Lawrence C. Mahan

Antigen Site Densities and Ultrastructural Distribution Patterns of Red Cell Rh Antigens

The ultrastructural distribution pattern and antigen site density of the major Rh antigens (C, c, E, e, and D) on red blood cell ghosts sensitized with IgG Rh antibodies were determined using electron microscopy and ferritin conjugated rabbit anti‐human IgG. The ferritin particle distribution pattern of all the Rh antigens studied was random and ranged from relatively isolated monodis‐persed clusters containing less than eight ferritin particles to large localized aggregates or masses of 20 or more ferritin particles. Evidence is presented to indicate that most of the antigen clustering was due to the conjugate during staining of the cell bound IgG on the stroma preparations. The conjugate‐induced antigen clustering was influenced by both the titer of the conjugate and the staining time. Whether a similar degree of antigen mobility exists in the native membrane remains to be determined. Estimates of the number of cell bound IgG molecules (number of antigenic determinants) based on ferritin particle scoring of IgG Rh antibody sensitized red blood cells under nonsaturating conditions were in the range of 20,000 to 32,000 for each of the Rh antigens. These findings are consistent with the view that the Rh antigen complex is associated with a single membrane component.

Use of superoxide dismutase and catalase to protect catecholamines from oxidation in tissue culture studies

A new enzymatic approach for the prevention of catecholamine oxidation that is particularly useful for studies with cultured cells is described. Catecholamine oxidation was assayed by acid alumina chromatography using a modified procedure that yields greater than or equal to 90% column recovery of catecholamine concentrations as low as 1.0 nM. Addition of superoxide dismutase and catalase (10-25 micrograms/ml each) results in virtually complete inhibition of catecholamine oxidation under a variety of experimental conditions. Although superoxide dismutase could prevent catecholamine oxidation, addition of catalase helped prevent the cytotoxicity of oxidative products. When used together, these enzymes have no effect on cell growth, hormonal response, or radioligand binding to membrane beta-adrenergic receptors in the murine S49 lymphoma cell, a widely used model system for studying catecholamine action. Combined use of superoxide dismutase and catalase offers nonperturbing, long-lasting protection of catecholamines in studies with cells in vitro. This method provides a useful alternative to ascorbic acid, chelators, or reducing agents which have previously been used to prevent catecholamine oxidation but which may have other effects on cultured cells and on membrane proteins.

Radioligand, agonists and membrane receptors on intact cells: data analysis in a bind

Abstract Radioligand binding experiments are being used to probe a variety of receptor types. Although most such experiments have been conducted with membrane preparations, many investigators are now using radioligand binding techniques to study the interaction of agonists with receptors on intact cells. Often these experiments have been analysed using techniques developed for studies of antagonist binding to membrane fragments. Harvey Motulsky , Lawrence Mahan and Paul Insel wish to point out several flaws in such analyses.

Certain beta-blockers can decrease beta-adrenergic receptor number: II. Down-regulation of receptor number by alprenolol and propranolol in cultured lymphoma and muscle cells.

We have used two different cultured cell lines—S49 lymphoma cells and BC3H-1 muscle cells—to examine the regulation of β-adrenergic receptors by receptor antagonists. Rather than an increase (“up-regulation”) of receptor number that such antagonists often produce, we found that certain β-blockers elidt a decrease (“down-regulation”) of beta-adrenergic receptors. Alprenolol and propranolol, but not sotalol or ICI 118,551, at concentrations of 10–100 nM down-regulated β-adrenergic receptors 20–70% following 16–20 hours of treatment of S49 or BC3H-1 cells. Several observations suggest that this phenomenon depends upon beta-receptor interaction, including stereoselectivity [(-)-enantiomers more potent than (+)-enantiomers], blockade of the effect by ICI 118,551, absence of down-regulation of α-adrenergic receptors hi BC3H-1 ceUs, and lack of a decrease in beta-adrenergic receptor-independent (forskolin-stimulated) cyclic AMP accumulation in S49 ceUs. The possibility of retained antagonist interfering with receptor measurement was precluded by the fact that the antagonist-induced decrease in receptor number required several hours incubation and occurred without a prominent change in receptor affinity. The ability of the β-blockers to elicit down-regulation did not correlate with hydrophobicity of the drugs. Antagonist-induced down regulation of beta-adrenergic receptors did not occur in S49 lymphoma cells that lack the α-subunit of Gg, the guanine nucleotide-binding regulatory protein, thus implying a requirement for receptor-α, interaction hi eliciting β-receptor down-regulation. The ability of certain antagonists to promote a down-regulation of β-adrenergic receptors provides a mechanism that may contribute to the pharmacological activity of these agents.

