The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane.

Human erythrocyte ghosts exhibit an inositol trisphosphate phosphomonoesterase activity that rapidly converts inositol 1,4,5-trisphosphate into inositol 1,4-bisphosphate and Pi. Degradation of the released inositol 1,4-bisphosphate is not observed. This activity is dependent on Mg2+ (or Mn2+) and it is not activated by Ca2+. Optimum activity is around pH 7 and activity is abolished by heat denaturation. The Km for inositol trisphosphate is approx. 25 microM. 2,3-bisphosphoglycerate is a competitive inhibitor, with a Ki of approx. 0.35 mM. Glycerophosphoinositol 4,5-bisphosphate is attacked at about one-eighth of the rate for inositol trisphosphate, but glycerophosphoinositol 4-phosphate is not a substrate. Incubation of 32P-labelled erythrocyte membranes with Mg2+ causes little breakdown of phosphatidylinositol 4,5-bisphosphate, the parent compound from which both glycerophosphoinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate are derived. On the basis of its substrate specificity and the inhibition by 2,3-bisphosphoglycerate, we suggest that this enzyme is selective for the 5-phosphate in those water-soluble phosphate esters of inositol that possess the vicinal pair of 4,5-phosphates but that it may also interact less strongly with other water-soluble compounds that have pairs of vicinal phosphates.     

The exon-intron organization of the human erythrocyte alpha-spectrin gene.

The human erythrocyte alpha-spectrin gene which spans 80 kbp has been cloned from human genomic DNA as overlapping lambda recombinants. The exon-intron junctions were identified and the exons mapped. The gene is encoded by 52 exons whose sizes range from 684 bp to the smallest of 18 bp. The donor and acceptor splice site sequences match the splice site consensus sequences, with the exception of one splice site where a donor sequence begins with -GC. The size and location of exons do not correlate with the 106-amino-acid repeat, except in three locations where the surrounding codons are conserved as well. The lack of correspondence between exons and 106-amino-acid repeat is interpreted to reflect the appearance of a spectrin-like gene from a minigene early in the evolution of eukaryotes. Since current evidence indicates that introns were present in genes before the divergence of prokaryotes and eukaryotes, it is possible that the original distribution of introns within the minigene has been lost by the random deletion of introns from the spectrin gene.     

The phylogenetic odyssey of the erythrocyte. II. The early or invertebrate prototypes

Freely existing hemoglobin-bearing cells suspended in a plasmic milieu (erythrocytes) are found in a relatively small number of taxanomically scattered invertebrates. These species include some annelids, echiurids, molluscs, phoronids, nemerteans and echinoderms, e.g. Pista pacifica, Urechis caupo, Noetia ponderosa, Phoronis australis, Lineus fuscoviridis and Cucumaria miniata respectively. The typical invertebrate erythrocyte (hemocyte, coelomocyte) can be described as permanently nucleated, considerably larger than the human red cell, oval or circular in configuration and spherical, biconvex or flattened in profile. The marginal band of the erythrocyte, a bundle of subplasmalemmal microtubules that circumscribes the periphery of the cell and lies in the plane parallel to its flat surface makes its first appearance in certain invertebrates. This structure in association with the cell surface-associated cytoskeleton is responsible for the flattened elliptical shape seen in some invertebrate erythrocytes and endows them with flexibility and resilience to mechanical forces. This in an evolutionarily persistent characteristic that is retained throughout the submammalian vertebrates. The erythrocytes of invertebrates are more morphologically and functionally diversified than the mammalian model. In addition to respiratory activities (oxygen storage and transport) they can sometimes function as vendors of nutrients and participate in other less obvious processes. These cells therefore frequently not only retain organelles that are usually discarded by vertebrate erythrocytes (ribosomes, golgi apparatus, etc.) but may also depending upon the species, manifest in their cytoplasm organelles and inclusions that are not a normal component of developing or mature submammalian vertebrate and mammalian erythroid cells. Examples of the latter are pigment granules, lipid droplets, extensive glycogen stores and prominent Prussian blue positive inclusions. Erythrocytes in the invertebrates, though presenting certain cytologic and functional features in common, are a heterogenous collection of cells, each tailored for a specific species or group of organisms.     

The reaction of chemical probes with the erythrocyte membrane.

Trinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB) and suberimidate have been reacted with intact human erythrocytes. TNBS does not penetrate the cell membrane significantly at 23 degrees C in bicarbonate-NaCl buffer, pH 8.6, as estimated by the labeling of the N-terminal valine of hemoglobin. Hence, under these conditions it can be used as a vectorial probe. However, at 37 degrees C, especially in phosphate buffer, at pH 8.6, TNBS does penetrate the cell membrane. FDNB and suberimidate both penetrate the erythrocyte membrane. The time course reaction of TNBS with intact erythrocytes over a 24-hr period at 23 degrees C is complex and shows transition zones for both membrane phosphatidylethanolamine (PE) and membrane proteins. No significant cell lysis occurs up to 10 hr. The fraction of total PE or phosphatidylserine (PS) which reacts with TNBS by this time period can be considered to be located on the outer surface of the cell membrane. Under these conditions it can be located on the outer surface of the cell membrane. Under these conditions it can be shown that 10 to 20% of the total PE and no PS is located on the outer surface of the membrane and hence these amino phospholipids are asymmetrically arranged. The pH gradient between the inside and outside of the cell in our system is 0.4 pH units. Nigericin has no effect on the extent of labeling of PE or PS by TNBS. Isotonic sucrose gives a slight enhancement of the labeling of PE by TNBS. Hence, the inability of PE and PS to react with the TNBS is considered not due to the inside of the cell having a lower pH. The extent of reaction of TNBS with PE is not influenced by changing the osmolarity of the medium or by treatment of cells with pronase, trypsin, phospholipase A or phospholipase D. However, bovine serum albumin (BSA) does protect some of the PE molecules from reacting with TNBS. Cels treated with suberimidate were suspended in either isotonic NaCl or in distilled water. In both cases the suberimidate-treated cells became refractory to hypotonic lysis. Pretreatment of cells with TNBS did not prevent them from interacting with suberimidate and becoming refractory to lysis. However, pretreatment of cells with the penetrating probe FDNB abolished the suberimidate effect. Electron-microscopic analysis of the cells showed a continuous membrane in the case of cells suspended in isotonic saline. The cells suspended in water did not lyse but their membranes had many large holes, sufficient to let the hemoglobin leak out. Since the hemoglobin did not leak out we know that the hemoglobin is cross-linked into a large supramolecular aggregate.     

The 'hollow cylinder' protein of erythrocyte membranes.

The 'hollow cylinder' protein (Harris, J.R. (1968) Biochim. Biophys. Acta 150, 534-537) has been purified from human erythrocyte membranes. The molecular weight of the native protein, as determined by analytical ultracentrifugation, was found to be 747,000. By means of sodium dodecyl sulphate gel electrophoresis, the purified protein was shown to be composed of three different low molecular weight polypeptides of average molecular weight 25,000. This study provides convincing evidence that the spectrin tetramer is not responsible for the characteristic electron microscopic appearance of the hollow cylinder protein.     

The insulin receptor of the turkey erythrocyte. Characterization of the membrane-bound receptor.

The insulin receptor of the turkey erythrocyte has previously been shown to be very similar to that of the mammalian insulin receptors. As a first step in the isolation of this receptor a highly purified plasma membrane fraction has been prepared. The binding characteristics of the purified membrane-bound receptor were identical to those found with intact erythrocytes, but the membrane preparation had very little insulin-degrading activity. Isolation of the membrane by the methods described gave a 100-fold purification of the insulin receptor with 67% yield.     

The similarity of the two high-molecular-weight polypeptides of erythrocyte spectrin.

1. The two major polypeptides (P1 and P2) of erythrocyte-membrane spectrin were isolated by preparative polyacrylamide-gel electrophoresis. 2. The two polypeptides were shown to possess similar amino acid compositions, both with the characteristically high glutamate and leucine contents of the parent spectrin. 3. The tryptic-peptide 'maps' of the two polypeptides were prepared by a combination of t.l.c. and electrophoresis. 4. Radioactive peptides were prepared by [14C]carboxymethylation and chloramine-T-catalysed [125I]iodination. 5. 'Maps' of both sets of peptides demonstrate a marked similarity between the two polypeptides. 6. These new data confirm earlier evidence for the similarity of the two chains. 7. The number of peptides in the 'maps' of carboxymethylated peptides suggest that polypeptides P1 and P2 are not aggregates.     

The phosphorylation of the major proteins of the human erythrocyte membrane.

