The organization of the major protein of the human erythrocyte membrane

The enzyme lactoperoxidase was used to catalyse the radioiodination of membrane proteins in intact human erythrocytes and in erythrocyte `ghosts'. Two major proteins of the erythrocyte membrane were isolated after iodination of these two preparations, and the peptide `maps' of each protein so labelled were compared. Peptides from both proteins are labelled in the intact cell. In addition, further mobile peptides derived from one of the proteins are labelled only in the `ghost' preparation. Various sealed `ghost' preparations were also iodinated, lactoperoxidase being present only at either the cytoplasmic or extra-cellular surface of the membrane. The peptide `maps' of protein E (the major membrane protein) labelled in each case were compared. Two discrete sets of labelled peptides were consistently found. One group is obtained when lactoperoxidase is present at the extra-cellular surface and the other group is found when the enzyme is accessible only to the cytoplasmic surface of the membrane. The results support the assumption that the organization of protein E in the membrane of the intact erythrocyte is unaltered on making erythrocyte `ghosts'. They also confirm previous suggestions that both the sialoglycoprotein and protein E extend through the human erythrocyte membrane.     

The Mr 93,000 polypeptide of the postsynaptic glycine receptor complex is a peripheral membrane protein

The glycine receptor of mammalian spinal cord is an oligomeric membrane protein that, after affinity purification on aminostrychnine-agarose or immobilized antibody, contains three polypeptides of Mr 48,000, 58,000, and 93,000. Here, the association and the properties of the polypeptides of the rat glycine receptor were investigated. Upon phase partitioning in the nonionic detergent Triton X-114, the three receptor polypeptides behaved as a hydrophilic protein complex exhibiting phospholipid binding. Sucrose gradient centrifugation or gel filtration in the presence of dithiothreitol and Triton X-100 separated the Mr 93,000 polypeptide from the Mr 48,000 and 58,000 polypeptides, which harbor the antagonist binding site of the glycine receptor. Alkaline or dimethylmaleic acid anhydride treatment of crude synaptic membrane fractions resulted in extraction of the Mr 93,000 polypeptide. Lectin binding was observed for the Mr 48,000 and 58,000 glycine receptor subunits but not the Mr 93,000 polypeptide. These results indicate that the Mr 93,000 polypeptide is a peripheral membrane protein that is located at the cytoplasmic face of the postsynaptic glycine receptor complex.     

Human erythrocyte protein 4.2, a high copy number membrane protein, is N-myristylated.

Band 4.2 is a major protein of the erythrocyte membrane which has been immunologically detected in a variety of cell types and is apparently essential for normal erythrocyte membrane function. Since band 4.2 has unusual solubility and membrane binding properties and has an N-terminal glycine following the initiating methionine, we explored the possibility that band 4.2 is myristylated. When Sf9 cells infected with a recombinant band 4.2 Baculovirus were incubated with [3H]myristic acid, label became incorporated into recombinant band 4.2 protein and resisted extraction with hydroxylamine. Consistent with N-terminal myristylation, the incorporation of label was dependent upon protein synthesis. The fatty acid covalently bound to recombinant band 4.2 was definitively identified as myristic acid by recovering the fatty acid after hydrolysis of band 4.2 and examining its migration relative to standards in thin layer chromatography. It was determined that native erythrocyte band 4.2 is an N-myristylated protein by reverse phase high performance liquid chromatography detection of an azlactone derivative of N-myristylglycine after mild acid hydrolysis and azlactone derivatization of the purified protein. Study of myristylation of band 4.2, an abundant normal cellular protein, and its role in membrane binding may produce insights relevant to other myristylated cellular proteins.     

The structure of the major protein of the human erythrocyte membrane. Characterization of the intact protein and major fragments.

Polypeptide 3, the major membrane-penetrating protein of the human erythrocyte membrane, was characterized, together with two major fragments derived by specific proteolysis of the native protein in the membrane. One fragment (fragment 3f) was obtained from thermolysin cleavage in the extracellular region of the protein, and the other (fragment T1) was derived from tryptic cleavage in the intracellular region of the protein. The results of N- and C-terminal group analysis suggest that fragment 3f contains the N-terminal region of polypeptide 3 and fragment T1 contains the C-terminal part of the molecule. The carbohydrate contents of the polypeptides suggest that carbohydrates are present in three regions of the molecule, much of this carbohydrate being present in the C-terminal part of the molecule. This region of the protein also contains the receptors for concanavalin and the lectins from Phaseolus vulgaris and Ricinis communis, and our results suggest that there is heterogeneity in the carbohydrate chains present in the C-terminal region of polypeptide 3. These data are related to the folding of polypeptide 3 in the erythrocyte membrane.     

