Determination of protein in conidia

Determination of protein in conidi

Precursors of Cytochrome Oxidase in Cytochrome-Oxidase-Deficient Cells of Neurospora crassa

Three different cell types of Neurospora crassa deficient in cytochrome oxidase were studied: the nuclear mutant cni-1, the cytoplasmic mutant mi-1 and copper-depleted wild-type cells. * 1. The enzyme-deficient cells have retained a functioning mitochondrial protein synthesis. It accounted for 12–16% of the total protein synthesis of the cell. However, the analysis of mitochondrial translation products by gel electrophoresis revealed that different amounts of individual membrane proteins were synthesized. Especially mutant cni-1 produced large amounts of a small molecular weight translation product, which is barely detectable in wild-type. * 2. Mitochondrial preparations of cytochrome-oxidase-deficient cells were examined for precursors of cytochrome oxidase. The presence of polypeptide components of cytochrome oxidase in the mitochondria was established with specific antibodies. On the other hand, no significant amounts of heme a could be extracted. * 3. Radioactively labelled components of cytochrome oxidase were isolated by immunoprecipitation and analysed by gel electrophoresis. All three cell types contained the enzyme components 4–7, which are translated on cytoplasmic ribosomes. The mitochondrially synthesized components 1–3 were present in mi-1 mutant and in copper-depleted wild-type cells. In contrast, components 2 and 3 were not detectable in the nuclear mutant cni-1. Both relative and absolute amounts of these polypeptides in the enzyme-deficient cells were quite different from those in wild-type cells. * 4. The components of cytochrome oxidase found in the enzyme-deficient cells were tightly associated with the mitochondrial membranes. * 5. Processes, which affect and may control the production of enzyme precursors or their assembly to a functional cytochrome oxidase are discussed

Requirement of a Membrane Potential for the Posttranslational Transfer of Proteins into Mitochondsria

Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocatio

Late Reading: Erich Auerbach and the Spätboot of Comparative Literature

Focusing in particular on Erich Auerbach’s seminal essay ‘Philology of World Literature’ (1952), this essay proposes to re-examine the conceptualization of comparative literature in the post-WWII period not only from the perspective of its philological, but also from that of its historical self-understanding. Its principal concern will be to consider what it means to view this comparative philology as historical, which is to say in the context of how it emerges from the particular ‘historical perspectivism’ of the immediate post-war period. The category that best characterizes this philology, it will be argued, is that of late reading, a term that the essay coins as the hermeneutic counterpart to the artistic concept of late style. Characterized by its consciousness of coming at the end of the tradition of European high culture, late reading – at least in Auerbach’s understanding of it –makes its very lateness a constituent element of its hermeneutics. Out of this sense of lateness emerges, the essay will argue, a view of comparative literature as defined by its distance from the normative maturity of classical European culture – by what one might term, in Frank Kermode’s phrase, its ‘sense of an ending’. Auerbach’s conception of world philology does not ignore the increasing obsolescence of the Eurocentric perspective, but rather makes this obsolescence the basis of its synoptic purview. As such, it continues to offer a model for how comparative literature may engage with the legacy of high European culture whilst acknowledging the limitations of its perspective

Functional and Biogenetical Heterogeneity of the Inner Membrane of Rat-Liver Mitochondria

Rat liver mitochondria were fragmented by a combined technique of swelling, shrinking, and sonication. Fragments of inner membrane were separated by density gradient centrifugation. They differed in several respects: electronmicroscopic appearance, phospholipid and cytochrome contents, electrophoretic behaviour of proteins and enzymatic activities. Three types of inner membrane fractions were isolated. The first type is characterized by a high activity of metal chelatase, low activities of succinate-cytochrome c reductase and of glycerolphosphate dehydrogenase, as well as by a high phospholipid content and low contents of cytochromes aa3 and b. The second type displays maximal activities of glycerolphosphate dehydrogenase and metal chelatase, but contains relatively little cytochromes and has low succinate-cytochrome c reductase activity. The third type exhibits highest succinate-cytochrome c reductase activity, a high metal chelatase activity and highest cytochrome contents. However, this fraction was low in both glycerolphosphate dehydrogenase activity and phospholipid content. This fraction was also richest in the following enzyme activities: cytochrome oxidase, oligomycin-sensitive ATPase, proline oxidase, 3-hydroxybutyrate dehydrogenase and rotenone-sensitive NADH-cytochrome c reductase. Amino acid incorporation in vitro and in vivo in the presence of cycloheximide occurs predominantly into inner membrane fractions from the second type. These data suggest that the inner membrane is composed of differently organized parts, and that polypeptides synthesized by mitochondrial ribosomes are integrated into specific parts of the inner membrane

