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The factors that determine the activity of the Na,K pump in vivo were investigated by measuring Na,K pump activity under in vivo conditions in human red cells and relating it to the intracellular content of sodium ([Na]i) and potassium ([K]i) and the number of pump units per cell (pump number). Na,K pump activity was measured as ouabain-sensitive K+ influx, pump number was determined from the maximal binding of 3H-ouabain to intact cells, and [Na]i and [K]i were measured by atomic absorption spectrophotometry in washed, packed cells. In the 81 samples studied, pump activity per cell was significantly correlated with pump number (r = .64, P less than 0.001), but was negatively correlated with [Na]i (r = -.28, P less than 0.02) and was not correlated with [K]i. An inverse relationship was found between pump number and [Na]i. When pump activity was expressed as activity per pump unit, rather than per cell, a significant relationship was seen between pump activity and [Na]i (r = .50, P less than 0.001), and a negative correlation existed between the activity per pump unit and [K]i (r = -.29, P less than 0.01). The effect of intracellular Na+ at physiologic levels on pump activity was not strong, with the activity per pump unit increasing only 25% with a doubling of [Na]i. These results indicate that pump number is the major determinant of pump activity in human red cells in vivo, while [Na]i and [K]i are of secondary importance.(ABSTRACT TRUNCATED AT 250 WORDS)

A procedure for purification of the bungarotoxin-binding fraction of sarcolemma from rabbit skeletal muscle is described. Muscle is homogenized in 0.25M sucrose without high salt extraction and membrane fractions separated initially by differential centrifugation procedures. An ultracentrifugation pellet enriched in cell surface and sarcoplasmic reticulum markers is further fractionated on a dextran gradient (density = 1.0 to 1.09). Two fractions are identified as sarcolemma according to high specific activities for lactoperoxidase-iodination, Na+, K+-ATPase and alpha-bungarotoxin-binding. No Ca++, Mg++-ATPase activity is found in these fractions. A third fraction, the dextran gradient pellet, is enriched in Ca++, Mg++-ATPase activity and lactoperoxidase iodinatable material and characterized by low bungarotoxin binding. This fraction represents a mixture of sarcoplasmic reticulum and transverse tubules with some sarcolemma contamination.

Malignant mammary epithelial cells (MMECs) were isolated from 8 human breast carcinomas and 1 adenoma as single cells or organoids and established in vitro. Depending on the cellularity of the tumor, between 9 X 10(4) and 4 X 10(6) cells were released per gram of tumor tissue. With the use of conditioned media and growth-promoting agents, a high proportion of cells (ranging from 0.5 to 11.4%) could be established in culture. A high degree of tumor cell heterogeneity in breast carcinomas was suggested by the observation that significantly different proliferative rates were found for 50 mammary epithelial cells cloned from 2 different tumors during the first subpassage in vitro prior to significant expansion of the cell colonies. The computed doubling times of these clones varied between 16 hours and more than 48 hours. The mammary epithelial nature of the cells was confirmed by their surface reactivity with monoclonal antibodies specific for MMECs. Studies on clones from 2 tumors revealed a positive correlation between proliferative rates of MMECs, their lactate production, and specific proteins synthesized as analyzed by two-dimensional macromolecular protein maps.

Throughout its erythrocytic cycle the plasmodial parasite modifies the plasma membrane of its host cell. Some changes derive from parasite metabolism. Intraerythrocytic forms use glucose at more than 10-fold normal red cell rates. The H+ accompanying the lactate end-product is exported into the host cell cytoplasm by an electrogenic proton pump in the parasite membrane. This maintains a pH greater than 7.0 in the parasite cytoplasm, but lowers erythrocyte cytoplasmic pH from approximately 7.2 to 6.5. Ca2+ transport across parasite membranes is coupled to the proton pump, possibly a Ca2+/H+ antiporter. The Ca2+, Mg2+-ATPase and Na+,K+-ATPase activities of erythrocyte membranes from schizont-infected erythrocytes have been studied. Under optimal assay conditions (pH = 7.0; [ATP] = 1 mM; +/- calmodulin) membranes from infected cells showed a 30% reduction in Ca2+,Mg2+-ATPase activity but no difference from normal in Na+,K+-ATPase activity. The calmodulin levels of infected cells were depressed by about 30%. The [ATP] in the cytoplasm of infected erythrocytes was only 0.2 mM (as against 1.3 mM in normals) and at this ATP concentration the activities of both ATPases are only 30% of normal. Shifting the pH from 7.0 to 6.5 decreases Na+,K+-ATPase activity by an additional 50% but is without effect on the Ca2+,Mg2+-ATPase. The results provide a partial explanation for the increased Ca2+ permeability and altered Na+/K+ content of plasmodia-infected erythrocytes.

Weak acid distribution methods demonstrate that mitogenic levels of concanavalin A induce an intravesicular alkalinization of isolated thymocyte membrane vesicles. Experiments with chemical reagents that crosslink the high affinity concanavalin A receptor and extensive correlation with known cellular events suggest that a "membrane Bohr effect" may participate in the initiation of mitogenesis.

