Evidence for two asymmetric conformational states in the human erythrocyte sugar-transport system.

6-O-methyl-, 6-O-propyl-, 6-O-pentyl- and 6-O-benzyl-D-galactose, and 6-O-methyl-, 6-O-propyl- and 6-O-pentyl-D-glucose inhibit the glucose-transport system of the human erythrocyte when added to the external medium. Penetration of 6-O-methyl-D-galactose is inhibited by D-glucose, suggesting that it is transported by the glucose-transport system, but the longer-chain 6-O-alkyl-D-galactoses penetrate by a slower D-glucose-insensitive route at rates proportional to their olive oil/water partition coefficients. 6-O-n-Propyl-D-glucose and 6-O-n-propyl-D-galactose do not significantly inhibit L-sorbose entry or D-glucose exit when present only on the inside of the cells whereas propyl-beta-D-glucopyranoside, which also penetrates the membrane slowly by a glucose-insensitive route, only inhibits L-sorbose entry or D-glucose exit when present inside the cells, and not when on the outside. The 6-O-alkyl-D-galactoses, like the other nontransported C-4 and C-6 derivatives, maltose and 4,6-O-ethylidene-D-glucose, protect against fluorodinitrobenzene inactivation, whereas propyl beta-D-glucopyranoside stimulates the inactivation. Of the transported sugars tested, those modified at C-1, C-2 and C-3 enhance fluorodinitrobenzene inactivation, where those modified at C-4 and C-6 do not, but are inert or protect against inactivation. An asymmetric mechanism is proposed with two conformational states in which the sugar binds to the transport system so that C-4 and C-6 are in contact with the solvent on the outside and C-1 is in contact with the solvent on the inside of the cell. It is suggested that fluorodinitrobenzene reacts with the form of the transport system that binds sugars at the inner side of the membrane. An Appendix describes the theoretical basis of the experimental methods used for the determination of kinetic constants for non-permeating inhibitors.     

Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use or paramagnetic quenching.

Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short range interaction we developed a spectroscopic method to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase. This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.     

Isolation of the porcine ileal intrinsic factor receptor by sequential affinity chromatography

An extract containing solubilised receptor was passed through four columns containing Sepharose to which had been covalently coupled anti-cobalophilin IgG, vitamin B-12-intrinsic factor, vitamin B-12, and free intrinsic factor, respectively. Following a wash the receptor was eluted with EDTA, then residual Triton X-100 micelles and vitamin B-12-intrinsic factor were removed by Sephadex G-200 filtration. The receptor was purified 84 000-fold, sodium dodecyl sulphate electrophoresis indicated two subunits and gel filtration of its vitamin B-12-inttrinsic factor complex resolved it into two molecular species.     

Isolation and in vitro expression of the ricin A-chain gene: effect of deletions on biological activity.

The gene encoding the ricin A-chain was isolated and subcloned into an in vitro expression vector downstream from the SP6 promotor. mRNA encoding the A-chain strongly inhibited the translational activity of reticulocyte lysates. The inhibition correlated with glycosylase activity on rRNA, and could be abolished by addition of antibodies specific for ricin. mRNA generated after linearization of the vector at unique restriction sites within the A-chain coding sequence did not inhibit, except after linearization with ScaI. Also mutants lacking the 28 N-terminal amino acids of native A-chain strongly inhibited the lysates. However, in both cases no glycosylase activity could be observed. We also show that the lack of a stop codon in mRNA does not affect the level of expression as assayed here.     

2-Amino-2-deoxygalacturonic acid in lipopolysaccharides from Pseudomonas aerugimosa.

2-Amino-2-deoxygalacturonic acid was identified as a component of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 8505. The compound probably occurs in the region of polysaccharide responsible for O-antigenic specificity.     

Culture of Clostridium pasteurianum in Defined Medium and Growth as a Function of Sulfate Concentration

Clostridium pasteurianum strain W-5 was selected as an anaerobe which may be grown from large inocula in defined media with sulfate as its primary sulfur source. Since it is important to keep inocula small in minimizing transfer of sulfur sources, culture conditions were optimized. The medium devised decreased lag period and generation time when compared with other media, but growth could not be induced consistently with 6 x 10(6) cells per ml or less. Addition of trace elements, chelating agents, reducing agents, metabolites, and spent medium from various stages of growth did not stimulate growth from small inocula. Generation time was 85 min on inoculation with 10(7) or more cells per ml taken from young stocks, but the lag period decreased somewhat with larger inocula. On the other hand, generation time and lag period increased with age of the inoculum. The total yield of cells increased when buffer capacity was increased. Growth of C. pasteurianum W-5 was dependent upon sulfate at relatively low sulfate concentrations, and the organism is thus suitable for study of sulfur metabolism. No evidence of a maintenance requirement for sulfate was detected.