Isolation and characterization of the rainbow trout erythrocyte band-3 protein

Rainbow trout (Salmo gairdneri) band-3 protein was isolated from trout erythrocyte plasma membranes by a combination of preparative SDS/PAGE and electroelution. High purity and recovery of the plasma membranes were achieved by a new method. This was demonstrated using 4,4'diiso-thiocyano[3H2]dihydro-stilbene 2,2'disulfonic acid (3H2DIDS) which specifically labels band-3 protein. On SDS/PAGE, band-3 protein yields a similarly diffuse pattern, as does mammalian band-3 protein, with an apparent Mr of 116,000. In situ chymotryptic cleavage/cross-linking experiments with 3H2DIDS reveal that the fragments cross-link as in human and mouse band-3 proteins but that there are minor differences. Treatment of trout erythrocytes with trypsin results in cleavage of the band-3 protein. Purified polyclonal antibodies raised against trout band-3 protein react with trout band-3 protein and do not crossreact with mouse or human band-3 protein. They react specifically with only one chymotryptic fragment of trout band-3 protein.     

The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes.

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1----4GlcNAc beta 1----3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.     

Cytosolic protein binding to band-3 protein inhibits endocytosis of isolated human erythrocyte membranes.

Recent studies of haemoglobin binding to the cytoplasmic side of the erythrocyte membrane have shown that the predominant high-affinity interaction occurs with the major integral membrane protein known as band-3 protein and that this interaction may occur within the intact erythrocyte in a manner regulated by cell pH. We report here that haemoglobin and glyceraldehyde 3-phosphate dehydrogenase binding to band-3 protein in isolated membranes can inhibit endocytosis during vesiculation in vitro. The specificity of this effect was demonstrated by showing that myoglobin, which has an affinity for the membrane fully one to two orders of magnitude lower than that for haemoglobin, does not inhibit endocytosis.     

Role of band 3 tyrosine phosphorylation in the regulation of erythrocyte glycolysis.

Previous studies demonstrated that the in vitro tyrosine phosphorylation of the human erythrocyte anion transporter, band 3, prevented the binding of various glycolytic enzymes to the N terminus of the cytoplasmic tail. Since these enzymes are inhibited in their bound state, the functional consequences of band 3 tyrosine phosphorylation in the red cell should be to activate the enzymes and elevate glycolysis. We searched for various enhancers of band 3 tyrosine phosphorylation using a novel assay designed to measure the phosphotyrosine levels at the band 3 tyrosine phosphorylation/glycolytic enzyme-binding site. This assay measures the extent of phosphorylation of a synthetic band 3 peptide entrapped within resealed red cells. Using this assay, three distinct compounds, all mild oxidants, were found to stimulate the tyrosine phosphorylation of band 3. All three compounds were also found to elevate glycolytic rates in intact erythrocytes. Moreover, the antitumor drug adriamycin was found to coordinately prevent these agents from stimulating both band 3 tyrosine phosphorylation and erythrocyte glycolysis. These results suggest a possible function for a protein tyrosine kinase in human erythrocytes, to regulate glycolysis through the tyrosine phosphorylation of band 3.     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Augmented phosphorylation of cardiac troponin I in hypertensive heart failure

An altered cardiac myofilament response to activating Ca(2+) is a hallmark of human heart failure. Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility and Ca(2+) sensitivity of cardiac muscle. cTnI can be phosphorylated by protein kinase A (PKA) at Ser(22/23) and protein kinase C (PKC) at Ser(22/23), Ser(42/44), and Thr(143). Whereas the functional significance of Ser(22/23) phosphorylation is well understood, the role of other cTnI phosphorylation sites in the regulation of cardiac contractility remains a topic of intense debate, in part, due to the lack of evidence of in vivo phosphorylation. In this study, we utilized top-down high resolution mass spectrometry (MS) combined with immunoaffinity chromatography to determine quantitatively the cTnI phosphorylation changes in spontaneously hypertensive rat (SHR) model of hypertensive heart disease and failure. Our data indicate that cTnI is hyperphosphorylated in the failing SHR myocardium compared with age-matched normotensive Wistar-Kyoto rats. The top-down electron capture dissociation MS unambiguously localized augmented phosphorylation sites to Ser(22/23) and Ser(42/44) in SHR. Enhanced Ser(22/23) phosphorylation was verified by immunoblotting with phospho-specific antibodies. Immunoblot analysis also revealed up-regulation of PKC-α and -δ, decreased PKCε, but no changes in PKA or PKC-β levels in the SHR myocardium. This provides direct evidence of in vivo phosphorylation of cTnI-Ser(42/44) (PKC-specific) sites in an animal model of hypertensive heart failure, supporting the hypothesis that PKC phosphorylation of cTnI may be maladaptive and potentially associated with cardiac dysfunction.     