Isolation of the major intrinsic transmembrane protein of the human erythrocyte membrane

Summary The major intrinsic protein of the human erythrocyte membrane commonly referred to as Band 3, was isolated by a multi-step procedure. Extraction of ghost membranes in dilute solutions of lithium diiodosalicylate removed most of the proteins considered to be extrinsic to the membrane. The resulting membrane fragments were solubilized in sodium dodecyl sulfate, and the major sialoglycoprotein (glycophorin A) was removed by wheat germ agglutinin-Sepharose affinity chromatography. Gel filtration in sodium dodecyl sulfate was used as the final step to yield the band 3 polypeptide in electrophoretically homogeneous form.     

cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)-blood-group-antigen expression.

Lactotransferrin was highly purified from lysates of human neutrophilic leucocytes by immuno-affinity chromatography. A comparative analysis of the molar carbohydrate compositions of human leucocyte lactotransferrin and human milk lactotransferrin reveals that the glycans of leucocyte lactotransferrin differ essentially by the absence of fucose residues. Structural analysis combining methylation-mass spectrometry and 400 MHz 1H-n.m.r. spectrometry of oligosaccharide alditols released from human leucocyte lactotransferrin shows the presence of two disialylated and non-fucosylated biantennary glycans of the N-acetyl-lactosaminic type. These results question a previously proposed mechanism for hyposideraemia in which the leucocyte lactotransferrin was involved and in which the fucose residues played a key role.     

Isolation of the major intrinsic transmembrane protein of the human erythrocyte membrane.

The major intrinsic protein of the human erythrocyte membrane commonly referred to as "Band 3", was isolated by a multi-step procedure. Extraction of ghost membranes in dilute solutions of lithium diiodosalicylate removed most of the proteins considered to be extrinsic to the membrane. The resulting membrane fragments were solubilized in sodium dodecyl sulfate, and the major sialoglycoprotein (glycophorin A) was removed by wheat germ agglutinin-Sepharose affinity chromatography. Gel filtration in sodium dodecyl sulfate was used as the final step to yield the band 3 polypeptide in electrophoretically homogeneous form.     

The neuroendocrine polypeptide 7B2 is a precursor protein

The neuroendocrine protein 7B2 is highly conserved and widely present in neurons and endocrine cells. It is coexpressed with the prohormone proopiomelanocortin (POMC) in the intermediate lobe of the pituitary gland of Xenopus laevis. To study the biosynthesis of 7B2 in this amphibian, an anti-7B2 monoclonal antibody was used in immunoprecipitation analysis of newly synthesized radiolabeled proteins, produced by pulse and pulse-chase-incubated neurointermediate lobes. Following a 15-min pulse incubation, a single immunoprecipitable protein of 25 kDa was synthesized. During subsequent chase incubation, this newly synthesized 7B2 protein was processed to an 18-kDa immunoprecipitable form. Analysis of the chase incubation medium revealed that only the 18-kDa processed product of 7B2, and not 7B2 itself, had been secreted. This secretion is a regulated process because it was blocked completely by the dopamine receptor agonist apomorphine. A study of protein biosynthesis in lobes treated with tunicamycin to prevent N-linked glycosylation showed that in contrast to POMC and an 18-kDa derivative of POMC, neither 7B2 nor its 18-kDa derivative was glycosylated. Chemical and enzymatic peptide mapping showed that processing of 7B2 occurs in the carboxyl-terminal region. The function of the 7B2 protein is unknown; the present results show that 7B2 itself is a precursor molecule and can only have an intracellular function whereas an extracellular function can only be attributed to 7B2-derived peptides.     

Automated purification of human protein band 3, the major integral protein of the erythrocyte membrane

Human erythrocyte protein band 3 was purified from a Triton X-100 extract of white ghosts. This purification, including an ion-exchange chromatography and a group-affinity chromatography, was automated. The apparatus was assembled from commercially available elements and allowed for the recovery of 2 to 3 mg pure band 3 in 2 hr. The purification could be repeated several times a day. The advantages of automation are discussed.     

The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide.

