Immunochemical Studies of Infectious Mononucleosis IX. Heterophile Antigen Associated with a Glycoprotein from the Bovine Erythrocyte Membrane

Abstract. A glycoprotein was isolated from the membrane of the bovine erythrocyte by refluxing the acetone- and ethanol-extracted stroma residue with 75% ethanol. The glycoprotein was purified by phosphocellulose chromatography, ethanol precipitation, lipid-solvent extraction and DEAE chromatography. The glycoprotein appeared to have two serological determinants, both reactive with antibodies present in the sera of patients with infectious mononucleosis. One of the determinants is similar to the Paul-Bunnell heterophile antigen found on sheep erythrocytes. It is dependent on carbohydrate, including sialic acid residues. Another specificity, seemingly not shared by sheep erythrocytes to any great extent, is resistance to neuraminidase and to alkaline borohydride treatment and thus it may be located either on the polypeptide portion of the molecule or on an alkali-stable oligosaccharide. The purified glycoprotein comprises 73% amino acids. Carbohydrate components and their molar ratios were sialic acid (1.0): galactose (1.5): N-acetylglucosamine (1.1): N-acetylgalactosamine (0.5): mannose (0.1).     

Amino acid and carbohydrate structural variants of glycoprotein products (M-N glycoproteins) of the M-N allelic locus.

Major glycoprotein of MgM, MM Miltenberger III (MiIII), and M-N erythrocyte membranes from individual donors were cleaved with CNBr and their amino-terminal octapeptides were examined with respect to amino acid and carbohydrate composition. The amino-terminal octapeptides from the heterozygous MgM donor were resolved into two types, A and A'. MgM A was identical to octapeptide A from MM glycoproteins in carbohydrate and amino acid compositions. MgM A' exhibited amino acid composition similar to NN peptide A except for a single substitution of an Asx for a Thr and, as a result, was not glycosylated. MM(MiIII) octapeptide A was identical to M peptide A in amino acid composition, but differed in carbohydrate content. This glycopeptide contained three O-glycosidically linked carbohydrate units, one of which contained GlcNAc bound to a core of NeuAc, Gal, and GalNAc. About two such units were also present in the CNBr glycopeptide B of the glycoprotein, and on the basis of studies with alkaline borohydride and alkaline sulfite degradations, these units are believed to have the following structure: (formula see text) The Mg is an allelomorph of the M-N locus, likely evolved from a single base substitution in the N gene. The resulting single amino acid substitution effects the posttranslational carbohydration of neighboring Ser and Thr residues. The MM(MiIII) appears to be a product of the M gene that undergoes sequences of posttranslational glycosylations different from those of the M-N glycoproteins.     

Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized, double-blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein-Barr virus vaccine in healthy young adults

BACKGROUND: To date, there is no commercially available vaccine to prevent infectious mononucleosis, a disease frequently induced by Epstein-Barr virus (EBV) infection in adolescents or adults devoid of preexisting immunity to the virus. METHODS: A total of 181 EBV-seronegative, healthy, young adult volunteers were randomized in a double-blind fashion to receive either placebo or a recombinant EBV subunit glycoprotein 350 (gp350)/aluminum hydroxide and 3-O-desacyl-4'-monophosphoryl lipid A (AS04) candidate vaccine in a 3-dose regimen. RESULTS: The vaccine had demonstrable efficacy (mean efficacy rate, 78.0% [95% confidence interval {CI}, 1.0%-96.0%]) in preventing the development of infectious mononucleosis induced by EBV infection, but it had no efficacy in preventing asymptomatic EBV infection. One month after receipt of the final dose of gp350 vaccine, 98.7% of subjects showed seroconversion to anti-gp350 antibodies (95% CI, 85.5%-97.9%), and they remained anti-gp350 antibody positive for >18 months. Furthermore, there were no concerns regarding the safety or reactogenicity of the gp350/AS04 vaccine. CONCLUSION: These data support the clinical feasibility of using an EBV vaccine to prevent infectious mononucleosis. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00430534.     

