Isolation and some properties of troponin T kinase from rabbit skeletal muscle.

A method for isolation of troponin T kinase (ATP-protein phosphotransferase, EC 2.7.1.37) from rabbit skeletal muscles in proposed. The method gives a 7000-10 000-fold purification and results in an enzyme with specific activity of 400-800-nmol x min-1 x mg-1 of protein. The molecular weight of tropin T kinase as determined by gel filtration exceeds 500 000. Electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate revealed that isolated preparations of the enzyme consisted of at least three distinct proteins with apparent mol.wt. of 50 000, 46 000 and 31 000. The enzyme phosphorylates isolated troponin T at a rate which exceeds the phosphorylation rates of casein, phosvitin, histones, phosphorylase b and protamine 5-30-fold. Within the whole troponin complex, only troponin T is phosphorylated by the enzyme. The enzyme phosphorylates only the N-terminal serine residue of troponin T, i.e. the site that is normally phosphorylated in the whole troponin complex isolated from rabbit skeletal muscles.     

Cloning and sequence analysis of a cDNA from human ovarian granulosa cells encoding the C-terminal part of human elongation factor 2

A cDNA clone, pHGR81, encoding 358 amino-acid residues of the C-terminal region of human elongation factor 2 (EF-2), was isolated from a human ovarian granulosa cell cDNA library. The deduced amino-acid sequence of pHGR81, when compared with the known identical amino-acid sequences of hamster as well as rat EF-2 revealed a substitution of a glutamine by an alanine residue in the partially determined human sequence. The 15 amino-acid-residue sequence comprising the histidine-715, supposed to be of importance for the biological function of EF-2, is preserved in human EF-2. The coding region of the cDNA insert of pHGR81 displays a homology of 87% to hamster and of 88% to rat EF-2 cDNA. In Northern-transfer analysis, pHGR81 specifically hybridizes with an mRNA species of 3.1 kb.     

Ubiquinone biosynthesis. Cloning of the genes coding for chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyl transferase from Escherichia coli.

Chorismate pyruvate-lyase activity was detected in extracts of Escherichia coli. 4-Hydroxybenzoate was identified as the product of the enzymatic reaction by chemical derivatization and GC-MS analysis. The ubiC gene, coding for the chorismate pyruvate-lyase, was cloned and sequenced. The molecular weight of the gene product was calculated as 18,776 Da and confirmed by expression of the protein in E. coli minicells. The ubiA gene, coding for the 4-hydroxybenzoate octaprenyl transferase, was identified by sequence homology and complementation of a ubiA- strain. It is located directly downstream of ubiC in a typical operon structure.     

Carbohydrate content and monosaccharide composition of Rapana thomasiana grosse (Gastropoda) hemocyanin and its structural subunits. Comparison with gastropodan hemocyanins

The hemocyanin of Rapana thomasiana grosse (marine snail, gastropod) is a glycoprotein with a carbohydrate content of 8.9% (w/w) and monosaccharide constituents xylose, fucose, 3-OOmethylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues. The two structural subunits of this oxygen carrier, RHSS1 and RHSS2, are unevenly glycosylated. On subtracting the carbohydrate contribution from the M_r values of 250 and 450 kDa attributed to the two subunits, values of 2.18 × 10⁵ daltons and 4.30 × 10⁵ daltons were calculated for the polypeptide part of the “light” and “heavy” subunits, respectively. Comparison of the monosaccharide compositions of gastropodan hemocyanins revealed qualitative similarities, as well as relationships between the quantities, of the individual monosaccharides: Man 3MeGal > GlcNAc GalNAc and Fuc Xyl     

Molecular Requirements for Bi-directional Movement of Phagosomes Along Microtubules

Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubuledependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with “late” phagosomes moving threefold more frequently than “early” ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dyneinmediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.     

The effect of bacteriolytic preparation lysoamidase on Staphylococcus aureus 209P cells

The effect of the bacteriolytic preparation "Lysoamidase" on Staphylococcus aureus 299 P was studied. The maximum activity of the preparation was observed at pH 8.0 ionic strength 0.01-0.02 M and 50-60 degrees of the incubation medium. The electron microscopic examination revealed that "Lysoamidase" hydrolyzed the cell wall in one or several points with the following osmotic shock and extrusion of the cytoplasm. In an isotonic solution (1 M sucrose) "Lysoamidase" caused protoplast formation.     

Analytical investigation of cilia induced mucous flow

This is an analytic study of mucous flow caused by ciliary motion. The computed flow data may be compared with that already found exprimentally. The effects of mucous density, viscosity and layer depth on flow phenomena are investigated. The effects of cilia diameter, length, spacing and oscillation frequency are determined from the equations governing the flow of the mucous blanket. A pertinent finding of the analysis is that the mucous flow in the airway tubes can satisfy physical constraints only through the assumption of a variable viscosity in the covering mucous blanket. The mucous viscosity must increase considerably from the low value at the cillium layer to a much higher value at the air-mucus interface.     

Sterol composition of Candida curvata yeast

Summary Six sterols were extracted from lipids produced by Candida curvata yeast. Ergosterol (main component) and five minor compounds: ergosta-7, 24(28)-dien-3 -ol, ergosta-5, 7-dien-3 -ol, ergosta-5, 7, 9, (11), 22-tetraen-3 -ol, ergosta 7, 22-dien-3 -ol, ergost-7-en-3 -ol were identified by gas chromafography/mass spectrometry (GCMS) and quantitated by GLC.