A quantitative method for separation of livingHydra cells

Summary We describe a rapid method for the isolation of large numbers of livingHydra cells of defined cell type in an isotonic cell medium (Gierer et al. 1972). Intact animals are enzymatically dissociated into a single cell suspension and the various cell types separated in less than one hour by counterflow centrifugation elutriation. Cell loss is minimal. RNA isolated from various fractions can be probed with cell type specific cDNA-clones.     

Counterflow efflux of thiamin in Escherichia coli

A resting cell of Escherichia coli lacking thiamin kinase incorporated external thiamin with an energy-dependent counterflow efflux (C-efflux). This C-efflux could be separated from an energy-dependent exit by a selective inhibition of exit by $\text{2 · 10}{}^{\mathrm{?2}}\text{M}$ NaN₃. The extracellular thiamin could be replaced by thiamin diphosphate, resulting in the same rate of C-efflux, but the rate of C-efflux of intracellular thiamin diphosphate against the external thiamin was markedly low. This low rate of C-efflux of thiamin diphosphate could explain the higher accumulation of the compound than that of free thiamin in the thiamin-kinase-defective mutant as well as in its wild-type parent. Basic characteristics of free thiamin uptake and exit in E. coli W mutant were compared with those reported in K 12 mutant: a marked difference existed in the rate of exit. The low rate of exit in E. coli W 70-23-102 was inferred as the reason for the absence of an overshoot phenomenon of thiamin uptake in this strain.     

Carrier-mediated acetate transport in Acetobacterium woodii

Intracellular and extracellular acetate concentrations of Acetobacterium woodii DSM 1030 were determined during growth or incubation of resting cell suspensions. The internal concentrations during growth decreased from initially 350 mM to 145 mM at the end of the experiment. The intracellular pH was lowered from 7.5 to 6.6 and the pH was enlarged from 0.2 to 0.6 units. Both, growing and resting cells of A. woodii showed no equilibrium between internal and external acetate concentrations during glucose consumption; the internal concentrations were always higher than expected assuming equal concentrations of the free acid inside and outside the cells. From counterflow experiments it is suggested that acetate does not only leave A. woodii cells by passive diffusion but also by carrier-mediated transport.     

Evidence for non-uniform distribution of d-glucose within human red cells during net exit and counterflow

The kinetic parameters of net exit of d-glucose from human red blood cells have been measured after the cells were loaded to 18 mM, 75 mM and 120 mM at 2°C and 75 mM and 120 mM at 20°C. Reducing the temperature, or raising the loading concentration raises the apparent K_m for net exit. Deoxygenation also reduces the K_m for d-glucose exit from red blood cells loaded initially to 120 mM at 20°C from 32.9 ± 2.3 mM (13) with oxygenated blood to 20.5 ± 1.3 mM (17) (P<0.01). Deoxygenation increases the ratio V_max/K_m from 5.29 ± 0.26 min⁻¹ (13) for oxygenated blood to 7.13 ± 0.29 min⁻¹ (17) for deoxygenated blood (P < 0.001). The counterflow of d-glucose from solutions containing 1 mM ¹⁴C-labelled d-glucose was measured at 2°C and 20°C. Reduction in temperature, reduced the maximal level to which labelled d-glucose was accumulated and altered the course of equilibration of the specific activity of intracellular d-glucose from a single exponential to a more complex form. Raising the internal concentration from 18 mM to 90 mM at 2°C also alters the course of equilibration of labelled d-glucose within the cell to a complex form. The apparent asymmetry of the transport system may be estimated from the intracellular concentrations of labelled and unlabelled sugar at the turning point of the counterflow transient. The estimates of asymmetry obtained from this approach indicate that there is no significant asymmetry at 20°C and at 2°C asymmetry is between 3 and 6. This is at least 20-fold less than predicted from the kinetic parameter asymmetries for net exit and entry. None of the above results fit a kinetic scheme in which the asymmetry of the transport system is controlled by intrinsic differences in the kinetic parameters at the inner and outer membrane surface. These results are consistent with a model for sugar transport in which movement between sugar within bound and free intracellular compartments can become the rate-limiting step in controlling net movement into, or out of the cell.