Heterozygosity of CDAN II (HEMPAS) gene may be detected by the analysis of erythrocyte membrane glycoconjugates from healthy carriers

BACKGROUND AND OBJECTIVES: Congenital dyserythropoietic anemia (CDA) type I, II, and III, is associated with abnormalities of erythrocyte membrane glycoconjugates that are most pronounced in type II CDA or hereditary erythroblastic multinuclearity with a positive acidified-serum test (HEMPAS). The abnormalities consist in hypoglycosylation of polylactoaminoglycans linked to proteins (as in band 3 glycoprotein) and ceramides (known under the name of polyglycosylceramides) as well as in accumulation of some oligoglycosylceramides: lactotriaosylceramide, neolactotetraosylceramide, and sometimes globotetraosylceramide. Glycophorin A is partially unglycosylated with respect to O-linked glycans. Types I and II of the disease are inherited in an autosomal recessive fashion. The aim of the present study was to investigate a possibility that heterozygosity with respect to CDAN2 gene in healthy carriers could be detected by analysis of erythrocyte membrane glycoconjugates. DESIGN AND METHODS: We examined a family which consisted of heterozygous parents and their two sons, one of whom was afflicted with CDA II (proband) while the other was healthy. In all family members the glycosylation status of band 3 glycoprotein, polyglycosylceramides and glycophorin A was evaluated from their carbohydrate molar composition. In addition we determined erythrocyte membrane contents of oligo- and polyglycosylceramides, and agglutinability of erythrocytes by anti-i antibody. RESULTS: We found that the heterozygous parents showed, but about 50% less pronounced, most of the typical abnormalities of erythrocyte membrane glycoconjugates that were present in the proband. These abnormalities included: hypoglycosylation of band 3, accumulation and hypoglycosylation of polyglycosylceramides, and accumulation of lactotriaosylceramide. The level of neolactotetraosylceramide in the erythrocyte membranes of the parents was, however, normal. Globotetraosylceramide content was elevated in erythrocytes from the proband and, surprisingly, even more so in the parents. Glycophorin A in the proband was only slightly abnormal. Erythrocytes from both the parents and the proband expressed increased agglutinability with anti-i antibody. All glycoconjugates examined were normal in erythrocytes from the healthy son. INTERPRETATION AND CONCLUSIONS: Individuals heterozygous with respect to CDAN2 gene can be identified through determination of the carbohydrate molar composition of band 3 and polyglycosylceramides as well as by an elevated erythrocyte content of polyglycosylceramides. In the parents these abnormalities show dosage effects. Determination of the carbohydrate molar composition of glycophorin A and of oligoglycosylceramides seems to be less promising. These findings indicate that the analysis of erythrocyte membrane glycoconjugates may be a valuable addition to the repertoire of methods used in studies on the genetics of CDA.     

Hepatitis in fatal infectious mononucleosis

A detailed clinicopathologic analysis of 30 patients with sporadic fatal infectious mononucleosis and 31 males with fatal infectious mononucleosis and the X-linked lymphoproliferative syndrome was performed to determine the extent of hepatic dysfunction in these cases. At death, the median age of patients with sporadic infectious mononucleosis was 10.7 yr vs. 2.4 yr for X-linked lymphoproliferative syndrome. The median survival time was 8 wk for sporadic infectious mononucleosis and only 4 wk for X-linked lymphoproliferative syndrome. The male to female ratio was 3:2 in sporadic infectious mononucleosis; all patients with X-linked lymphoproliferative syndrome were males. Fever, sore throat, lymphadenopathy, hepatomegaly, and splenomegaly were prominent findings. Hepatic dysfunction was uniformly present and caused death in 13 of 30 sporadic infectious mononucleosis cases and 18 of 31 X-linked lymphoproliferative syndrome cases. Diagnosis of infectious mononucleosis was confirmed by heterophile antibody titers or Monospot, Epstein-Barr virus antibody studies, viral culture, molecular hybridization studies, clinical and histologic findings, and pedigree analysis.     

A rapid slide test for heterophile antibody in infectious mononucleosis

The sera of 473 individuals were examined for sheep cell agglutinins both by theslide test and the Paul-Bunnell method. In this group there were 46 patients withpositive slide tests and 35 of these individuals also had a diagnostic serum dilutiontest for heterophile antibody. In 11 cases the slide test was positive but the Paul-Bunnell test gave very low serum dilution values. However, when the slide testwas carried out at 37 C, it was negative in 9 of the 11 cases. In the remaining 2instances, one patient had a Forssman type of antibody which gave a 1:64 titer insaline and the slide test was positive at 37 C. In the other case no studies were madeon the effect of temperature and the nature of the agglutination reaction wasunfortunately not determined.

