Poul Kristensen

Comparison of the decameric structure of peroxiredoxin-II by transmission electron microscopy and X-ray crystallography.

The decameric human erythrocyte protein torin is identical to the thiol-specific antioxidant protein-II (TSA-II), also termed peroxiredoxin-II (Prx-II). Single particle analysis from electron micrographs of Prx-II molecules homogeneously orientated across holes in the presence of a thin film of ammonium molybdate and trehalose has facilitated the production of a >/=20 A 3-D reconstruction by angular reconstitution that emphasises the D5 symmetry of the ring-like decamer. The X-ray structure for Prx-II was fitted into the transmission electron microscopic reconstruction by molecular replacement. The surface-rendered transmission electron microscopy (TEM) reconstruction correlates well with the solvent-excluded surface of the X-ray structure of the Prx-II molecule. This provides confirmation that transmission electron microscopy of negatively stained specimens, despite limited resolution, has the potential to reveal a valid representation of surface features of protein molecules. 2-D crystallisation of the Prx-II protein on mica as part of a TEM study resulted in the formation of a p2 crystal form with parallel linear arrays of stacked rings. This latter 2-D form correlates well with that observed from the 2.7 A X-ray structure of Prx-II solved from a new orthorhombic 3-D crystal form.

Isoelectric point of native and sialidase-treated human-serum cholinesterase

1. 1. The electrophoretic mobility of native and sialidase-treated human-serum cholinesterase was determined by paper electrophoresis over the pH range 2.8–9.6; the determination included corrections for electro-osmosis, evaporation, apper-structure and adsorption of protein to the paper. The performance of the method was controlled with proteins of known mobility, orosomucoid and coeruloplasmin. 2. 2. The isoelectric point of native cholinesterase was found to be 2.9–3.0 and that of sialidase-treated cholinesterase 6.7–7.0. This is an agreement with previous findings that human-serum cholinesterase contains a large number of sialic acid residues in a terminal position. 3. 3. At pH 8.6 the electrophoretic mobility of native cholinesterase was −3.1·10−5 cm2/V/sec and that of sialidase-treated cholinesterase −0.2·10−5 cm 2/V/sec. 4. 4. Between pH 4 and 9.6 the mobility of sialidase-treated cholinesterase was 2.9·10−5 units above that of native cholinesterase. 5. 5. At pH 2.8 this difference decreased to about 2·10−5 units. 6. 6. It is suggested that the mobility-pH curve might be of value in the differentiation between closely related types of cholinesterases.

Mitochondria-rich cells as experimental model in studies of epithelial chloride channels

The mitochondria-rich (mr) cell of amphibian skin epithelium is differentiated as a highly specialised pathway for passive transepithelial transport of chloride. The apical membrane of mr cells expresses several types of Cl(-) channels, of which the function of only two types has been studied in detail. (i) One type of channel is gated by voltage and external chloride concentration. This intriguing type of regulation leads to opening of channels only if [Cl(-)](o) is in the millimolar range and if the electrical potential is of a polarity that secures an inwardly directed net flux of this ion. Reversible voltage activations of the conductance proceed with long time constants, which depend on V in such a way that the rate of conductance activation increases when V is clamped at more negative values (serosal bath grounded). The gating seems to involve processes that are dependent on F-actin localised in the submembrane domain in the neck region of the flask-shaped mr cell. (ii) The other identified Cl(-) pathway of mr cells is mediated by small-conductance apical CFTR chloride channels as concluded from its activation via beta-adrenergic receptors, ion selectivity, genistein stimulation and inhibition by glibenclamide. bbCFTR has been cloned, and immunostaining has shown that the gene product is selectively expressed in mr cells. There is cross-talk between the two pathways in the sense that activation of the conductance of the mr cell by voltage clamping excludes activation via receptor occupation, and vice versa. The mechanism of this cross-talk is unknown.

Subunit stoichiometry of human proteasomes.

Subunits from human placental proteasomes were separated by two-dimensional polyacrylamide gel electrophoresis. The amino acid composition of proteins from individual spots were determined. Some of the spots had identical amino acid compositions, confirming that they contain isoforms of the same subunit. Proteasomes from HeLa cells, labelled with 3H-leucine, were precipitated with an antibody and similarly separated into subunits. The radioactivity in each subunit was measured. The subunit stoichiometry was then calculated from these data and the leucine contents in the subunits. Each of the 14 major subunits of human proteasomes are apparently present in equal amounts.

Identification of the major annexins in Ehrlich ascites tumor cells

1. Three calcium-binding proteins have been purified from Ehrlich ascites tumor cells. 2. They were identified by amino acid sequence analysis on selected fragments obtained by tryptic digestion. 3. The proteins belong to the annexin family and were identified as annexins II, III and V. 4. Antibodies raised against the proteins were used to examine for their presence in a number of murine tissues. 5. The occurrence was found to be in reasonable accordance with earlier reports.

The influence of anaerobic conditions on sodium transport and adenine nucleotide levels in the isolated skin of the frog Rana temporaria.

Abstract 1. 1. An apparatus is described in which frog skins can be studied under anaerobic conditions, while short-circuit current is recorded until the moment of sampling. 2. 2. An interdependence was found between the ATP ADP ratio and short-circuit current under anaerobic conditions. 3. 3. The efflux of sodium was increased under anaerobic conditions. 4. 4. On the basis of the present experiments it is suggested that ATP may be involved directly in the process leading to active transport of sodium in the frog skin and that the sodium pump of that tissue may have some features in common with the sodium pump of erythrocytes.

The role of glycolysis in energy production in the isolated skin of the brown frog (Rana temporaria, L.)

Abstract 1. Total amounts of glycogen and the time course of the disappearance of glycogen were measured in frog skin under aerobic conditions. The calculated maximum energy production from the metabolism of glycogen to CO2 and water was found to be insufficient to account for Na+ transport. 2. Lactate production under anaerobic conditions was correlated with anaerobic Na+ transport. The calculated energy production was found to be large enough to account for the transport recorded. 3. A significant Pasteur effect was observed.