Electrophoretic characterization of the protein and glycoprotein content of purified Kurloff cell cytosol

Proteins and glycoproteins from Kurloff cells (KC) were analyzed by SDS-polyacrylamide electrophoresis, isoelectric focusing, and two-dimensional electrophoresis, and major cytosolic glycoproteins of Mr 30,000-35,000 and pHi 5.7-6.7 were characterized. After incubation with radiolabeled amino acids (L-35S) methionine and L-(U14C) leucine) and gel autoradiography, all the proteins seemed to be labeled. D-(U14C) glucosamine-labeled proteins and periodic-acid-Schiff(PAS)-positive proteins focalized at the same pH. These data suggest that the major glycoprotein are synthesized by the KC themselves and that the PAS-positive Kurloff body has an endogenous origin. Whereas estrogens increase the KC number, 10(-6) M estradiol had no effect on the KC protein electrophoretic pattern and protein biosynthesis, in agreement with the lack of estradiol receptor in the KC cytosol.     

Perinuclear organelles of the sensory cell of the Grandry cutaneous corpuscle. Ultrastructure and formation

Perinuclear organelles are found in the sensory cell of the Grandry cutaneous corpuscle in the duck. They are ovoid, fusiform or conical. They measure up to 5 µ length and l µ in diameter. They are formed by regular alternation of granular layers (mornolayers of RNA-rich granules which can be interpreted as ribosomes) and fibrous layers (generally formed by two sublayers of parallel fibrils). These fibrils (60-80 Å diameter) are in continuity with the intra-cytoplasmic fibrils which are very abundant in the Grandry cell. The central part of the organelles is devoid of RNA-rich granules.The formation of these organelles begins about one week after hatching, in the cytoplasmic perinuclear area where abundant granular endoplasmic reticulum and very numerous ribosomes and fibrils are present. The function of these perinuclear organelles remains unknown.     

Heterogeneite des variations spectrales photoinduites vers 700 nm observees sur les membranes chlorophylliennes et les complexes chlorophylle-proteines isoles de divers organismes photosynthetiques

A heterogeneity of photoinduced spectral variations near 700 nm is observed at −196°C, by comparison of difference spectra obtained from several materials (two blue-green algae, Spirulina and Fremyella; a red alga Porphyridium and a higher plant, Nicotiana tabacum). The maximum of complexity is seen with Spirulina chlorophyllous membranes where three bands are detected at 718, 705 and 695 nm. These bands are recovered in relatively different amounts in two chlorophyll-protein complexes prepared after sodium dodecyl sulfate electrophoresis of Spirulina membranes.

The photoinduced changes near 700 nm with Spirulina membranes are reversible and seem to be synchronous and related to the bleaching of three different pigments. This spectral diversity for photosensizable pigments is comparable with the spectral heterogeneity of the major forms of the bulk chlorophyll. It is assumed that these pigments may play the same functional role. This assumption may be related to a possible heterogeneity of Photosystem I centres recently detected by Breton using linear dichroism method (Breton, J. (1977) Biochim. Biophys. Acta 459, 66–70).     

Kinetic studies of the reaction mechanism of rat liver phosphoglycerate kinase in the direction of ADP utilization

The kinetic properties of rat liver phosphoglycerate kinase were investigated in the forward direction of the reaction (utilization of ADP). The kinetic studies were performed in an assay system using combined hexokinase/glucose-6-phosphate dehydrogenase as an ATP trap. The Km values for Mg ADP1- and 1,3-diphospho-D-glycerate were approximately 0.11 and 0.006 mM, respectively. Reciprocal plots of 1/v versus 1/ (Mg ADP1-) at different fixed concentrations of 1,3-diphospho-D-glycerate and 1/v versus 1/ (1,3-diphospho-D-glycerate) at different fixed concentrations of Mg ADP1- were apparently parallel. However, product inhibition studies (3-phospho-D-glycerate), dead-end inhibition studies (2,3-diphospho-D-glycerate), and adenosine and AMP inhibition patterns yielded results consistent with a rapid equilibrium random mechanism in which the binding of one substrate greatly decreases the affinity of the enzyme for the second substrate. Existence of two sites for 3-phospho-D-glycerate is suggested.     