Using human and bovine albumen, sheep serum and human AB serum absorbedwith sheep cells as a diluent no evidence for blocking or hyperimmune antibodywas discovered in the cases of infectious mononucleosis studied in this series.Moreover, of the 6 patients with negative serology but with strong clinical andhematological evidence for the disease, no blocking or hyperimmune antibodywas disclosed by the slide test or by the use of absorbed human AB serum. Theconclusion seems justified that blocking, incomplete or hyperimmune heterophileantibody must be rather uncommon in infectious mononucleosis.

In the use of the rapid slide test it has been pointed out that cold agglutinins,(which may be abolished by warming to 37 C) and Forssman antibodies (whichmay be absorbed by guinea pig kidney) can give positive results. However, diseases in which cold agglutinins are strong enough to give a positive slide testare relatively rare and the occurrence of Forssman antibodies of a strength likely togive a positive slide test would be decidedly uncommon. In any event unless furtherexperience reveals more serious discrepancies, the rapid slide test as described inthis paper seems to offer a practical screening test to detect clinically significantamounts of heterophile antibody in cases of infectious mononucleosis.

     

Vaccination with‘concealed’antigens: myth or reality?

Cattle infested with the tick Boophilus microplus produce antibodies to intrinsic membrane glycoproteins of the tick, as well as to Bm86, a well characterized antigen from the tick gut. Several factors explain how cattle could produce antibody to such antigens, which one would expect to be 'concealed' from the host's immune system, during natural infestation. It has been shown that the carbohydrate determinants on many tick glycoproteins are cross-reactive immunologically and that the reaction of bovine antibodies with intrinsic membrane glycoprotein is at least partially blocked by low molecular weight carbohydrate. Further, antisera from cattle exposed to ticks react with a glycosylated, native Bm86 but not with a non-glycosylated, recombinant Bm86. Thus the reaction of concealed antigens with antibodies produced as a result of tick infestation appears to be due to a relatively non-specific reaction with carbohydrate determinants on tick glycoprotein. Evidence is also presented that antibodies directed against carbohydrate determinants of Bm86 are not protective. Care must therefore be exercised in interpreting the results of antibody reaction with glycoproteins in such complex organisms.     

Elevated Toxoplasma IgG antibody in patients tested for infectious mononucleosis in an urban emergency department

There are many infectious causes of fatigue, sore throat, and fever, including mononucleosis and toxoplasmosis. Toxoplasma antibody testing is rarely performed in most emergency departments; as a result, toxoplasmosis is diagnosed infrequently. We obtained Toxoplasma IgG IFA titers on ED patients who had mononucleosis testing performed to determine the frequency of toxoplasmosis in this population. Two hundred sixty patients were included in our study. Eleven (4.2%) had a positive mononucleosis test, and 14 (5.4%) had a positive Toxoplasma titer. In the detection of toxoplasmosis, Toxoplasma IgG titers of 1:1,024 or greater have been shown to be a sensitive means of detecting infection in the first six months. Further testing with IgM titers is needed to establish a positive diagnosis when necessary. We found more patients with elevated Toxoplasma IgG titers than with positive heterophil antibody titers in an ED population tested for mononucleosis over a two-year period. We conclude that toxoplasmosis may be as common as mononucleosis in our ED and that clinicians should consider this pathogen when working up patients with appropriate symptoms.     

Primary infection with Epstein-Barr virus in infants in the United States: clinical and serologic observations.

Previous studies in Ghana had shown that primary infections with Epstein-Barr virus in infants under the age of two years remain silent and evoke antibody responses different from those seen in infectious mononucleosis. In order to determine whether or not these observations were limited to Africa, 80 American infants presenting with minor infectious complaints were studied serologically; 14 (17.5%) showed evidence of recent or current primary infections with Epstein-Barr virus. The clinical features of these 14 infants were similar to those of the other 66 and did not suggest a diagnosis of infectious mononucleosis, nor were there histories of a recent infectious mononucleosis-like illness. Ten (72%) had antibodies to the early antigen complex induced by Epstein-Barr virus; however, these antibodies were directed, as in the Ghanaian infants, against the restricted rather than the diffuse components, in contrast to the pattern generally observed in infectious mononucleosis. Possible reasons for the differences between the responses of infants and those of older individuals to primary infection with Epstein-Barr virus and to the early antigen complex are discussed.     