Calcium-calmodulin dependent phosphorylation of erythrocyte pyruvate kinase

In the red cell incubated with ortho-[32P] phosphate, CaCl₂ and calcium ionophore A 23187, phosphorylation of erythrocyte pyruvate kinase was demonstrated using the double antibody technique and autoradiography. Phosphorylation was inhibited by calmodulin inhibitors, trifluoperazine or ZnCl₂. In the presence of purified erythrocyte calmodulin, CaCl₂ and [γ-³²P] ATP, the partially purified erythrocyte pyruvate kinase containing cytozol protein kinases was phosphorylated. This was also inhibited by trifluoperazine or ZnCl₂. From these results, it was concluded that erythrocyte pyruvate kinase is phosphorylated by a calcium-calmodulin dependent process.     

Cyclic AMP-dependent phosphorylation of erythrocyte variant pyruvate kinase

Cyclic AMP-dependent phosphorylation of a variant erythrocyte pyruvate kinase (PK; EC 2.7.1.40) was studied. This variant PK shows a faster electrophoretic mobility than the normal enzyme. The decreased enzyme activity observed in this variant is associated with a quantitative decrease of enzyme protein. Other parameters are within normal ranges. The partially purified variant PK is phosphorylated with a subsequent increase of k0.5s (phosphoenolpyruvate) similar to the normal control, suggesting that the structural abnormality of the variant enzyme has no influence on the phosphorylation-deactivation mechanism. On the other hand, the variant PK in the erythrocyte was less extensively phosphorylated than PK in normal erythrocytes. This may be the result of abnormal metabolism in the patient's red cells, including increased 2,3-diphosphoglycerate and decreased adenosine triphosphate levels.     

Calcium ion-dependent phosphorylation of human erythrocyte membranes.

Calcium ions promote the rapid transfer of the terminal phosphate of ATP to a protein of human erythrocyte membranes. The concentration of Ca2+ for half-maximal effect is 7 muM. At nonlimiting ATP concentrations the level of 32P incorporated by the membranes is independent of the presence or absence of Mg2+. The number of phosphorylating sites in a single erythrocyte membrane is about 700. The influence of pH on the rate of hydrolysis of the bound phosphate and its rapid release on exposure to hydroxylamine are both consistent with an acylphosphate bond. The phosphate in the protein undergoes rapid turnover. Enzymatic splitting of the phosphate is stimulated by Mg2+ but not by Ca2+. It is proposed that Mg2+ accelerates the splitting of the phosphate by favoring the conversion of the phosphoprotein from a state of low reactivity to a state of high reactivity towards water. The reactions described probably are intermediate steps in the hydrolysis of ATP catalyzed by the Ca2+-dependent ATPase of human erythrocyte membranes.     

Spectrin phosphorylation in intact neonatal and adult erythrocyte membranes.

The erythrocyte of the human neonate exhibits clustering and endocytosis of membrane receptors in response to the plant lectin concanavalin A, but erythrocytes from adults do not. Because the phosphorylation of spectrin has been postulated to influence protein mobility in human erythrocyte membranes, the phosphorylation of spectrin was compared in intact neonatal and adult human erythrocytes. No difference in spectrin phosphorylation was seen. The addition of concanavalin A under conditions which produce protein mobility resulted in no change in spectrin phosphorylation.     

Augmented responses to ozone in obese carboxypeptidase E-deficient mice

We reported previously that mice obese as a result of leptin deficiency (ob/ob) have enhanced ozone (O3)-induced airway hyperresponsiveness (AHR) and inflammation compared with wild-type (C57BL/6) controls. To determine whether this increased response to O3 was independent of the modality of obesity, we examined O3-induced AHR and inflammation in Cpe(fat) mice. These mice are obese as a consequence of a mutation in the gene encoding carboxypeptidase E (Cpe), an enzyme important in processing prohormones and proneuropeptides involved in satiety and energy expenditure. Airway responsiveness to intravenous methacholine, measured by forced oscillation, was increased in Cpe(fat) vs. wild-type mice after air exposure. In addition, compared with air exposure, airway responsiveness was increased 24 h after O3 exposure (2 ppm for 3 h) in Cpe(fat) but not in wild-type mice. Compared with air-exposed controls, O3 exposure increased bronchoalveolar lavage fluid (BALF) protein, IL-6, KC, MIP-2, MCP-1, and soluble TNF receptors (sTNFR1 and sTNFR2) as well as BALF neutrophils. With the exception of sTNFR1 and sTNFR2, all of these outcome indicators were greater in Cpe(fat) vs. wild-type mice. Serum sTNFR1, sTNFR2, MCP-1, leptin, and blood leukocytes were elevated in Cpe(fat) compared with wild-type mice even in the absence of O3 exposure, similar to the chronic systemic inflammation observed in human obesity. These results indicate that increased O3-induced AHR and inflammation are consistent features of obese mice, regardless of the modality of obesity. These results also suggest that chronic systemic inflammation may enhance airway responses to O3 in obese mice.     

