A comparison of intact human red blood cells and resealed and leaky ghosts with respect to their interactions with surface labelling agents and proteolytic enzymes

Resealed ghosts and intact red blood cells were directly compared with respect to their interactions with surface probes and to digestion by pronase. The amount and pattern of labelling of surface proteins by 4.4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) and by pyridoxal phosphate-borohydride (as seen after sodium dodecylsulfate/acrylamide gel electrophoresis) was substantially the same in cells and resealed ghosts under conditions in which a relatively small change would be apparent. In each membrane system, DIDS labels a protein component of apparent molecular weight 95 000 and pyridoxal phosphate labels the same protein plus three glycoprotein components. The sensitivity of surface proteins and of DIDS and pyridoxal phosphate-labelled sites to pronase was also similar in the cells and resealed ghosts. The glycoproteins were digested, in each case, and the 95 000 (molecular weight) protein was largely split into two portions of apparent molecular weights 65 000 and 35 000, with both portions containing DIDS and pyridoxal phosphate binding sites. The pattern of labelling of “leaky” ghosts by pyridoxal phosphate in the presence of hemoglobin was similar to the labelling of intact cells, provided that the pyridoxal phosphate was present on both the outside and inside of the cells. Virtually all of the major protein components visible by staining on acrylamide gels were labelled. It is concluded that none of the probes could detect any substatial differences in reactivity of proteins of the outer surface of the membrane of the ghosts as compared to the cells and that no irreversible changes in membrane protein conformation or arrangement occur as a consequence of lysis and resealing of ghosts, that are detectable by the reported procedures.     

LYSOSOMAL PHYSIOLOGY IN TETRAHYMENA : III. Pharmacological Studies on Acid Hydrolase Release and the Ingestion and Egestion of Dimethylbenzanthracene Particles

The ingestion of ¹⁴C-labeled 9,10-dimethyl-1,2-benzanthracene particles, the extracellular release of acid phosphatase, ribonuclease, and α-glucosidase, and the egestion of preingested dimethylbenzanthracene particles by Tetrahymena taken from logarithmically growing cultures and resuspended in a dilute salt solution were followed in the presence of several pharmacologic agents. Serotonin, caffeine, and, to a lesser extent, dibutyryl cyclic AMP increased the rate of particle ingestion, but did not alter the rate of release of the three acid hydrolases studied. Added catecholamines did not affect either particle ingestion or acid hydrolase release, but particle ingestion was inhibited by the catecholamine antagonists, dichloroisoproterenol, desmethylimipramine, reserpine, and phenoxybenzamine. These drugs also increased the release of acid phosphatase and ribonuclease in 5-h incubations. Desmethylimipramine acted within 1 h to increase acid hydrolase release, but the effect of dichloroisoproterenol developed more slowly and was secondary to a change in cellular content of the hydrolases. Desmethylimipramine increased the energy of activation for the release of acid phosphatase, while dichloroisoproterenol did not. Both of these drugs enhanced the egestion of preingested dimethylbenzanthracene particles, supporting the view that acid hydrolase release occurs through a cytoproct egestion mechanism. Particle ingestion was also inhibited by colchicine, vinblastine, and cytochalasin B, but these agents had no effect on acid hydrolase release, thus further differentiating the properties of the ingestion mechanism from those of the egestion mechanism. It appears that both microtubules and microfilaments play a role in the ingestion process and that this process may be controlled in part by a cyclic AMP-mediated serotoninergic and adrenergic system.     

