Biogenesis of erythrocyte membrane proteins. In vivo studies in anemic rabbits.

To study the process of red cell membrane protein synthesis we have followed the time course of [3-H]leucine appearance in total protein and individual peptides of the erythrocyte membrane following injection of the amino acid into phenylhydrazine-anemic rabbits. Multiple peripheral blood samples were taken from single animals over a 5-week period. Erythrocyte membrane proteins were separated by polyacrylamide gel electrophoresis in sodium dodecylsulfate and dithiothreitol; incorporation of radioactivity was determined by gel slicing and liquid scintillation spectrometry. Appearance of [3-H]leucine in circulating erythrocytes reached a peak at 1-3 days, with a steady decline thereafter. The radioactive amino acid appeared first in the lowest molecular weight peptides and last in the largest peptides; at the earliest time point (8 h), little radioactivity was observed in any of the four largest peptides present in the membranes (bands A, 1, 2 and 3). Certain smaller peptides (bands 4, 5 and 9) were the predominant species labeled at this time. By 24 h all peptides showed significant incorporation. With maturation of the red cells, label largely disappeared from bands A, 9 and several smaller peptides; this was confirmed by finding that the peptides are virtually absent from mature circulating erythrocytes. These data are interpreted as showing that red cell membrane proteins are synthesized asynchronously during the life cycle of the erythrocyte; the largest peptides are made predominantly in the earlier marrow stages of development, while certain of the smaller peptides are still being synthesized in the reticulocyte stage. Several membrane proteins appear to be specific to the reticulocyte and are lost during the process of cell maturation in the circulation.

Biogenesis of erythrocyte membrane proteins. In vitro studies with rabbit reticulocytes.

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [3-H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polyacrylamide gel electrophoresis of labeled membranes on large (19 mm) gels which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [3-H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removable of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.

The binding and processing of monoclonal human IgG1 by cells of a human macrophage-like cell line (U937).

The goal of these experiments was to assess the relationship between the binding and processing of IgG by Fc-receptor-bearing cells. Cells of the U937 human macrophage-like cell line were incubated with 125I-labeled monomers, dimers, oligomers (composed of 2-4 IgG1 subunits), and HP (heavy polymers composed of 5 or more subunits per polymer) of monoclonal human IgG1 in vitro. Binding was assessed by spinning cells through a layer of phthalate oils. Internalization of IgG1 was assessed by quantitating residual binding to cells after surface-bound IgG was removed by a brief treatment with a solution containing 0.25 M acetic acid and 0.5 M sodium chloride. Catabolism was assessed by measuring the release of radioactive fragments of IgG1, which were not precipitated by 10% trichloroacetic acid. Unstimulated U937 bound about 10,000 molecules per cell of IgG1 monomer, with an equilibrium binding constant (Ka) of 5 X 10(8) M-1. After stimulation with a conditioned medium in vitro, binding per cell was increased 3-7--fold, and the Ka was decreased 2-4--fold. Both unstimulated and stimulated cells internalized and catabolized labeled IgG1 HP, but stimulated cells internalized and digested much more IgG1 HP per cell than unstimulated cells. Both monomers and dimers of IgG1 were internalized and degraded very slowly by stimulated cells, even though both preparations readily bound to cells. In contrast, oligomers and (to an even greater extent) IgG1 HP were internalized and degraded much more rapidly. Internalization of IgG1 HP was markedly inhibited by incubation at 4 degrees C, but not by incubation with a variety of metabolic inhibitors. Catabolism was inhibited by chloroquine and monensin (inhibitors of lysosomal acidification) and by cytochalasin (an inhibitor of microfilament polymerization). Binding to the surface of cells was not markedly inhibited by any agent tested. The capacity of cells to bind labeled IgG1 was markedly reduced by prior incubation in the presence of unlabeled IgG1. This reduction was in part due to the steric blockade of receptors caused by the avid, but reversible, binding of IgG1. In addition, IgG1 oligomers or HP (but not IgG1 monomers or dimers) also caused an irreversible reduction in the number of Fc receptors by a process analogous to receptor down-regulation, as observed in other receptor--ligand systems.