A minor sialoglycoprotein of the human erythrocyte membrane

The sialoglycoprotein periodate fuchsin sulfite 2 has about 8% of the sialic acid contained in the sialoglycoproteins of the human erythrocyte membrane. This polypeptide appears to have an apparent monomeric molecular weight of 35,000, somewhat smaller than the monomer of the major sialoglycoprotein (periodate fuchsin sulfite 1) as judged by sodium dodecyl sulfate-polyacry lamide gel electrophoresis, and has frequently been confused with the monomer of the major sialoglycoprotein. Periodate fuchsin sulfite 2 is not labeled by the lactoperoxidase procedure in the intact cell, although it is accessible to neuraminidase and other hydrolases. On the other hand, this component can be labeled by lactoperoxidase on the cytoplasmic surface of open membranes or resealed ghosts. Thus, it is a trans membrane protein. Although most of the other transmembrane proteins of the human erythrocyte membrane are extracted from the membrane by 0.1% Triton X-100 in 7 mm phosphate buffer, pH 7.4, this component is not removed and may be a cytoskeletal component. Trypsin, chymotrypsin, and thermolysin peptides, as well as cyanogen bromide fragments, clearly indicate that the primary sequence of this polypeptide can be distinguished from dimeric or monomeric forms of the major sialoglycoprotein (periodate fuchsin sulfite 1).     

Abnormal bovine erythrocyte membrane proteins and glycoproteins during and after infection with Anaplasma marginale

The erythrocyte membrane proteins from normal and Anaplasma-infected bovine blood have been compared. Two distinct new polypeptides were present in membranes from acutely infected cells. The glycoprotein pattern was also altered: in addition to the three main bands observed in normal cells, there were four new bands present which were glycosylated. The normally found membrane glycolypeptide (250000 D) was missing. The role of these protein alterations in relation to the infectious process is discussed.     

A novel mass spectrometric procedure for the rapid determination of the types of carbohydrate chains present in glycoproteins: application to alpha-galactosidase I from Vicia faba seeds

A fast atom bombardment mass spectrometric protocol has been developed to determine the type of oligosaccharide chain present in glycoproteins. The procedure is based on acetolysis of the intact glycoconjugate, extraction of the peracetylated carbohydrate fragments and analysis by fast atom bombardment mass spectrometry. The molecular ions present in the FAB spectra uniquely define the composition of the oligosaccharides with respect to hexose, aminohexose and sialic acid content. High mannose oligosaccharides yield a series of peracetylated hexose oligomers whereas complex-type oligosaccharides afford a series of N-acetyl-lactosamine containing species. Fucosylation is usually not detected but sialylated oligosaccharides are readily identified and the type of sialic acid is also defined. The method has been tested on three glycoproteins of known structure - fetuin, ribonuclease B and erythrocyte Band 3 - and on a glycoprotein of unknown structure - alpha-galactosidase I, an enzyme lectin from Vicia faba. The latter is shown to contain high mannose carbohydrate chains.     

Biosynthesis of glycoproteins in human placenta: Processing of oligosaccharides

The processing of the high-mannose asparagine-linked oligosaccharides synthesized by first-trimester human placenta has been investigated. Tissue was pulsed for 1 h with [2-³H]mannose and chased for zero, 45, 90, and 180 min in media containing unlabeled mannose. Glycopeptides, prepared by Pronase digestion of the delipidated membrane pellets at each time point, were treated with endo-β-N-acetylglucosaminidase-H to release the high-mannose asparagine-linked oligosaccharides. The largest major processing intermediate isolated was Glc₁Man₉GlcNAc, which was converted into Man₉GlcNAc, and then into Man₈GlcNAc, Man₇GlcNAc, Man₆GlcNAc, and Man₅GlcNAc. There was also a minor pathway in which mannosyl residues were removed prior to the glucose. By carrying out the detailed structural characterization of the individual processing intermediates, it was possible to demonstrate that processing of the Man₉GlcNAc to Man₅GlcNAc proceeded by the nonrandom removal of the α1,2-linked mannosyl residues. Specifically, of 12 possible sequences of removal of the four α1,2-linked mannosyl residues present in Man₉GlcNAc, first-trimester human placenta utilized only two of these in the processing of asparagine-linked oligosaccharides. It is suggested that the limited number of processing pathways reflects a high degree of specificity of these reactions in human placenta.     

