Participation of cytochrome P-450 in the steroid 17alpha-hydroxylase of testis microsomes.

The effect of metyrapone on the activity of the steroid 17alpha-hydroxylase from rat testis was evaluated. A competitive pattern of inhibition was observed after analysis of data using a least mean squares computer analysis. The substrate for the hydroxylase induced a Type I difference spectrum in an active suspension of Triton treated microsomes. The magnitude of this spectral change was dependent on steroid concentration and was diminished by metyrapone. The effect of metyrapone was abolished at infinite steroid concentration. These results confirm the participation of cytochrome P-450 as a reactant in the 17alpha-hydroxylase reaction.     

Metabolism of the obligatory aerobic yeast Rhodotorula gracilis

Summary D-Glucose and D-xylose addition to not-growing Rhodotorula gracilis cells brings about alterations in pyruvate kinase and phosphoenolpyruvate carboxykinase activities characteristic for glycolysis and gluconeogenesis, respectively.Abbreviations used PK Pyruvate kinase (EC 2.7.1.40) - PEPCK Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) - PFK Phosphofructokinase (EC 2.7.1.11)     

Sheathed Cells in Cultures of Clostridium sporogenes

Many strains of Clostridium sporogenes were shown to contain two types of cells which exhibited strikingly different growth habits. Over 99% of the population of most strains were motile bacilli which occurred singly or in short chains. Infection by any of several C. sporogenes bacteriophages lysed most of these cells and revealed a minority population component consisting of cells which grew in extremely long chains. Each chain was surrounded by and contained in a long tubular polysaccharide sheath which was ultrastructurally quite separate and distinct from the cell walls of the enclosed cells. The sheathed cells were identical to "normal" cells of C. sporogenes in anaerobiosis, Gram reaction, sporulation, deoxyribonucleic base composition, general morphology, and ultrastructure. They differed from the "normal" cells in having a sheath, in being nonmotile, and in that they were infected by C. sporogenes bacteriophages but not usually lysed by them. The sheathed cells appeared spontaneously in cultures cloned from single colonies and were demonstrably present in cultures before bacteriophage infection. Thus, they were not contaminants but were normal, although inconspicuous, growth forms of C. sporogenes which were selected but not induced by bacteriophage infection.     

Evidence for active chloride accumulation in normal and denervated rat lumbrical muscle

Intracellular Cl- activity (aiCl) was measured with Cl(-)-sensitive microelectrodes in normal and denervated rat lumbrical muscle. In normal muscle bathed in normal Krebs solution, aiCl lay close to that predicted by the Nernst equation. The addition of 9-anthracene carboxylic acid, which blocks Cl- conductance, caused aiCl to increase far above that predicted by a passive distribution. Furosemide (10 microM) reversibly blocked this accumulation. After muscle denervation, aiCl progressively increased for 1-2 wk. The rise occurred in two stages. The initial stage (1-3 d after denervation) reflected passive Cl- accumulation owing to membrane depolarization. At later times, aiCl continued to increase, with no further change in membrane potential, which suggests an active uptake mechanism. This rise approximately coincided with the natural reduction in membrane conductance to Cl- that occurs several days after denervation. Na+ replacement, K+ replacement, and furosemide each reversibly blocked the active Cl- accumulation in denervated muscle. Quantitative estimates suggested that there was little difference between Cl- flux rates in normal and denervated muscles. The results can be explained by assuming that, in normal muscle, an active accumulation mechanism operates, but that Cl- lies close to equilibrium owing to the high membrane conductance to Cl-. The rise in aiCl after denervation can be accounted for by the membrane depolarization, the reduction in membrane Cl- conductance, and the nearly unaltered action of an inwardly directed Cl- "pump."     

Orientation of cultured arterial smooth muscle cells growing on cyclically stretched substrates

Arterial smooth muscle cells from rabbit aortic media were grown in first subcultures on hydrophilized and collagen-coated silicone membranes which were then subjected to directional cyclic stretches and relaxations at a frequency of 50 times/min. The membranes were stretched 2, 5 and 10% beyond their resting length. Cells on unstretched and stationary membranes in the same chamber served as controls. The cells which were stretched with an amplitude of 2% remained in random orientation after 14 days of continuously performed cyclic stretching. The cells which were stretched 5% for 12 days orientated at an angle of 61 +/- 9 degrees to the direction of stretching, while the cells which were stretched with an amplitude of 10% for 6 days orientated at an angle of 76 +/- 5 degrees. The cells on the stationary and unstretched membranes remained in random orientation. We were able to confirm that the angle of orientation is reversible, i.e. preorientated cells changed their orientation during application of another stretching amplitude. The results suggest that stretching of the artery wall by blood pulsation may be a factor influencing the orientation of smooth muscle cells within the media of the artery wall and of those smooth muscle cells which proliferate into the subendothelial space after mechanical injury of the endothelium or electrical stimulation of the artery wall. An apparatus is presented which produces cyclic and directional mechanical stimuli similar to those which may occur in the artery wall.     

