model of Cyt b559-quinone redox interaction revealed that diminution of R_HP in a range from 80 to 10% reflects a shift in redox equilibrium between the heme group of Cyt b559 and the interacting quinone, due to a gradual decrease of 90?mV in E_m of the heme group at the virtually unchanged E_m of the quinone component.     

Probing the role of Valine 185 of the D1 protein in the Photosystem II oxygen evolution

In Photosystem II (PSII), the Mn₄CaO₅-cluster of the active site advances through five sequential oxidation states (S₀ to S₄) before water is oxidized and O₂ is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824–6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV–visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking Tyr_Z and the halide. The V185 contributes to the stabilization of S₂ into the low spin (LS), S?=?1/2, configuration. Indeed, in the V185T mutant a high proportion of S₂ exhibits a high spin (HS), S?=?5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S₁Tyr_Z?→?S₂^HSTyr_Z transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S₂^LSTyr_Z?→?S₃Tyr_Z transition was observed and we propose that it occurs in the S₂^LSTyr_Z?→?S₂^HSTyr_Z intermediate step before the S₂^HSTyr_Z?→?S₃Tyr_Z transition occurs. The dramatic slowdown of the S₃Tyr_Z?→?S₀Tyr_Z transition in the V185T mutant does not originate from a structural modification of the Mn₄CaO₅ cluster since the spin S?=?3?S₃ EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.