Loss of insulin receptors on maturation of reticulocytes

The insulin receptors in rabbit reticulocytes have been studied; their properties are undistinguishable from those of classical receptors for insulin. During the maturation of the reticulocytes the number of hormone binding sites decreased 10 fold. Their calculated value was of 1600–2000 sites per reticulocyte compared with 110–260 sites per mature red cell.     

Analysis of the tetrahydrobiopterin synthesizing system during maturation of murine reticulocytes

The enzymes of tetrahydrobiopterin synthesis have been studied in murine bone marrow, in spleen, in erythrocytes, and in reticulocytes. Mice with chemically induced and with genetically conditioned reticulocytosis as found in the lactate dehydrogenase deficient strain (Ldh-1c/Ldh-1c) were used for analysis of reticulocytic enzyme activities. The activity of the biopterin synthesizing system is highest in bone marrow even though it amounts to only about 10% as compared with liver. The first enzyme of the biosynthetic pathway, GTP-cyclohydrolase, virtually disappears during the final maturation step of reticulocytes. In contrast, the activities of 6-pyruvoyltetrahydropterin synthase and of sepiapterin reductase of erythrocytes are only reduced to about one half of the reticulocyte level. The absence of biopterin in erythrocytes is therefore caused by the loss of the enzyme that initiates the pterin biosynthetic pathway.     

Pig reticulocytes. IV. In vitro maturation of naturally occurring reticulocytes with permeability loss to glucose

Naturally occurring reticulocytes of week old piglets were used to characterize the maturation process under in vitro conditions. When the reticulocytes were suspended in tissue culture medium fortified with metabolic substrates, nearly all cells were viable after 24 hours incubation and usually more than 85% of the initial cell population survived after an 80 hour period. In cells maintained as long as a week in incubation, an adequate level of total adenine nucleotide with a large accumulation of IMP was found. In most cases, reticulocytes lose their reticular materials within two days and assume normal erythrocyte configuration. Concomitant with the morphological change, the cell volume decreases toward normal erythrocyte size, the extent of which can be accounted for by the intracellular loss of salt and accompanying water. As in the in vivo reticulocyte maturation process, reticulocytes undergoing in vitro maturation lose their membrane permeability to glucose. These findings suggest that the process of reticulocyte maturation occurring in cell culture approaches that which naturally occurs in vivo. Thus, these cells may be used to delineate the mechanism of the loss of membrane transport of glucose which normally occurs in the adult pig cells.     

Alteration in the protein components of catecholamine-sensitive adenylate cyclase during maturation of rat reticulocytes

The maturing rat reticulocyte was used as a model system in which to study developmental changes in the protein components of hormone-sensitive adenylate cyclase. Plasma membranes from rat erythrocytes display 10 to 20% of the adenylate cyclase activity and 30 to 50% of the beta-adrenergic receptors which are measured in membranes from rat reticulocytes, as noted by others. Reticulocyte membranes also display equal activities in response to (-)-isoproterenol in the presence of either GTP or GTP gamma S, whereas erythrocyte membrane adenylate cyclase is twice as active in the presence of isoproterenol plus GTP gamma S as in the presence of isoproterenol plus GTP. We have studied this system in greater detail by developing or applying independent assays for the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of adenylate cyclase. C was assayed in membranes by its intrinsic Mn2+-stimulated activity. It was also measured by reconstituting membranes with saturating amounts of GTP gamma S-activated G/F, yielding an operationally defined Vmax for the catalyst. By either assay, reticulocytes display about 3-fold greater C activity than do erythrocytes. G/F was assayed by its ability to confer GTP gamma S-stimulated activity upon C (which was supplied by membranes of cyc- S49 lymphoma cells). This assay indicates that reticulocyte membranes contain about 3 times as much G/F as do erythrocyte membranes. Cholera toxin and [32P]NAD were used to [32P]ADP-ribosylate the 45,000- and 52,000-dalton subunits of G/F. Total incorporation of 32P into these subunits decreased 3- to 4-fold with reticulocyte maturation. The ratio of label in the 52,000-dalton peptide to that in the 45,000-dalton peptide decreased from 0.29 in reticulocyte membranes to 0.14 in erythrocyte membranes. The apparently coordinate decrease in the amounts of C, G/F, and beta-adrenergic receptors suggest that the stoichiometry between these components is maintained during maturation, and may account for the decrease in adenylate cyclase in the membranes. However, the qualitative changes in responsiveness to hormones in the presence of GTP or GTP gamma S may be related to loss or proteolysis of the 52,000-dalton subunit of G/F.     