We have already reported that the protein tyrosine kinase (PTK) activity in the dog prostate is distributed in cytosolic (75%) and particulate (Triton X-100-solubilized) fractions and that upon gel filtration, both PTKs migrate as entities of Mr 44,000 [(1991) Biochem. Cell. Biol. 69, 146-153]. Herein we demonstrate by immunoprecipitation with anti-phosphotyrosine antibodies that the soluble PTK has the ability to undergo self-phosphorylation. In addition, the polypeptide responsible for that enzymatic activity has been identified by 2 approaches: (1) a two-dimensional electrophoresis, in which the first dimension performed in non-denaturing conditions allowed the localization of the native enzyme, while the second dimension (SDS-PAGE) permitted the analysis of alkali-resistant phosphoproteins corresponding to the activity; (2) protein renaturation after SDS-PAGE followed by in situ phosphorylation (with [gamma-32P]ATP) of polyGT electrophoresed together with the enzyme preparation; the exclusive presence of the radiolabeled phosphotyrosine in the renatured protein confirmed its enzymatic nature. Using these methods, the major form of PTK in the dog prostate was shown to be expressed by a 50 kDa polypeptide which possesses autophosphorylation sites and which is present in the cytosol as an active monomer.     

Characterization of major glycolipids in bovine erythrocyte membrane

Several neutral glycolipids and gangliosides were isolated from bovine erythrocyte stroma. Their structures were determined by partial acid hydrolysis, methylation analysis, periodate oxidation and CrO3 oxidation. Two major neutral glycolipids were characterized as lactosylceramide and galactosyl(alpha1--3)galactosyl(beta1--4)N-acetylglucosaminyl(beta1--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide. Two major gangliosides were N-glycolylneuraminosyl(2--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide and N-glycolylneuraminosyl(2--3)galactosyl(beta1--4)N-acetylglucosaminyl(beta1--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide. Minor glycolipids were glucosyl- and galactosylceramide, glucosamine-containing tri- and tetraglycosylceramide, glucosamine-containing disialosylhexaglycosylceramide, and gangliosides containing N-acetylneuraminic acid. The ceramide moiety of each glycolipid contained perdominantly d18:1 sphingosine, and normal fatty acids of C16:0, C22:0, C24:0, and C24:1.     

The major outer membrane protein of Acidovorax delafieldii is an anion-selective porin.

The major outer membrane protein (Omp34) of Acidovorax delafieldii (formerly Pseudomonas delafieldii) was purified to homogeneity and was characterized biochemically and functionally. The polypeptide has an apparent molecular weight (Mr) of 34,000, and it forms stable oligomers at pH 9.0 in the presence of 10% octylpolyoxyethylene or 2% lithium dodecyl sulfate below 70 degrees C. The intact protein has a characteristic secondary structure composition, as revealed by Fourier transforming infrared spectroscopy (about 60% beta sheet). These features and the amino acid composition are typical for porins. The purified Omp34 is associated with 1 to 2 mol of lipopolysaccharide per mol of the monomer. Pore-forming activity was demonstrated with lipid bilayer experiments. Single-channel and selectivity measurements showed that the protein forms highly anion-selective channels. The unusual dependence of the single-channel conductance on salt concentration suggests that the porin complexes bear positive surface charges, accumulating negatively charged counterions at the pore mouth.     

The E protein is a multifunctional membrane protein of SARS-CoV

The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and α-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin.     

Glycosylation of a membrane protein is restricted to the growing polypeptide chain but is not necessary for insertion as a transmembrane protein.

The membrane glycoprotein of vesicular stomatitis virus (VSV), synthesized in vitro in the presence of pancreatic microsomes, is glycosylated in two distinct steps while its polypeptide chain is nascent (Rothman and Lodish, 1977). We show here that unglycosylated glycoprotein, which accumulates in vivo following treatment of cells with tunicamycin and in vitro as a result of translation in the presence of detergent-treated microsomal membranes, is inserted normally as a transmembrane protein. This means that glycosylation, while normally occurring concurrently with insertion, is not required for insertion. Our experiments also show that the two steps in glycosylation correspond to the sequential transfer of preformed “core” oligosaccharides of typical structure to two Asn residues in the growing chain. The accumulation of unglycosylated glycoprotein in vitro is due to the fact that the completed transmembrane polypeptide cannot be glycosylated. The detergent treatment of microsomes impairs their rate of glycosylation so that chains are frequently completed before they can be glycosylated. This provides a simple explanation for certain types of heterogeneity often found in glycoproteins. We believe that the detergent treatment procedure results in the solubilization of the microsomal membrane followed by reconstitution. This is a prerequisite for the eventual purification of the membrane proteins and lipids involved in insertion and glycosylation of this model membrane protein.