Inhibition of anion transport associated with chymotryptic cleavages of red blood cell band 3 protein.

Right-side-out vesicles derived from red blood cells treated with chymotrypsin retain specific anion transport function (defined as transport sensitive to the specific inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), even though the transport protein, band 3, is cleaved into two segments of 60 and 35 kdaltons. In contrast, vesicles derived from alkali-stripped ghosts treated with relatively high concentrations of chymotrypsin retain almost no specific anion function. The loss of function appears to be related to additional cleavages of band 3 protein that occur in treated ghosts, the 60-kdalton segment being reduced first to a 17- and then to a 15-kdalton segment and the 35-kdalton segment being reduced to a 9-kdalton segment plus a carbohydrate containing fragment. The chymotryptic cleavages of band 3 protein of ghosts are preferentially inhibited by high ionic strength, the production of the 9-kdalton segment being somewhat slower than that of the 15-kdalton segment. Vesicles derived from ghosts treated with chymotrypsin at different ionic strengths show a graded reduction in specific anion transport activity, but it was not possible to determine, definitively, which of the additional cleavages was inhibitory. In the light of these data and other information, the functional role of the segments of band 3 is discussed.

State of water and electrolytes in mammalian cells during maturation and differentiation.

The state of water and electrolytes was examined 1) in the rat erythroblastic leukemic cell, as a model of a maturing erythrocyte; 2) in the mouse Ehrlich ascites tumor cell during the cell cycle as a model of the uninhibited proliferating cell; and 3) in a clonal population of proliferating and differentiating precursor cells cultured from the bone marrow of the rat. Methods used were phenomenological and included assessments of the volumes of osmotically active water, content of K+, Na+, and Cl-, volumes of distribution for Na+, urea, and ethylene glycol, and the thermodynamics of transport of water and solutes into and out of the cell. The erythroblastic leukemic cell provided evidence for compartments of osmotically active and inactive water and solute. The synchronized ascites tumor cell indicated that these compartments varied during the cell cycle. Membrane function as defined by its permeability to water also varied during the cell cycle. When proliferating cells matured, changes in membrane permeability to water and to nonelectrolytes also correlated with successive stages of differentiation.

Electrolyte and non-electrolyte distribution in the Ehrlich ascites tumor cells during the cell cycle.

In a previous study, evidence was presented for changes in the state of water and osmotically active solutes during the cell cycle. Total water was constant at 82% (w/w), while the fraction of water that was osmotically active decreased from a maximum during S to a minimum at mitosis. Total Na+, K+, and C1- in milliequivalents per liter of cell water remained constant. Therefore, electrolytes are sequestered in the osmotically inactive water. Evidence is now presented that Na+ exists primarily as one compartment, with a second, slower compartment appearing during S and disappearing during G2. Na+ is completely exchangeable during the entire cell cycle. The distribution of other penetrating solutes was also investigated. When placed in hyperosmotic ethylene glycol solutions, cells first shrink, then swell to their original volumes. 14C-ethylene glycol distributes in 89% of cell water throughout the cell cycle. However, 14C-urea distributes in anywhere from 86-100% of the cell water, depending on the stage in the cell cycle. Both solutes are at chemical equilibrium in water in which they are distributed, but they differ in their effects on cell volume. The final volume at which cells equilibrate in urea varies with the concentration of urea in the environment and with time into the cell cycle. Results suggest a loss of osmotically active particles or decreased osmotic activity of urea.

Osmotic properties of Ehrlich ascites tumor cells during the cell cycle.

Ehrlich ascites tumor cells were grown and maintained in continuous spinner culture. The population of dividing cells was synchronized by a double thymidine block technique. Cell cycle phases were determined graphically by plotting mitotic index, cell number, and DNA synthesis against time. Changes in the osmotic properties of Ehrlich ascites tumor cells during the cell cycle are described. Permeability to water is highest at the initiation of S and progressively decreases to its lowest value just after mitosis. Heats of activation for water permeability vary during the cell cycle, ranging from 9-14 kcal/mole. Results may imply changes in the state of water in the membrane during the cycle. The volume of osmotically active cell water is highest during S and early G2 and decreases during the mitotic phase, as cells undergo division. Total water content remains stable at 82% (w/w) during the cycle. Total concentration of the three major ions (Na, K, Cl), expressed as mEq/liter total cell volume, does not change. The fraction of total cell water which is osmotically active (Ponder's R) decreased gradually from 0.75 at S to about 0.56 following mitosis. Findings suggest that a fraction of the total water within the cell exists in a "bound" form and is, therefore, incapable of being shifted under the driving force of osmotic pressure. This fraction of bound water increases during the cell cycle. Possible alterations in membrane fluidity and the state of water in the cell are discussed.