Cloning and characterization of several dominant-negative and tight-binding mutants of lac repressor

Using both general recombination and molecular cloning techniques, 13I-, I-d and Itb missense mutations in the lacI gene were transferred from F'lacIq episomes to ColE1 derivative plasmids. Two deletion derivatives of the lacI genes encoding the wild-type (wt) and the tight-binding (Itb) B3 and B5 repressors were also constructed. The mutant repressors were examined for polypeptide size and stability, and for binding to the inducer isopropyl-beta-D-thiogalactoside (IPTG). Several of the I-d repressors were shown to be partially degraded in vivo, in confirmation of earlier results based on [14C]IPTG binding [Miwa and Sadler, J. Mol. Biol. 117 (1977) 843-868]. The sizes of polypeptides produced by lacI deletion derivatives were consistent with expectations based on the extent of deletion and the location of termination sites within the plasmid sequence. The first 400 bp of several mutant lacI genes were sequenced. Our wt lacI gene differs from another wt lacI sequence (Farabaugh, 1978), containing a single bp change that results in an Ala to Thr substitution at amino acid (aa) 109. We identified bp substitutions and the resultant aa changes for two Itb and two I-d genes; the positions correlated with prior genetic mapping data. Three of these new changes were in the N-terminal domain (headpiece) of repressor, with one change in the core domain at aa 99.     

Base substitution mutants of the lac operator: in vivo and in vitro affinities for lac repressor

16 single-site mutations and a 1-bp deletion in the lac operator have been cloned and examined with regard to repressor binding. A 13-bp, central ‘core’ operator sequence, bp 5–17 of the natural operator, was also synthesized and cloned. Repressor affinity was assessed in vivo by quantitating the level of β-galactosidase activity resulting from chromosomal operon derepression and in vitro by measuring the stability of repressor-operator complexes. Our results support the general conclusion that the repressor-operator interaction is asymmetric, particularly across the center of the operator sequence, with little or no specific contact at position 12. Some sequence changes in the right side of the operator markedly reduced repressor affinity, indicating that although binding to this half of the sequence has been suggested to be less important than the left half, it still significantly contributes to the binding affinity.     

The effect of metyrapone on the 17,20-lyase from rat testis microsomes

A new and simple method was presented to isolate purified holoenzyme of $\text{E. coli}$ RNA polymerase. When a purified enzyme preparation was chromatographed on a DNA-cellulose column equilibrated with a buffer containing 10mM MgCl₂, holoenzyme was separated from core enzyme. Thus holoenzyme was eluted at 0.15M KCl and core enzyme at 0.25M KCl.     

Zur Atmung Wachsender Wurzelspitzen

Zusammenfassung Es werden Daten über den Protein-Stickstoffgehalt und die Atmung isolierter Wurzelabschnitte vonZea Mays undPisum sativum mitgeteilt und nach Prüfung ihrer Zuverlässigkeit festgestllt, daß auf den Protein-N bezogen die Meristeme beider Objekte weniger intensiv atmen als die Streckungszonen, welch letztere bei der Erbse sogar noch von der jüngsten Zone ausgewachsener Zellen übertroffen wird.Wie schon vonRuhland undUllrich (1936) undRuhland undRamshorn (1938) festgestellt wurde, scheiden isolierte Wurzelspitzen mehr CO₂ aus, als ihrer gleichzeitigen O₂-Aufnahme entspricht, d. h. sie gären. Dieses Extra-CO₂ stammt nicht aus einer früheren Phase partieller Anaerobiose, muß also während des Versuches gebildet werden. Längere Abschnitte liefern weniger Extra-CO₂, als ihrem Anteil embryonaler Gewebe entspricht. Neben einer Steigerung der Gärung durch die Präparation, besonders in den jüngst ausgewachsenen Partien, ist aus dem Gaswechsel auf einen Austausch von Intermediärprodukten zu schließen, deren weitere Verarbeitung zu dem nahezu ausgeglichenen Gaswechsel längerer Wurzelspitzen führen dürfte. Die CO₂-Produktion sinkt, während der Versuchszeit ganz erheblich ab, die Ausscheidung von Extra-CO₂ mit ähnlicher Geschwindigkeit auch in Proben verschiedener Ausgangslänge. Möglicherweise vorhandene Beziehungen zwischen diesem Verhalten und dem bekannten Sinken der meristematischen Aktivität isolierter Organteile werden diskutiert.Mit 5 Textabbildungen.     

The 93-kDa glycine receptor-associated protein binds to tubulin.

A peripheral membrane protein with a relative molecular mass of 93,000 Da is associated with cytoplasmic domains of the inhibitory glycine receptor of mammalian spinal cord. Here, evidence is given that this 93-kDa protein binds to polymerized tubulin. First, tubulin cofractionated with the 93-kDa protein upon affinity purification of the glycine receptor. Second, tubulin bound to the isolated 93-kDa protein in an overlay procedure. Third, in assays containing the purified glycine receptor, the 93-kDa protein as well as the glycine receptor alpha and beta subunits coassembled with tubulin and microtubules. The interaction of the 93-kDa protein with tubulin displayed high affinity (KD approximately 2.5 nM) and significant cooperativity (Hill coefficient approximately 2.1) and approached a stoichiometry of approximately 1:4 under saturating conditions. These data suggest that the 93-kDa protein anchors the glycine receptor at postsynaptic sites via binding to subsynaptic tubulin.