Relationship of Cation Influxes and Effluxes in Yeast

The Na⁺ efflux from Na⁺-rich yeast cells into a cation-free medium is largely balanced by the excretion of organic anions. In the presence of Rb⁺, K⁺, or high levels of H⁺ (pH 3–4), the Na⁺ efflux is increased and the organic anion excretion is suppressed so that stoichiometric cation exchanges occur. H⁺ participates in the exchanges, moving into or out of the cells depending on the external pH and on the concentration of external Rb⁺(K⁺). The total cation efflux is dependent on the external Rb⁺ concentration in a "saturation" relationship, but the individual cations in the efflux stream are not. The discrimination factor in the efflux pathway between H⁺ and Na⁺ is very large (of the order of 10,000), and between Na⁺ and K⁺ considerable (of the order of 50). For the latter pair, the recycling of K⁺ from the cell wall space is an important factor in the discrimination. In addition, the Na⁺ efflux as a function of Na⁺ content follows a sigmoidal curve so that the discrimination factor is increased at high levels of cellular Na⁺. Although the influx and efflux pathways behave as a tightly coupled system, the mechanism of coupling is not entirely clear. A single system with different cation specificities and kinetic behaviors on the inside and outside faces of the membrane could account for the data.     

The solubility of fibrinogen in dilute salt solutions

Highly purified human fibrinogen was dialyzed versus eleven different sodium salts at ionic strengths of 0.005–0.3 and pH values of 4.5–8.0. After equilibration and centrifugation of the protein solutions, fibrinogen solubilities were determined spectrophotometrically and were analyzed as functions of pH, ionic strength, and specific anion. Bell-shaped curves are obtained when fibrinogen solubility is plotted as a function of pH. The solubility exhibits a minimum at a given pH and rises at acid and alkaline values. As the ionic strength is increased, the solubility curves shift toward more acid pH values. At constant pH values between 6 and 7, fibrinogen solubility increases with an increase in ionic strength. At constant pH values below pH 6, a decrease in solubility occurs as the ionic strength is increased. The isoionic pH of a saturated aqueous fibrinogen solution has been determined to be 6.25, meaning that fibrinogen in pure water behaves as a weak acid with a mean net charge of ?0.9. At pH values acid to 6.25, the anions solubilize fibrinogen in the following order of increasing efficacy: thiocyanate, perchlorate, sulfate, citrate, bromide, nitrate, phosphate, chloride, acetate, fluoride, and formate. This order is reversed at pH values alkaline to 6.25. Anion binding parameters calculated from the solubility data indicate that those anions which most effectively solubilize fibrinogen at alkaline pH and precipitate it at acid pH have the highest apparent binding affinities for the protein. Anions with less pronounced solubility effects have lower binding affinities.     

Membrane proteins related to anion permeability of human red blood cells

Summary (³H)DIDS (4,4-diisothiocyano-2,2-ditritiostilbene-disulfonate) was used as a convalent label for membrane sites involved in anion permeability. The label binds to a small, superficially located population of sites, about 300,000 per cell, resulting in almost complete inhibition of anion exchange. The relationship of biding to inhibition is linear suggesting that binding renders each site nonfunctional. In the inhibitory range less than 1% of the label is associated with lipids but at higher concentrations of DIDS, the fraction may be as high as 4%. In ghosts, however, treatment with (³H)DIDS results in extensive labeling of lipids. In cells, a protein fraction that behavens on SDS acrylamide gels as thought its molecular weight is 95,000 daltons (95K) is predominatly labeled by (³H)DIDS. The only other labeled protein is the major sialoglycoprotein which contains less than, 5% of the total bound (³H)DIDS. Because of the linear relationship of binding to inhibition and the unique architecture of the site, it is suggested that the (³H)DIDS-binding site of the 95K protein is the substrate binding site of the anion transport system. The 95K protein is asymmetrically arranged in the membrane with the sites arranged on the outer face accessible to agent in the medium. In leaky ghost, only a few additional binding sites can be reached from the inside of the membrane in the 95K protein, in contrast to the extensive labeling of other membrane proteins in ghosts as compared to cells.Abbreviations DADS 4,4-Diamino-2,2-dihydrostilbene disulfonic acid - DIDS 4,4-Diisothiocyano-2,2-stilbene disulfonic acid - (³H)DADS 4,4-Diamino-2,2-ditritiostilbene disulfonic acid - (³H)DIDS 4,4-Diisothiocyano-2,2-ditritiostilbene disulfonic acid     

Regulation of Neurotransmitter Aspartate Metabolism by Glial Glutamine Synthetase

Aspartate levels and release from rat striatal slices following the inhibition of glutamine synthetase (GS) by methionine sulfoximine (MSO) were studied. Striatal levels of aspartate and glutamine were decreased over time in a manner that correlated with GS inhibition. Ca2+-dependent, K+-stimulated aspartate release was diminished in striatal tissue slices from animals pretreated with MSO. The decreased release of aspartate correlated over time with the inhibition of GS. The addition of glutamine to the perfusion medium completely reversed the effects of MSO on calcium-dependent aspartate release. It is suggested that glutamine is a major precursor for transmitter aspartate.     