A comparative study of the major glycoprotein isolated from normal and neoplastic gastric mucosa

The isolation and composition of glycoproteins from mucosae of normal stomachs, of stomachs with gastric ulcer, and of stomachs with carcinoma is described.The glycoproteins from the mucosae of normal stomachs and with gastric ulcer showed virtually the same carbohydrate and amino acid content as the principal gastric glycoprotein isolated from gastric aspirates. They all revealed a common basic carbohydrate composition: galactose, fucose, glucosamine, and galactosamine were present in approximate molar ratios of 4:3:3:1.THE RESULTS SUGGEST THAT THE GLYCOPROTEINS ISOLATED FROM GASTRIC ASPIRATES FROM NORMAL AND NEOPLASTIC GASTRIC MUCOSAE SHARE A NUMBER OF STRUCTURAL FEATURES: (1) a protein core with a characteristic amino acid composition; (2) the range of sugars forming the carbohydrate side chains; (3) galactosamine approximately equimolar with the sum of threonine and serine; (4) galactose approximately equimolar with the sum of glucosamine and galactosamine; (5) absence of mannose; (6) a high carbohydrate content (80-85%); and (7) blood group activity.The neoplastic glycoproteins differed from the normal glycoproteins in that the quantitative relationships of the carbohydrate components of the neoplastic glycoproteins showed variations dividing the extracts investigated into groups, each group with a distinctive and constant carbohydrate composition. The blood group specificity of 15 out of 24 cases investigated differed from that of the hosts' red cells.     

The "Infectious Mononucleosis Cell" A Cytochemical Study

The abnormal cells in the peripheral blood of patients with infectious mononucleosis were studied by available histochemical technics and with immunofluorescent and autoradiographic technics. The findings indicated a closerelationship of these cells to lymphocytes, although the strong acid phosphatase reaction observed in the infectious mononucleosis (IM) cells wasnot usually seen in normal lymphocytes, but rather in monocytes and reticulum cells. There was no evidence of antibody production by the IM cells ofthe peripheral blood.

Studies of the lymph nodes showed two abnormal cell types: one, similarto the IM cells in the peripheral blood but more immature, and the second,a primitive cell with various indications of immunologic competence.

Submitted on February 28, 1963 Accepted on July 8, 1963     

Decrease in generation of reactive oxygen species by neutrophils from patients with infectious mononucleosis: role of suppressor T lymphocytes

We assessed the generation of reactive oxygen species (ROS: O2-, H2O2, OH . , chemiluminescence) by neutrophils and monocytes from six patients with infectious mononucleosis, ten patients with other viral diseases, and ten normal controls. Neutrophils from infectious mononucleosis patients showed markedly decreased generation of all reactive oxygen species, compared with the two control groups; this abnormality persisted for four to eight weeks after disease onset. Monocytes from these patients generated normal levels of ROS. Normal neutrophils incubated with T lymphocytes from infectious mononucleosis patients generated significantly less of each ROS than did those incubated with T cells from either control group. T cell-mediated suppression of ROS generation required both OKT4+ cells from infectious mononucleosis patients and OKT8+ cells from either patients or normals. We conclude that the generation of reaction oxygen species in neutrophils is suppressed in patients with infectious mononucleosis, at least in part, by interacting subsets of T lymphocytes.     

Characterization of murine monoclonal antibodies directed against the Kell blood group glycoprotein

Murine monoclonal antibodies (MoAbs) were produced against the blood group KEL1 glycoprotein (93 kD component) immunopurified from human erythrocytes. One monoclonal antibody, 5A11 (IgGa, kappa), detects by immunoblotting a 93 and 184 kD component from KEL: 1,-2 or KEL: -1,2 red cell membrane preparations, separated by SDS polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions. The 184 kD component was not detected under reducing conditions, suggesting that it represented a dimer of the 93 kD KEL glycoprotein. Neither the 93 nor the 184 kD could be detected from K0 or McLeod erythrocyte membrane preparations, indicating that the monoclonal antibody reacts with the KEL glycoprotein previously identified as a 93 kD species. Since this antibody does not agglutinate native or protease-treated erythrocytes, it is likely that it reacts with the cytoplasmic domain of the KEL glycoprotein. This was also substantiated by showing that 5A11 could immunoprecipitate the 93 kD component from intact membranes and inside-out vesicles but not from right-side-out vesicles. Immunostaining of membrane proteins prepared from human blood cells (platelets, lymphocytes, monocytes and granulocytes) and non-human erythrocytes revealed that the 93 kD molecule was only present on human red cells. Several other murine monoclonal antibodies obtained from the same fusion experiment gave identical results, but competition analyses on immunoblots indicated that these antibodies reacted with distinct epitopes on the KEL glycoprotein.     