Different Transport Pathways of Individual Precursor Proteins in Mitochondria

Transport of mitochondrial precursor proteins into mitochondria of Neurospora crassa was studied in a cellfree reconstituted system. Precursors were synthesized in a reticulocyte lysate programmed with Neurospora mRNA and transported into isolated mitochondria in the absence of protein synthesis. Uptake of the following precursors was investigated: apocytochrome c, ADP/ATP carrier and subunit 9 of the oligomycin-sensitive ATPase. Addition of high concentrations of unlabelled chemically prepared apocytochrome c (1–10 μM) inhibited the appearance in the mitochondrial of labelled cytochrome c synthesized in vitro because the unlabelled protein dilutes the labelled one and because the translocation system has a limited capacity [apparent V is 1–3 pmol × min−1× (mg mitochondrial protein)−1]. Concentrations of added apocytochrome c exceeding the concentrations of precursor proteins synthesized in vitro by a factor of about 104 did not inhibit the transfer of ADP/ATP carrier or ATPase subunit 9 into mitochondria. Carbonylcyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation, inhibited transfer in vitro of ADP/ATP carrier and of ATPase subunit 9, but not of cytochrome c. These findings suggest that cytochrome c and the other two proteins have different import pathways into mitochondria. It can be inferred from the data presented that different 'receptors' on the mitochondrial surface mediate the specific recognition of precursor proteins by mitochondria as a first step in the transport process

Biosynthesis of Mitochondrial Porin and Insertion into the Outer Mitochondrial Membrane of Neuruspora crassa

Mitochondrial porin, the major protein of the outer mitochondrial membrane is synthesized by free cytoplasmic polysomes. The apparent molecular weight of the porin synthesized in homologous or heterologous cell-free systems is the same as that of the mature porin. Transfer in vitro of mitochondrial porin from the cytosolic fraction into the outer membrane of mitochondria could be demonstrated. Before membrane insertion, mitochondrial porin is highly sensitive to added proteinase; afterwards it is strongly protected. Binding of the precursor form to mitochondria occurs at 4°C and appears to precede insertion into the membrane. Unlike transfer of many precursor proteins into or across the inner mitochondrial membrane, assembly of the porin is not dependent on an electrical potential across the inner membrane

Cell-Free Synthesis of the Mitochondrial ADP/ATP Carrier Protein of Neurospora crassa

ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence

On the magnetism of Ln{2/3}Cu{3}Ti{4}O{12} (Ln = lanthanide)

The magnetic and thermodynamic properties of the complete Ln$_{2/3}$Cu$_3$Ti$_4$O$_{12}$ series were investigated. Here $Ln$ stands for the lanthanides La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, and Yb. %Most of the compounds were prepared as single phase polycrystalline powder %without any traces of impurities. Marginal amounts of %impurities $(< 2$ were detected $Ln=$ Gd, Er, and Tm. %Significant amounts of impurity phases were found for $Ln=$ Ce and Yb. All the samples investigated crystallize in the space group $Im\bar{3}$ with lattice constants that follow the lanthanide contraction. The lattice constant of the Ce compound reveals the presence of Ce$^{4+}$ leading to the composition Ce$_{1/2}$Cu$_3$Ti$_4$O$_{12}$. From magnetic susceptibility and electron-spin resonance experiments it can be concluded that the copper ions always carry a spin $S=1/2$ and order antiferromagnetically close to 25\,K. The Curie-Weiss temperatures can approximately be calculated assuming a two-sublattice model corresponding to the copper and lanthanide ions, respectively. It seems that the magnetic moments of the heavy rare earths are weakly coupled to the copper spins, while for the light lanthanides no such coupling was found. The $4f$ moments remain paramagnetic down to the lowest temperatures, with the exception of the Tm compound, which indicates enhanced Van-Vleck magnetism due to a non-magnetic singlet ground state of the crystal-field split $4f$ manifold. From specific-heat measurements we accurately determined the antiferromagnetic ordering temperature and obtained information on the crystal-field states of the rare-earth ions. The heat-capacity results also revealed the presence of a small fraction of Ce$^{3+}$ in a magnetic $4f^1$ state.Comment: 10 pages, 10 figure