The basis for EDTA-stimulation of methemoglobin reduction in hemolysates of human erythrocytes.

We have studied the stimulation by EDTA of methemoglobin reduction in hemolysates of human erythrocytes. The EDTA effect has been shown not to be the result of an allosteric interaction of EDTA with hemoglobin or the result of a photochemical reduction. The effect does not appear to be due to a direct interaction of free EDTA with either of the catalytic components of the erythrocyte methemoglobin reduction system. The EDTA stimulation seen in hemolysates is due to the formation of an iron-EDTA complex, which transfers electrons from the reductase to methemoglobin.     

Methylation analysis in glycoprotein chemistry: a comparative study of the carbohydrate structures of three hormone-binding glycoproteins from human serum

The structures of the carbohydrate moieties of three hormone-binding glycoproteins from human serum, namely, thyroxine-binding globulin, transcortin, and sex hormone-binding globulin, have been characterised using quantitative g.l.c. of the methylated monosaccharide derivatives obtained after methanolysis of the methylated glycoproteins.     

Passive shedding of erythrocyte antigens induced by membrane rigidification

Rigidification of the cell membrane lipid bilayer can lead to an increase in the degree of exposure of membrane proteins to either side of the membrane. It is shown in this study that excess increase of the membrane lipid microviscosity (‘hyper-rigidification’) in intact human erythrocytes can cause the release of Rh₀(D) and A blood group antigens from the cell surface which can then be collected from the supernatant by affinity chromatography. The most efficient antigen shedding was achieved upon incorporation of cholesteryl hemisuccinate (CHS) (incubation for 2 h at 37 °C in a mixture of 200 μg/ml CHS, 3.5% polyvinylpyrrolidone 1% bovine serum albumin, 0.5% glucose in phosphate-buffered saline) followed by application of hydrostatic pressure (1 500 atm, 5 min) which increases the lipid microviscosity by about 2-fold. This technique can be of general application for isolation of membrane proteins without disruption of the cells or the use of detergents.     

Iron-sulfur clusters and cysteine distribution in a ferredoxin from Azotobactervinelandii

In 80% dimethyl sulfoxide/H₂O, $\text{Azotobacter}$ ferredoxin FeS clusters can be extruded with benzene thiol. The extruded clusters have an absorption spectra maximum at 458 nm which is characteristic of 4Fe4S centers. The amino terminal sequence of the $\text{Azotobacter}$ ferredoxin has 7 of the 8 Cys residues at residue numbers 8, 11, 16, 20, 24, 39 and 42. Except for Cys 24, all of these residues can be correlated to homologous Cys residues in other bacterial ferredoxins. Although two thirds of the first 45 residues are identical to or conservative replacements for the first 43 residues of other bacterial ferredoxins, the insertion of Cys-24 indicates a major change in the environment of one of the two 4Fe4S clusters.     

Evidence for the presence of the trimannosyl-di-N-acetyl-chitobiose core in the carbohydrate chains of human-parotid,proline-rich glycoprotein

The carbohydrate chains of the human-parotid, proline-rich glycoprotein are linked through a single type of carbohydrate-peptide linkage (asparaginyl-N-acetyl-glucosamine). The structure of the internal part of the carbohydrate chains, determined by chemical, enzymic, and g.l.c.-m.s. methods, includes the trimannosyl-di-N-acetylchitobiose core involved in the carbohydrate-peptide linkage. Furthermore, an L-fucose residue is linked to the 2-acetamido-2-deoxy-d-glucosyl residue linked to the L-asparaginyl residue. The sequence of the peripheral part of the chains has also been determined as α-L-Fucp→β-d-Galp→β-d-GlcpNAc→α-d-Manp, suggesting a double-branched, basic carbohydrate structure.     