Chemical Characterization and Surface Orientation of the Major Glycoprotein of the Human Erythrocyte Membrane

The major glycoprotein of the human erythrocyte membrane has been isolated by treatment with lithium di-iodosalicylate and found to be a single polypeptide chain with a molecular weight of about 50,000. This molecule, which is 60% carbohydrate and 40% protein, carries multiple blood-group antigens, the receptors for influenza viruses, and various plant agglutinins. Four unique carbohydrate-containing peptides (alpha-1, alpha-2, alpha-3, and beta) are produced by tryptic digestion of the isolated glycoprotein; their order in the molecule has been determined by sequential tryptic digestion of intact erythrocyte membranes and partially digested glycoprotein fragments. Cleavage of the native protein with cyanogen bromide produces five fragments; two of these (C-5 and C-1) contain most of the carbohydrate in the molecule and are derived from the N-terminal half of the polypeptide chain. The nonpolar amino acids of this glycoprotein are located predominantly in the C-terminal fragment (C-2).Phytohemagglutinin conjugated to ferritin has been used to map the distribution of glycoprotein receptors over the surfaces of intact erythrocytes by freeze-etching and electron microscopy. This label localizes to sites on the membrane that overlie the intramembranous particles. These findings suggest that the glycoprotein is oriented at the cell surface with its oligosaccharide-rich N-terminal end exposed to the exterior, while its C-terminal segment interacts with other components in the interior of the membrane to form intramembranous particles.     

Participation of spectrin in Sendai virus-induced fusion of human erythrocyte ghosts.

Fusion of washed human erythrocyte ghosts could be induced by the addition of Sendai virus after they were loaded with bovine serum albumin and resealed. Antispectrin antibody purified on a spectrin-Sepharose column and sequestered in the ghosts at 4-5 mg/ml together with the albumin was highly inhibitory for the virus-induced cell fusion, whereas Fab fragments prepared from the same antibody were without effect. The virus-induced aggregation of intramembrane particles of human erythrocytes was also inhibited by the same concentrations of the antispectrin antibody. The virus-induced agglutinations of the ghosts and release of bovine serum albumin from the ghosts (which might be caused by fusion of the viral envelope to the erythrocyte membrane) were not inhibited by the sequestered antibody. Therefore, the antibody seems to inhibit fusion at the last step--i.e., fusion between adjacent erythrocyte membranes. Similarities and differences of the mode of participation of spectrin in the virus-induced fusion and in other membrane-linked phenomena of human erythrocytes are discussed.     

Abrogation of cell-mediated immunity by a serum blocking factor isolated from patients with infectious mononucleosis.

Lymphocytes from 80% of patients with infectious mononucleosis in this study failed to produce macrophage migration-inhibition factor in response to partially purified early antigen of Epstein-Barr virus or to tetanus toxoid, whereas lymphocytes from normal subjects did produce this lymphokine. Subsequent analysis of serum from the patients with infectious mononucleosis revealed a serum factor that completely abrogated antigen-specific inhibition of migration by human leukocytes as well as lymphocyte blastogenesis. The serum blocking factor was present in sera from 11 (73%) of 15 patients with infectious mononucleos but only in sera from two (13%) of 15 normal subjects. Samples of serum from five of the patients with infectious mononucleosis and five normal subjects were fractionated with use of Sephadex G-200 gel filtration, and the eluants were assayed for several substances known to inhibit cell-mediated immunity. Serum blocking factor activity could be demonstrated only in fractionated sera from patients with infectious mononucleosis. The serum blocking factor is postulated to be either a soluble immune complex or some as yet unidentified immunoregulatory globulin contained in the IgG fraction of human serum.     

Increased Activity of Some Folic Acid Enzyme Systems in Infectious Mononucleosis

Increased levels of activity for the formate activating enzyme and N⁵,N¹⁰-methylene FH₄ dehydrogenase have been found in the leukocytes from patients with infectious mononucleosis; in addition dihydrofolic reductase, anenzyme not found in mature circulating leukocytes, has been detected in infectious mononucleosis cells. Glucose-6-phosphate dehydrogenase activity isless in infectious mononucleosis leukocytes than in normal leukocytes.

These findings are similar to the results obtained in acute and chronic myelogenous leukemia and indicate that the leukocytes seen in infectious mononucleosis have the enzymatic apparatus associated with synthesis of DNA.