Both the major sialoglycoprotein (PAS-1) and the component designated by Fairbanks et al. (G. Fairbanks, T. L. Steck, and D. F. H. Wallach, 1971, Biochemistry10, 2606–2617) as Band 3 are shown to be bonafide phosphoproteins by virtue of the presence of covalently bound serine and threonine phosphate residues. In agreement with the findings of others, PAS-1 does not seem to be phosphorylated when ghosts are incubated with [γ-³²P]ATP, but the phosphorylation is significant (about 0.15 mol/mol) when the cells are incubated in the presence of ³²P_i. Band 3 is phosphorylated to the extent of 0.90 mol/mol, and these sites are apparently distributed in several places along the polypeptide chain. Spectrin is also a phosphoprotein containing approximately four molecules of phosphate per 450,000 daltons of protein. The phosphorylation of these three polypeptides is not stimulated by the presence of cAMP.     

The phylogenetic odyssey of the erythrocyte. III. Fish, the lower vertebrate experience.

The piscine erythrocyte can be considered the prototype of the red cells that are distributed among inframmalian vertebrates. It is a permanently nucleated, hemoglobin-ladened, oval, flattened, biconvex disc. Ultrastructurally it demonstrates a cytoskeleton comprised of a marginal band and a membrane skeleton which are responsible for the erythrocyte's conversion to an ellipsoid during morphogenesis and endow it with resilience to physical trauma. Erythropoiesis initiates in the yolk sac, followed in many fishes, by the intermediate cell mass. These sites are the sources of the transitory, primitive generation red cells which apparently make their first phylogenetic appearance in fishes and which are subsequently represented in all classes of vertebrates including mammals. Production of definitive generation erythrocytes is centered in evolutionary "pre-splenic" tissue of the gastrointestinal tract or in the spleen in cyclostomes, dipnoi, and chondrichthyes while in teleosts it is typically located in the kidneys with or without splenic participation. The blood is a major site of erythrocyte maturation in the lower fishes and exhibits significant numbers of immature erythroid cells plus occasional mitotic figures. Some teleosts also circulate developing erythroid cells. Certain fishes have occasional circulating erythroplastids, conceptually a portent of phylogenetic changes in higher vertebrates. Remarkably, some bristlemouths have denucleated erythrocytes exclusively in the circulation. The largest piscine erythrocytes are found in the dipnoi, myxines, and chondrichthyes. Primitive fish with the exception of the endothermic sharks tend to have lower hemoglobin concentrations than the modern teleosteans. The very highest hemoglobin concentrations are attained by the endothermic scombrids. Erythrocyte-based data have a broad extent and are variably affected by age, sex, season and environment. This report includes a substantial selection of illustrations (fish species and rbc micrographs).     

The phylogenetic odyssey of the erythrocyte. I. Hemoglobin: the universal respiratory pigment

Hemoglobin is a molecular entity that is capable of reversibly binding and releasing oxygen in either extra- or intracellular milieus. It is present in scattered invertebrates in physical solution or in cellular sites while in vertebrates it is universally located in circulating erythrocytes. These cells serve as the vehicle for and otherwise foster the optimum utilization hemoglobin. Hemoglobin's variable sphere of respiratory activities can be viewed as reflecting the specific requirements for each organism in which it is observed. Once these concepts have been established and the advantages and limitations of its cytologic packaging recognized, the study of the erythrocyte as expressed in its dimensions, colligative aspects, geometry, internal morphology and pathologic variations can be approached in a purposeful manner.     

On erythrocyte morphology.

The significance of the distinctive morphological difference between mammalian and non-mammalian vertebrate erythrocytes (disc versus ellipse) is discussed in the light of mammalian erythrocyte rheology.     

The role of spectrin in erythrocyte ghost endocytosis.

Endocytosis in white ghosts prepared from human erythrocytes was induced by three methods: incubation with Mg-ATP, incubation with 0.1 mM EDTA, and digestion with 20 nanograms (ng.) per ml. of trypsin. In each case the endocytic vacuoles that were produced when separated and analyzed on SDS-polyacrylamide gel electrophoresis were found to be depleted of spectrin. This observation suggested that a requirement for endocytosis is the establishment of spectrin-free domains in the membrane. This hypothesis was tested by pre-incubating ghosts with anti-spectrin antibodies. Pre-incubation with anti-spectrin antibody blocked white ghost endocytosis produced either by Mg-ATP, EDTA, or trypsin. Therefore, it is proposed that spectrin has a key role in the endocytosis process.