Adaptation of mouse leukemic cells (L5178Y) to anisotonic media : II. Cell growth

Mouse lymphoma cells (L5178Y) exposed to hypertonic media for 1 h behave as osmometers, but in hypotonic media, after initial swelling, they shrink back to normal volume and maintain it for long periods of time. The lower limit of osmolarity at which this “volume adaptation” will occur lies between 140 and 185 mosM. The “volume adaptation” is associated with a loss of cellular K⁺ probably due to a transient increase in K⁺ permeability and to loss of associated anions and osmotically obliged water. Partial dissipation of the large gradient of K⁺ between cells and medium by pre-exposure to ouabain or to K⁺-free medium results in a diminished capacity to adapt. After the shrinking phase is completed, a new steady state is established with a reduced cellular K⁺ content, normal Na⁺, normal K⁺-permeability, and a reduced activity of the Na⁺ − K⁺ transport system. When adapted cells are returned to normal medium, an initial shrinking is followed by a re-swelling to normal size, associated with a gain in K⁺ content, presumably due to the return to normal activity of the Na⁺ − K⁺ transport system.     

Chemical Modification of Membranes : II. Permeation paths for sulfhydryl agents

The amino-reactive reagent, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS),¹ considerably reduces the uptake of the sulfhydryl agent, parachloromercuriphenylsulfonic acid (PCMBS), but does not reduce its effects on cation permeability and on cation transport. These data indicate that PCMBS enters the membrane by at least two channels, one sensitive and the other insensitive to SITS, with only the latter leading to the cation-controlling sulfhydryl groups. Substitution of phosphate or sulfate for chloride results in an inhibition of PCMBS uptake via the SITS-insensitive pathway. These and other data lead to the conclusion that the SITS-sensitive pathway is the predominant one for anion permeation, and the insensitive one for cation permeation. Parachloromercuribenzoate (PCMB), an agent that is more lipid-soluble than PCMBS, penetrates faster but has a smaller effect on cation permeability. Its uptake is less sensitive to SITS. These and other observations suggest that the cation permeation path involves an aqueous channel in the membrane.     

Chemical Modification of Membranes : 1. Effects of sulfhydryl and amino reactive reagents on anion and cation permeability of the human red blood cell

Four different amino-reactive reagents, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS),¹ 1-fluoro-2,4-dinitrobenzene (FDNB), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and 2-methoxy-5-nitrotropone (MNT) decrease the anion permeability of the human red blood cell, as measured by sulfate fluxes, whereas the sulfhydryl agent, parachloromercuriphenyl sulfonic acid (PCMBS), does not. In contrast, PCMBS increases the cation permeability as measured by K⁺ leakage, whereas SITS does not. Of the other agents, FDNB increases the cation permeability to the same extent as PCMBS but MNT and TNBS produce smaller increases. PCMBS does not protect against FDNB as it does against other sulfhydryl agents (X-irradiation) and the FDNB effect on cations is attributed to amino groups. Studies of the binding of SITS indicate that it does not penetrate into the membrane and its failure to influence cation permeability is attributed to its inability to reach an internal population of amino groups. It is concluded that two ion permeability barriers, both involving proteins, are present in the red blood cell. The more superficial barrier contains amino groups and controls anion flow; the more internal barrier contains sulfhydryl and amino groups and controls cation flow. The amino groups contribute to the control of permeability by virtue of their positive charges, but the role of sulfhydryl groups is not clear. Only a small fraction of the membrane protein amino and sulfhydryl is involved in the barriers.