Two modes of inhibition of the Ca2+ pump in red cells by Ca2+.

Two different and independent modes of inhibition of the Ca2+ pump by Ca2+ can be detected measuring active Ca2+ extrusion from resealed ghosts of human red cells: one requires extracellular and the other requires intracellular Ca2+. Ki for inhibition by extracellular Ca2+ is about 10 mM. Extracellular Mg2+ replaces Ca2+ in inhibiting Ca2+ transport but with an apparent affinity for inhibition about 3-times less than that for Ca2+. Inhibition by external Ca2+ is not affected by Na+ or K+ at both surfaces of the cell membrane, external EGTA, internal Ca2+ or ATP. The apparent affinity for external Ca2+ progressively raises as pH increases. The effects of extracellular Ca2+ and Mg2+ are consistent with the idea that for Ca2+ pumping to proceed, external sites in the pump must be protonated and not occupied by extracellular Ca2+ or Mg2+. Inhibition by intracellular Ca2+ takes place with a Ki of about 1 mM and is independent of external Ca2+. The inhibitory effects of intracellular Ca2+ can be accounted for if Ca2+ and CaATP were competitive inhibitors of the activation of the pump by Mg2+ and MgATP, respectively.

The effects of alkali metal ions on active Ca2+ transport in reconstituted ghosts from human red cells.

1. K+, Rb+ or Na+ increase from 30 to 90% the maximum rate of Ca2+ transport from resealed ghosts, leaving unaltered the apparent affinity of the Ca2+ pump for Ca2+. Li+ is ineffective as activator of Ca2+ transport. 2. K+ does not change the temperature dependence of Ca2+ transport. 3. The effects of K+ and Na+ are half-maximal at 4.6 mM and 24 mM, respectively. 4. The site(s) at which K+ or Na+ combine are accessible only from the cytoplasmic surface of the cell membrane. 5. Under conditions in which Na+ activates the Ca2+ pump no Na+ efflux coupled to Ca2+ efflux is apparent.

Evaluation of the proteolytic potential of in vitro-cultivated hybridoma and recombinant mammalian cells.

The proteolytic potential of culture supernatants derived from recombinant baby hamster kidney (BHK) 21 and mouse-mouse hybridoma cells have been characterized. Several assays using enzyme specific chromogenic artificial peptides, as well as a radioactive test for the detection of the total activity, have been established and were adapted to the special conditions existing in culture media of mammalian cells. Proteolytic activity was detected in human serum albumin containing media which was specific for peptides ending with a terminal arginine. The addition of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer to the culture media resulted in a significant peptide cleavage potential, supporting the fact that this compound is not recommended as a supplement in animal cell culture media. Medium shock protease activity has been detected in culture supernatants of BHK cells when medium was changed completely, caused by a switch from a serum containing state of growth to a serum-free state of growth which is often used in processes with microcarriers. However, this proteolytic activity showed a transient behaviour whereby its secretion stopped when the cells had adapted to the serum-free medium conditions. Characterization of the proteolytic activities using different specific inhibitors and activators supported the assumption that the proteolytic activity reflects a cell specific composition of proteases which can also change dependent on the culture conditions used.

Cultivation of recombinant baby hamster kidney cells in a fluidized bed bioreactor system with porous borosilicate glass.

Dense cell cultivation of the recombinant cell line BHK 21 pSVIL2 was performed in a fluidized bed bioreactor system containing porous borosilicate glass carriers. Experiments were carried out with different medium formulations for a period of 48 days. Due to an effective immobilization of the cells in the reactor, continuous operation was easy to perform. Maximal cell densities and product yields could be maintained, even when protein-free medium was perfused exceeding 2 reactor volumes per day. Final cell densities of magnitude 7.1 x 10(7) mL-1 intrasphere volume were reached, while the interleukin-2 production rate was 0.70 mg day-1. The cell specific productivity reached a value of 1.3 x 10(-10) mg day-1. The first results were presented with a cell line that grows under glutamine-free medium conditions. The use of a glutamine-free medium for the cultivation of the cells resulted in a drastic decrease in cell metabolism. Furthermore, the amino acids lysine and histidine were produced and secreted into the culture supernatant, although these metabolites normally are considered to be essential for animal cells grown in vitro. However, no lethal effect on the cells has been detected, and the total number of cells in the reactor remained constant. The metabolism of threonine has been detected to be directly dependent on the presence of glutamine. Cells grown in glutamine-free culture medium produced glycine yields 6 times higher than those grown in glutamine-containing medium. A bead-to-bead transfer of the cells has also been detected when the cells immobilized within the intrasphere volume of the borosilicate carriers reached the stationary phase.

Evaluation of production of recombinant human interleukin-2 in fluidized bed bioreactor.

The production of recombinant human interleukin-2 in a fluidized bed bioreactor containing porous glass carriers is described. Cultivations were carried out with different medium formulations over 80 days. Maximal cell densities and product yield could be maintained even when protein free medium was perfused, with less than 10% cell washout. Due to this effective immobilization of the cells in the reactor, continuous operation was easy to perform. Final cell densities on the order of 3.8 x 10(8) mL(-1) intrasphere volume were reached while the interleukin-2 production rate was 0.75 mg L(-1) d(-1). The production rate showed a maximum of a 1.9 fold decrease compared with a homogeneous stirred bubble-free aerated system. This result was in contrast to that achieved with hybridoma cell lines, where better performance was obtained with the fluidized bed bioreactor. The situation may reflect the problems caused by the dense cell culture with adherent cells, as previously shown in a hollow-fiber bioreactor with the same cell line.