Spectrin phosphorylation and shape change of human erythrocyte ghosts

Human erthrocyte membranes in isotonic medium change shape from crenated spheres to biconcave disks and cup-forms when incubated at 37 degrees C in the presence of MgATP (M. P. Sheetz and S. J. Singer, 1977, J. Cell Biol. 73:638-646). The postulated relationship between spectrin phosphorylation and shape change (W. Birchmeier and S. J. Singer, 1977, J. Cell Biol. 73:647-659) is examined in this report. Salt extraction of white ghosts reduced spectrin phosphorylation during shape changes by 85-95%. Salt extraction did not alter crenation, rate of MgATP-dependent shape change, or the fraction (greater than 80%) ultimately converted to disks and cup-forms after 1 h. Spectrin was partially dephosphorylated in intact cells by subjection to metabolic depletion in vitro. Membranes from depleted cells exhibited normal shape-change behavior. Shape-change behavior was influenced by the hemolysis buffer and temperature and by the time required for membrane preparation. Tris and phosphate ghosts lost the capacity to change shape after standing for 1-2 h at 0 degrees C. Hemolysis in HEPES or N- tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid yielded ghosts that were converted rapidly to disks in the absence of ATP and did not undergo further conversion to cup-forms. These effects could not be attributed to differential dephsphorylation of spectrin, because dephosphorylation during ghost preparation and incubation was negligible. These results suggest that spectrin phosphorylation is not required for MgATP-dependent shape change. It is proposed that other biochemical events induce membrane curvature changes and that the role of spectrin is passive.     

Augmented lipopolysaccharide-induction of the histamine-forming enzyme in streptozotocin-induced diabetic mice

Disorders of the microcirculation and reduced resistance to infection are major complications in diabetes. Histamine enhances capillary permeability, and may also reduce cellular immunity. Here we demonstrate that streptozotocin (STZ)-induced diabetes in mice not only enhances the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), but also augments the lipopolysaccharide (LPS)-induced elevation of HDC activity in various tissues, resulting in a production of histamine. The augmentation of HDC activity occurred as early as 2 days after STZ injection, but was not seen in nondiabetic mice. When given to STZ-treated mice, nicotinamide, an inhibitor of poly(ADP-ribose) synthetase, reduced both the elevation of blood glucose and the elevations of HDC activity and histamine production. These results suggest that hyperglycemia may initiate a sequence of events leading not only to an enhancement of basal HDC activity, but also to a sensitization of mice to the HDC-inducing action of LPS. We hypothesize that bacterial infections and diabetic complications may mutually exacerbate one another because both involved an induction of HDC.     

The phosphorylation and isolation of two erythrocyte membrane proteins in vitro

Two proteins of bovine erythrocyte ghost membrane have been phosphorylated with γ-³²P-ATP and isolated by SDS polyacrylamide gel electrophoresis. One of the two proteins (MW 98,000) has been identified here as the phosphorylated intermediate of the Na⁺ + K⁺ activated ATPase. The other phosphorylated protein (MW 220,000) is apparently unrelated to the Na-K ATPase, but may be involved in other energy requiring membrane processes.     

Augmented ciliary reorientation response and cAMP-dependent protein phosphorylation induced by glycerol in triton-extracted Paramecium

In the presence of 30% glycerol, the cilia of a permeabilized cell model from Paramecium exhibit dynamic orientation changes while displaying only a restricted cyclic beating with a very small amplitude. The direction of cilia under these conditions corresponds to the direction of the effective power stroke of cilia beating in the absence of glycerol, i.e., pointing posteriorly in the absence of Ca2+ and anteriorly at > 10(-6) M Ca2+. Ciliary reorientation toward the posterior in response to the removal of Ca2+ is particularly conspicuous; all the cilia become predominantly pointing to the posterior end all through their beating phases. Previous studies suggested that the effect of glycerol is caused through modification of cAMP-dependent protein phosphorylation. To determine whether glycerol in fact affects ciliary reorientation through changes in protein phosphorylation, here we examined protein phosphorylation in the axonemes. Glycerol stimulated cAMP-induced phosphorylation of 29-kDa and 65-kDa proteins. The stimulation of phosphorylation was found to be partly due to the inhibition of endogenous phosphodiesterase (PDE), and partly due to the inhibition of the dephosphorylation of the 29-kDa and 65-kDa phosphoproteins within the axoneme. Thus glycerol appears to cause predominant posterior orientation of cilia by stimulating cAMP-dependent phosphorylation on those proteins. In addition, glycerol appears to inhibit ciliary beating through inhibition of dynein ATPase.