The IgG binding function of the normal red cell plasma membrane: identification of integral polypeptides that bind IgG

Summary. Non‐immune IgG binds to red cell integral membrane proteins obtained by mild alkaline extraction of ghosts. Detergent gel chromatography and electrophoretic analyses of IgG‐membrane protein complexes obtained by nonionic detergent solubilization and affinity binding to protein A indicate that three polypeptides participate in the binding of IgG. These have apparent molecular weights of 90 000, 44 000 and 22 000 and are present in a 1:3:0.9 stoichiometry. Evidence obtained indicates that the major sialoglycoproteins are not involved in this type of binding.

Some properties of alkali-extracted red cell ghost membranes

The properties of integral membrane proteins obtained by dilute alkali extraction of red cell ghosts were examined. A variety of conditions promoted the disulfide-mediated aggregation of integral membrane proteins, principally band 3. Procedural modifications which minimized aggregation were the use of EDTA and S-alkylation. Integral membrane proteins were solubilized under non-denaturing conditions using Brij 36T, a lauryl polyoxyethylene ether with an NMR-determined average chain length of 8.2 (oxyethylene) units. Detergent gel filtration revealed a chromatographic shoulder due to aggregated band 3 when membrane proteins were not alkylated. Analyses of the column profile also revealed a discrete peak for sialoglycoproteins and two phosphate peaks, an early one due to phospholipid and a later one not identified, but probably due to phosphoinositide.

Estimation of anti-D IgG in red blood cell eluates using the specific radioactivity of 125I-labeled IgG: effect of unlabeled, cytophilic IgG

The specific radioactivity of conventionally prepared 125I IgG anti‐D eluates is significantly less (from 1/5 to 1/20) than that of the 125I IgG fraction used to prepare the eluate. This discrepancy is due to the release of unlabeled, cytophilic IgG from normal red blood cells during eluate preparation and does not represent an underestimation of the eluate anti‐D IgG content. Cytophilic IgG content of eluates plays an important role in reducing the nonimmunologic binding of labeled antibody IgG. The results justify the assumption used in numerous studies that the specific radioactivity of 125I IgG fractions can be used to provide a valid estimate of the anti‐D IgG content of eluates.

Proteolysis of red cell membrane proteins by immunoglobulin G preparations.

Red cell integral membrane proteins obtained by mild alkaline extraction were degraded by isolated IgG preparations. Erythrocyte or ghost membranes were not significantly affected under comparable conditions. The IgG appeared pure following 125I-labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis by both densitometric scanning and radioelectrophoretic profiling. In addition, no proteolytic activity was detectable with azocoll under standard conditions. Despite this, the integral protein fraction, which consisted predominantly of band 3 and the major sialoglycoproteins, showed extensive degradation following incubation at 37°C for 1.5 hr with the IgG. The process was partially inhibitable with phenylmethylsulfonyl fluoride and totally inhibited by Trasylol. Proteolysis of band 3 led to the formation of fragments retained in the membranes of apparent mol. wt of 65,000 and lower fragments appearing in the 4.5 region. Band 7 present in the starting membrane fraction appeared resistant to degradation. The problems when working with purified membrane fractions are illustrated by data showing the IgG-binding membrane components that are obtained with IgG preparations having different levels of protease activity. Proteolytic susceptibility appears to be a general characteristic of isolated membrane proteins or receptors. UnIike soluble proteins which are resistant to proteolysis in the native state, enhanced protease degradation may be inherent in isolated membrane proteins even when obtained under mild non-denaturing conditions. Protease-sensitive sites on membrane proteins or receptors which in native membranes are protected by protein-protein or protein-lipid interactions probably become protease-vulnerable following isolation and purification.

Cell surface labeling by iodine monochloride

Abstract 1. 1. Human red cell membranes were trace iodinated with [ 125 I]ICI and the distribution of label in membrane components examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. 2. With intact erythrocytes over 84% of the bound iodine was associated with the membrane. 3. 3. Two membrane components accounted for almost all of the label, band 3 and PAS-1. 4. 4. Spectrin was not labeled in resealed ghosts.

Heat-induced Electrophoretic Anomalies of Red Cell Membrane Nonsialoglycoproteins

Abstract The effects of heating at 100° in the presence and absence of dithiothreitol has been examined in SDS-solubilized ghost membranes and isolated integral membrane proteins from human red cells. A significant increase in electrophoretic mobility and broadening of the major polypeptide band 3 was found. The anomalous behavior of band 3 alone could explain the apparent disappearance of band 4 which has occasionally been reported in hereditary spherocytosis red cells.