In vitro maturation of reticulocytes. Behavior of RNA and inorganic pyrophosphatase

A simple system of incubation of reticulocytes with a small degree of haemolysis has been developed. It was possible to test the influence of inorganic phosphate, pH-values in the range of 7.0--9.0 and anaerobic conditions on the in vitro maturation of reticulocytes. As main criteria of the maturation the RNA-content and the activity of PPase has been tested. The decrease of the RNA-content and the activity of the PPase is highly significant during maturation. The decrease of RNA and the reduction of reticulocytes are stimulated by inorganic phosphate.     

Hexokinase microheterogeneity in rabbit red blood cells and its behaviour during reticulocytes maturation

Hexokinase in rabbit reticulocytes is present in two molecular forms (hexokinase Ia and Ib) separable by ion-exchange chromatography on DE-52 columns. By the use of ion-exchange HPLC we have been able to show that the isozymic form we previously called hexokinase la can be resolved into two peaks of activity one of which is (Ia) soluble, the other (Ia*) particulate. Hexokinase Ia* can be solubilized by detergents like saponine and Triton X-100 and disappears during ‘in vivo’ reticulocytes maturation. This new hexokinase micro-heterogeneity is not caused by different oxidized forms of the enzyme nor influenced by the presence of proteolytic inhibitors during lysate preparation.     

Concanavalin A-induced endocytosis in rabbit reticulocytes, and its decrease with reticulocyte maturation

Concanavalin A (Con A) was taken up to a limited extent by endocytosis in rabbit reticulocytes but not in rabbit erythrocytes. This process was observed by the use of ferritin-labeled Con A and transmission electron microscopy of thin sections of plastic-embedded cells. Furthermore, the extent of endocytosis among the reticulocytes decreased with the extent of their maturation, reticulocyte age being measured by ribosome configurations. These results are consistent with the proposal that there are domains in the membranes of reticulocytes in which the Con A receptors are laterally mobile, and can be clustered and endocytosed. These mobile domains exist, or are formed, within a larger framework of immobile membrane. During reticulocyte maturation, these domains are gradually eliminated, eventually disappearing upon formation of the mature erythrocyte. Possible molecular mechanisms for this proposed elimination process are discussed.     

In vitro maturation of rabbit reticulocytes: oxygen consumption reaction]

With a simple experimental system the changes of endogenous, antimycin A-suppressed, oligomycin-suppressed and antimycin A-resistant oxygen consumption are studied during the maturation of intact cells of the 6th day of bleeding. All functional characteristics of oxygen consumption decrease during maturation. The rate of decrease is strongly increased by high inorganic phosphate concentrations (125 mM). This effect is most obvious for the oligomycin-suppressed and the endogenous respiration. The degree of uncoupling of non-incubated cells is 14%. During 24 h incubation it rises to 75%. Inorganic phosphate accelerates the increase of uncoupling during maturation. Reticulocytes of the 4th day of bleeding are characterized by a higher respiratory capacity and also by a higher rate of maturation of antimycin A-suppressed and endogenous respiration. The degree of uncoupling does not increase during maturation. This may be attributed to the low lipoxygenase activity of these cells. 25% of the endogenous oxygen consumption of unmatured cells are antimycin A-resistant. This type of respiration declines by 50% in 4 h incubation irrespective of inorganic phosphate concentrations and day of bleeding. In nitrogen all functional characteristics of respiration during the maturation decline more rapidly than in oxygen. The antimycin A-resistant respiration, however decreased more slowly and reached 50% after 12 h. A pH dependence of maturation (maximum at pH 8.4) was found for the endogenous and the antimycin A-suppressed respiration. The degree of uncoupling rises most quickly at pH 7.4. This is possibly related to the pH maximum of lipoxygenase.