Étude polarographique de la mobilisation du fer de la ferritine

Polarographic study of the mobilization of ferritin ironPolarographic study allows to propose a model for mobilization of ferritin iron: an equilibrium exists between iron core and small quantities of iron outside the protein.These iron atoms would be lying on electron acceptor sites including SH groups. The number of sites is dependent on iron content of ferritin.Therefore, the iron could be removed by the action of reducing agents such as xanthine oxidase or ascorbic acid, and then chelated by a complexing agent.     

Structural analysis of a cDNA clone from Xenopus laevis containing a repetitive sequence element

Total polysomal RNA from Xenopus laevis stage 40 embryos was probed for the presence of repetitive sequences by Northern blot analysis with a genomic DNA fragment which had previously been shown to contain several repetitive sequence elements (Spohr et al., 1981). The analysis revealed that various presumptive mRNAs contain sequences complementary to the repetitive probe. Consequently, a cDNA library was constructed and screened with the same probe. Forty-eight positive recombinants containing eucaryotic inserts of 300–700 base pairs were isolated and one such clone was characterized in detail. Analysis of its nucleotide sequence revealed the presence of an open reading frame for 118 amino acids. Comparison of nucleotide sequences located 3′ to this presumptive protein coding region with the sequence of the genomic DNA fragment used as a probe clearly identifies and allows one to define the exact location of the repetitive element in the cloned cDNA. This analysis shows furthermore that one portion of the repeated sequence is highly conserved in the two members of this repetitive sequence family, whereas the other part is more divergent. In this area blocks of oligonucleotides are scattered between nonhomologous DNA stretches. The occurrence frequency of the presumptive mRNAs which carry repetitive elements homologous to the used repetitive probe is suggested to be close to that of rare mRNAs.     

Construction of non-symmetric substitution matrices derived from proteomes with biased amino acid distributions

Automatic comparison of compositionally biased genomes, such as that of the malarial causative agent Plasmodium falciparum (82% adenosine + thymidine), with genomes of average composition, is currently limited. Indeed, popular tools such as BLAST require that amino acid distributions be similar in aligned sequences. However, the P. falciparum genome is so biased that six amino acids account for more than 50% of the protein composition. One reason for the comparison methods failure lies in the compositional difference between the query and the subject proteomes, which is not taken into account in the amino acid substitution matrices. This paper introduces a method to derive substitution matrices, in particular BLOSUM 62, in the frame of the information theory. It allows the construction of non-symmetrical matrices, taking into account the non-symmetric amino acid distributions. The dirAtPf family of matrices allowing the comparison of P. falciparum and A. thaliana is given as an example. This paper further provides an analysis of the obtained matrices in the frame of the information theory, supporting the discrimination advantage they bring.     

Actin-based motility as a self-organized system: mechanism and reconstitution in vitro

Site-directed actin polymerisation in response to signalling is responsible for the formation of cell protrusions. These elementary 'actin-based motility processes' are involved in cell locomotion, cell metastasis, organ morphogenesis and microbial pathogenesis. We have reconstituted actin-based propulsive movement of particles of various sizes and geometries (rods, microspheres) in a minimum motility medium containing five pure proteins. The ATP-supported treadmilling of actin filaments, regulated by Actin Depolymerizing Factor (ADF/cofilin), profilin and capping proteins provides the thermodynamic basis for sustained actin-based movement. Local activation of Arp2/3 complex at the surface of the particle promotes autocatalytic barbed end branching of filaments, generating a polarized arborescent array. Barbed end growth of branched filaments against the surface generates a propulsive force and is eventually arrested by capping proteins. Understanding the mechanism of actin-based movement requires elucidation of the biochemical properties and mode of action of Arp2/3 complex in filament branching, in particular the role of ATP binding and hydrolysis in Arp2/3, and a physical analysis of the movement of functionalised particles. Because the functionalisation of the particle by an activator of Arp2/3 complex (N-WASP or the Listeria protein ActA) and the concentrations of effectors in the medium are controlled, the reconstituted motility assay allows an analysis of the mechanism of force production at the mesoscopic and molecular levels.