Hydrophobic interactions of the apo and holo forms of human cobalamin binding proteins

The interactions of transcobalamin II (TC II), intrinsic factor (IF) and R-type binding protein of cobalamin (Cb1, vitamin B₁₂) with the hydrophobic chromatography matrix Phenyl-Sepharose CL-4B were investigated. IF-Cb1 and R-Cb1 complexes were not adsorbed on Phenyl-Sepharose at room temperature or at 4°C with buffer containing 50 mM sodium phosphate, pH 7.4 containing 150 mM sodium chloride. The TC II-Cb1 complex adsorbed and could be eluted with buffer containing 50% $\text{v}\text{v}$ glycerol. IF without Cb1 adsorbed and was eluted with 50% glycerol at room temperature and 4°C. At room temperature, R binder without Cb1 eluted with buffer, but later than the R-Cb1 complex. At 4°C, R binder completely adsorbed to the matrix. TC II-without Cb1 bound to the matrix at 4°C and room temperature and could not be eluted with glycerol. These results suggest that Cb1 binding proteins can be separated and identified based on their hydrophobic properties. In addition, upon binding Cb1, TC II, IF and R-type binders undergo a conformational change such that the protein-Cb1 complex shows reduced hydrophobicity.     

Nature of the water channels in the internodal cells ofNitellopsis

Summary The hydraulic resistance was measured on internodal cells ofNitellopsis obtusa using the method of transcellular osmosis. The hydraulic resistance was approximately 2.65 pm^–1 sec Pa, which corresponds to an osmotic permeability of 101.75 m sec^–1 (at 20°C).p-Chloromercuriphenyl sulfonic acid (pCMPS) (0.1–1mm, 60 min) reversibly increases the hydraulic resistance in a concentration-dependent manner.pCMPS does not have any effect on the cellular osmotic pressure.pCMPS increases the activation energy of water movement from 16.84 to 32.64 kJ mol^–1, indicating that it inhibits water movement by modifying a low resistance pathway.pCMPS specifically increases the hydraulic resistance to exosmosis, but does not influence endosmosis. By contrast, nonyltriethylammonium (C₉), a blocking agent of K⁺ channels, increases the hydraulic resistance to endosmosis, but does not affect that to exosmosis. These data support the hypothesis that water moves through membrane proteins in characean internodal cells and further that the polarity of water movement may be a consequence of the differential gating of membrane proteins on the endo- and exoosmotic ends.     

Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.     

Transport of methotrexate in L1210 cells: Mechanism for inhibition by p-chloromercuriphenylsulfonate and N-ethylmaleimide

Methotrexate transport in L1210 cells is highly sensitive to inhibition by p-chloromercuriphenylsulfonate (CMPS) and, to a lesser extent, by N-ethylmaleimide. A 50% reduction in the methotrexate influx rate occurred upon exposure of cells to 3 μM CMPS or 175 μM N-ethylmaleimide, while complete inhibition was achieved at higher levels of these agents. Dithiothreitol reversed the inhibition by CMPS, suggesting that a sulfhydryl residue is involved. This residue is apparently not located at the substrate binding site of the transport protein, since methotrexate failed to protect the system from inactivation by either CMPS or N-ethylmaleimide, and the transport protein retained the ability to bind substrate (at 4°C) after exposure to these inhibitors (at 37°C). Methotrexate efflux was also inhibited by CMPS (50% at 4 μM), indicating that both the uptake and efflux of methotrexate in L1210 cells occur via the same transport system. High concentrations of CMPS (greater than 20 μM) increased the efflux rate, apparently by damaging the cell membrane and allowing the passive diffusion of methotrexate out of the cell.     

Retinoic acid-binding protein: a plasma membrane component

Soluble protein extracts from lyophilized plasma membranes prepared from chick embryo skin and transplantable murine carcinomas were found to contain a specific retinoic acid-binding component. This binding component showed high affinity for biologically active analogs of retinoic acid. The plasma membrane component exhibited similar physicochemical properties to those of the retinoic acid-binding protein described earlier in the cytosol. The protein exhibited mercurial-sensitive thiol functions in ligand binding; the mercurial-inhibition was reversed on treatment with thiol compounds. The plasma membrane binding component may be involved in the cellular uptake of retinoic acid.