A study was undertaken using differential centrifugation methods to isolate from rabbit cerebral arteries the subcellular microsomal protein fractions capable of actively sequestering Ca++. One isolated protein fraction displayed a relatively large adenosine triphosphate (ATP)-dependent Ca++-accumulating capacity that was completely inhibited by NaN3, and was therefore designated the "mitochondrial fraction." Electron microscopy confirmed that this fraction consisted of numerous mitochondrial elements. Another isolated membrane fraction possessed a Ca++-accumulating capacity dependent on ATP and oxalate and only partially sensitive to NaN3. In the presence of mersalyl acid or the Ca++ ionophore, A23187, Ca++ uptake by this fraction was inhibited 98.0% and 87.4%, respectively. Electron microscopy revealed that this fraction consisted of numerous membrane vesicles, and measurements of Na+-K+-ATPase (adenosine triphosphatase) activity indicated minimal plasma membrane contamination. It was concluded that this microsomal fraction consisted primarily of sarcoplasmic reticulum. At physiological free [Ca++] levels, Ca++ uptake by this fraction was inhibited by norepinephrine through a process sensitive to tolazoline but not propranolol. The effects on Ca++ uptake of added cyclic adenosine monophosphate (cAMP) alone or with rabbit or bovine protein kinase were inconclusive. The organic Ca++ channel blockers, nifedipine and verapamil, significantly inhibited Ca++ uptake by sarcoplasmic reticulum.

(1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone.

The effects of temperature in the fever range (37-42 degrees) on the membrane potentials of normal and simian virus 40-transformed hamster lymphocytes were analyzed. The transmembrane distributions of radiolabeled triphenylmethylphosphonium and thiocyanate were measured, and they provide upper and lower limits for the normal cell membrane potential at 37 degrees of -48 +/- 6 (S.D.) and -31 +/- 5 mV and for the tumor cells, -36 +/- 4 and -19 +/- 2 mV. The mitochondrial contribution to the triphenylmethylphosphonium-measured membrane potential, 5 to 10 mV for both splenocytes and simian virus 40-transformed lymphocytes, was estimated by utilizing antimycin A and carbonylcyanide-m-chlorophenylhydrazone to inhibit generation of a mitochondrial membrane potential. Incubation for 1 to 2 hr at 38-42 degrees resulted in a 6- to 15-mV depolarization of normal cells and a 2- to 6-mV hyperpolarization of tumor cells. Both depolarization and hyperpolarization were fully reversible by subsequent incubation at 37 degrees and insensitive to antimycin A and carbonyl-cyanide-m-chlorophenylhydrazone. The membrane potential of normal splenocytes when measured with triphenylmethylphosphonium at 37 degrees was depolarized by 35% with 1 mM ouabain and thermally induced depolarization was blocked. The membrane potential of tumor cells at 37 degrees was insensitive to ouabain; however, the hyperpolarization at 40 degrees was inhibited. The membrane potential of normal lymphocytes stimulated with phytohemagglutinin was depolarized relative to that of nonstimulated control cells and assumed the thermal response characteristics of tumor cells.

The relationship between cation transport and cell volume in human erythrocytes was investigated by measuring ouabain-sensitive K+ influx, ouabain-resistant, furosemide-sensitive K+ influx, and ouabain + furosemide-resistant K+ influx, and maximal ouabain binding in microcytic, normocytic and macrocytic red cells. A significant correlation was found between the mean corpuscular volume and furosemide-sensitive K+ influx normalized either to cell number (r = 0.636, P less than 0.001) or to cell volume (r = 0.488, P less than 0.001). No relationship was seen between mean corpuscular volume and ouabain-sensitive K+ influx, and the number of ouabain-binding sites per cell was only weakly correlated with mean corpuscular volume (r = 0.337, P less than 0.05). A slight, negative relationship existed between mean corpuscular volume and ouabain + furosemide-resistant K+ influx expressed per volume of cells (r = -0.359, P less than 0.01), and an apparent relationship between furosemide-sensitive K+ influx and mean corpuscular hemoglobin concentration (r = 0.446, P less than 0.01) disappeared when microcytic samples were excluded from analysis. Furosemide-sensitive transport, including Na+ influx and K+ and Na+ efflux, was completely absent in microcytic cells from one patient with alpha-thalassemia minor. In addition, these cells exhibited a furosemide-resistant, Cl(-)-dependent K+ influx. Exposure of normal erythrocytes to hypotonic conditions (196 mosM) increased furosemide-sensitive K+ influx by a mean of 45% (P less than 0.05), while exposure to hypertonic conditions (386 mosM) had no significant effect. The results indicate that furosemide-sensitive transport and cell volume are interrelated in human erythrocytes. However, the inability to fully recreate this relationship with in vitro manipulation of cell volume suggest that this relationship is established prior to red cell maturation.

1. Suspension of human erythrocytes in halothane-saturated physiological media induces major cell deformation. 2. Release of halothane from erythrocyte suspensions after equilibrium with the anaesthetic produces complete hemolysis. 3. The membrane fragments isolated after halothane release are in the form of biconcave ghosts, impermeable to macromolecules (lactoperoxidase) and small molecules (ATP). 4. The protein composition of the membranes differs from that of ghosts produced by hypotonic lysis in the lack of components previously shown to be adsorbed at low ionic strength. 5. A hypothesis is presented explaining the action of halothane in terms of both its action on membranes and its capacity to modify water structure. 6. Halothane-induced hemolysis constitutes a simple method for the large-scale production of hemoglobin-depleted, sealed erythrocyte ghosts under physiological ionic conditions.