Reductive cleavage of Xaa-proline peptide bonds by mild alkaline borohydride treatment employed to release O-glycosidically linked carbohydrate units of glycoproteins

During the deglycosylation reaction of fish egg polysialoglycoproteins under the conditions of 1 M NaBH4 in 0.1 M NaOH at 37 degrees C for 48 h, a marked loss of the glycine content has been encountered, besides the serine and threonine residues to which the carbohydrate units are linked. The chemical basis behind this phenomenon has been elucidated by amino acid analysis first of the major glycopeptides (carbohydrate-(O)Thr-Gly-Pro-Ser) derived from desialylated polysialoglycoproteins and subsequently six proline-containing peptides before and after treatment under similar conditions. It has thus been established that -Xaa-Pro- sequences are remarkably susceptible to reductive cleavage under such mild aqueous conditions. In view of the finding that the reductive cleavage of insulin B-chain, which contains a single proline residue adjacent and C-terminal to a threonine residue, led to about 80% loss of the threonine residue, deglycosylation with alkaline borohydride reagents warrants a special comment. The decreased amounts of serine or threonine residues cannot be related simply to the degree of glycosylation of these residues. The above results are therefore discussed in the relation to other work.     

Chemical and physical properties of human bronchial mucus glycoproteins.

Human bronchial mucus glycoproteins of different chemical types were isolated by Ecteola and gel exclusion chromatography. Chemical analysis indicated polydispersity with regard to content of sulfate and sialic acid. No blood group A, B or H activity was found in these glycoproteins. Compositions are reported for amino acid and sugar residues for several fractions obtained from both cystic fibrotic and chronic bronchitic mucus. It is noteworthy that glycoproteins extracted from a single subject contain molecules with different acid groups as well as significant differences in carbohydrate chain length.     

The interaction of human erythrocyte band 3 with cytoskeletal components

Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of ³⁵SO₄. was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 n NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 n NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.     

Irreversible inactivation of human erythrocyte pyruvate kinase by 2,3-butanedione

Human erythrocyte pyruvate kinase was found to be irreversibly inactivated by butanedione in the dark. The second-order rate constants for inactivation at pH 8.0 and 25 degrees C were 2.14 and 2.74 M-1 min-1 in the absence and presence of 50 mM borate, respectively. The pH profile of the inactivation indicated the involvement of a residue with an apparent pK alpha of 8.1-8.3. ADP and phosphoenolpyruvate acted as partial inhibitors of the inactivation process. Certain details of the inactivation, spectral studies, and fluorometric determinations gave evidence for arginine as the only target residue. A total of 23 +/- 3 residues per subunit were modified within the period required for inactivation. In the same period the presence of 4 mM ADP reduced the extent of inactivation by 70% and the number of modified residues to 18 +/- 4. The number of the arginine residues protected by ADP from butanedione modification was 5.0 +/- 1.3 per subunit.     

Rapid purification of human placental aldose reductase

Sixty percent methanol is widely used for the extraction of nucleotides from lymphocytes for quantitation by high-performance liquid chromatography. In the course of such studies, we noted that these extracts analyzed on an anion-exchange column showed a major “unknown” uv-absorbing peak which eluted after the nucleosides and before the nucleotides. The material cochromatographed with and had the spectral properties of ascorbic acid. This compound was identified as ascorbic acid by chemical and enzymatic assays. The ascorbate content of human lymphocytes determined by high-performance liquid chromatography, $\text{42.2 ± 3.3 nmol}\text{10}{}^8$ cells (mean ± SEM), agreed closely with the levels obtained by standard less sensitive methodology. Evidence is presented that this technique can be used to determine the ascorbate content of lymphocytes where only scanty material or very low levels are found.     

Carbohydrate-peptide linkage in glycoproteins

The secondary structure of the peptide segment around the carbohydrate-peptide linkage in glycoproteins was predicted by using the Chou and Fasman determination. Such a study was carried out for 9 O-glycosidically linkages and 28 N-glycosidically linkages. In the case of O-glycosidically linkages, the residue Ser or Thr involved in the linkage always belongs to a β-turn. In the case of N-glycosidically linkages, 19 out of the 28 Asn studied belong to a β-turn. A predicted determination concerning the whole protein moiety of 9 glycoproteins in order to obtain some information concerning the spatial organization of the entire glycoprotein was carried out also. It seems that carbohydrate moiety takes place outside the glycoprotein.