Submitted on November 6, 1961 Accepted on January 12, 1962     

Epstein-Barr virus-induced diseases in boys with the X-linked lymphoproliferative syndrome (XLP): update on studies of the registry

Analyses of 100 subjects with the X-linked lymphoproliferative syndrome (XLP) in 25 kindreds revealed four major interrelated phenotypes: infectious mononucleosis, malignant B-cell lymphoma, aplastic anemia, and hypogammaglobulinemia. Eighty-one of the patients died. Two male subjects were asymptomatic but showed immunodeficiency to Epstein-Barr virus (EBV). Seventy-five subjects had the infectious mononucleosis phenotype and concurrently, 17 subjects of this group had aplastic anemia. All subjects with aplastic anemia died within a week. Aplastic anemia did not accompany hypogammaglobulinemia or malignant lymphoma phenotypes. Hypogammaglobulinemia had been detected before infectious mononucleosis in three subjects, after infectious mononucleosis in five subjects, and was not associated with infectious mononucleosis in 11 boys with hypogammaglobulinemia. In nine subjects infectious mononucleosis appeared to have evolved into malignant lymphoma; however, the majority of patients with malignant lymphoma showed no obvious antecedent infectious mononucleosis. One subject had infectious mononucleosis following recurrent malignant lymphoma. Twenty-six of 35 lymphomas were in the terminal ileum. Results of immunologic and virologic studies of 15 survivors revealed combined variable immunodeficiency and deficient antibody responses to EBV-specific antigens. Mothers of boys with XLP exhibited abnormally elevated titers of antibodies of EBV. Subjects of both sexes with phenotypes of XLP should be investigated for immunodeficiency to EBV. Persons with inherited or acquired immunodeficiency may be vulnerable to life-threatening EBV-induced diseases.     

Production of a New Murine Monoclonal Antibody with Fy6 Specificity and Characterization of the Immunopurified N-Glycosylated Duffy-Active Molecule

A murine monoclonal antibody (mAb i3A; IgG1, kappa light chain) was obtained using human red blood cells as immunogen. The antibody showed Fy6 specificity since it agglutinated all but Fy(a-b-)-untreated red cells and failed to agglutinate chymotrypsin-treated cells. An erythrocyte membrane protein of 42–46 kD was revealed as the major component recognized by the antibody on immunoblots. The antibody also bound to 92- to 95- and 200-kD proteins, tentatively identified as oligomers of the 42- to 46-kD monomeric form. The affinity-purified Fy6-active protein was converted to a sharp band of 35 kD after N-glycanase treatment. The molecule appeared as a slightly broadly band after neuraminidase treatment but was not further altered by O-glycanase. The i3A mAb bound to 6,000±1,000 receptor sites on either Fy(a-b+), Fy (a+b+) and Fy(a+b-) red cells with an affinity constant in the range of 3–6 times 10⁸ M^-1. No binding was observed to other blood cells nor to several cells (B, T, myelomonocytic and erythro-leukemia cell lines). Also, the bulk of i3A-Fy6 immune complexes could be dissociated from the red cell membrane with as low as 0.2% Triton X-100, showing that the Fy6-active glycoprotein is not tightly associated with the membrane skeleton. Our data obtained with a new monoclonal antibody directed to the Fy6 antigen demonstrate that the blood group Duffy-active component is a red cell-specific glycoprotein carrying one or more N-linked oligosaccharides.     

Evidence that two forms of bovine erythrocyte cytochrome b5 are identical to segments of microsomal cytochrome b5.

Homogeneous preparations of two forms of soluble cytochrome b5 have been obtained from bovine erythrocytes by successive chromatography on DEAE-cellulose, Bio-Gel P-60, and DEAE-Sephadex. Although the two forms could be separated on disc gel electrophoresis, they appeared to have similar molecular weights of approximately 12,000 and identical visible absorbance spectra. The tryptic hemepeptides derived from the two forms of bovine erythrocyte cytochrome b5 are electrophoretically indistinguishable from each other and from the tryptic core hemepeptide derived from liver microsomal cytochrome b5. The bovine erythrocyte tryptic hemepeptide was purified to homogeneity; its amino acid composition was shown to be identical to that of tryptic hemepeptide from liver microsomal cytochrome b5. The amino acid compositions of the two isolatable forms of erythrocyte cytochrome b5 correspond well to the compositions of the 97- and 95-residue segments of native liver microsomal cytochrome b5 that begin at the NH2 terminus. These results agree with the hypothesis that soluble erythrocyte cytochrome b5 is derived from microsomal protein by proteolysis during erythroid maturation.