Proteins and glycoproteins of the erythrocyte membrane

Erythrocyte membranes from several species were prepared by three different methods of hypotonic hemolysis and examined for variations in protein and glycoprotein content by acrylamide gel electrophoresis in sodium dodecyl sulfate. Significant variations were noted in morphology of the membranes prepared by the different methods without attendant variations in protein patterns of the major membrane proteins for most cases observed, which show a similar pattern of nine common bands for all of the species observed. The significant difference in protein pattern which was noted was attributed to proteolytic digestion of membranes which were fragmented during preparation. Failure to remove white blood cells from membrane preparations was shown to be a significant source of the problem with proteolytic digestion. Glycoproteins were analyzed by acrylamide gel electrophoresis or by column chromatography. Each species appears to have a different major glycoprotein (or group of closely related glycoproteins). Molecular weights of glycoproteins calculated from acrylamide gel electrophoresis were found to vary with the percentage of acrylamide in the gel, indicating that these proteins do not behave in a normal fashion in this electrophoresis system. The molecular weight calculated from gel filtration data for the human membrane glycoproteins (26,000) was quite disparate from those calculated from gel electrophoresis (88,000 to 62,000 in 5 to 10% gels).     

Analysis of purified tRNA species by polyacrylamide gel electrophoresis

Six purified amino acid acceptor tRNA species were examined by polyacrylamide gel electrophoresis. Small differences in migration were observed under conditions that preserve the conformation of tRNA. When tRNA was heated in the presence of either 10 mM acetate or EDTA at 60° a change in migration was observed for tRNA^Glu. No difference in migration was seen between Val-tRNA^Val and tRNA^Val. When tRNA was denatured by heating in 4M urea and applied to a gel containing the same amount of urea, all tRNA species migrated approximately the same distance with the exception of tRNA^{Leu V}, which showed an appreciable slower migration. From the difference in migration of tRNA^{Leu V} as compared to tRNA^Val and 5 S RNA, the difference in chain length between tRNA^Val and tRNA^{Leu V} was estimated to be approximately 9 nucleotides.     

Resolution of a complex ionic mixture of an apparently homogenous protein preparation by preparative electrophoresis

Preparations of recombinant envelope glycoprotein E2 of hepatitis C virus (r-HCV E2), found to be homogeneous by N-terminal amino acid sequencing and mass spectrometry, resolved into multiple ionic species (isoforms) when analysed by isoelectric focusing (IEF) gel electrophoresis in the p1 range of 3-10. These isoforms possessed pI values in the range of 4.5-8.2. The major isoform with p1 value of approximately 7.1 was separated from the rest of them by employing a method developed on Gradiflow BF 200, a device based on preparative electrophoresis. This isoform was adjudged to be homogenous by IEF and by native polyacrylamide gel electrophoresis (PAGE).     

Differences between individuals in high-molecular weight glycoproteins from mammary epithelia of several species

Milk fat globules are secreted by envelopment in plasma membrane of the lactating cell. SDS-gel electrophoresis of proteins from this membrane has revealed differences between milk donors in two mucin-like glycoproteins. One of these glycoproteins resolves in 3% acrylamide stacking gel and the other in 4% running gel. The proteins vary in number of bands (one or two) and band mobilities. This polymorphism arises, at least in part, from expression of hypervariable genes. In this study, gel electrophoretic evidence of similar polymorphism in glycoproteins from cow, chimpanzee, horse and human milks is presented. In distinction to the other species, the cow expressed only one of these proteins which was detected in the running gel at Mr 180,000 to 200,000. The electrophoresis pattern for this protein from six cows was highly varied with respect to number (one or two) and position of bands. Peanut agglutinin, wheat germ agglutinin and concanavalin A all were bound specifically by bands of the bovine glycoprotein. Binding of concanavalin A distinguishes the bovine protein from the two human glycoproteins. Further studies of species differences should help shed light on the evolution of these unique glycoproteins and their possible functions in mother and young.     

Rabies mRNA translation in Xenopus laevis oocytes.

Two rabies virus-specific mRNA species were identified by analysis of their encoded proteins after translation of the partially purified species in Xenopus laevis oocytes. One of these coded for the virion surface glycoprotein (G protein), and the other coded for the major structural protein of the virion nucleocapsid (N protein). The G-mRNA sedimented in a sucrose density gradient at about 18S, and the N-mRNA had a sedimentation coefficient of approximately 16S. Their respective translation products were identified in a radioimmunoassay with specific monoclonal antibody probes that recognized only G or N proteins. Immunoprecipitates formed between the radiolabeled viral antigens synthesized in programmed oocytes and their respective monoclonal antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The glycoprotein antigen translated from G-mRNA in oocytes migrated in the gel ahead of the virion G protein with a migration rate that was similar to that of nonglycosylated intracellular glycoproteins from virus-infected cells. The results suggested that the branched-chain carbohydrate of G protein was not required for recognition by the particular monoclonal antibody used. The nucleocapsid antigen translated from N-mRNA in oocytes migrated to the same position in the gel as marker virion N protein. Both the electrophoretic mobility of virus-specific antigens in sodium dodecyl sulfate-polyacrylamide gel and the antibody concentration dependence for immunoprecipitations were criteria for identifying the individual viral proteins encoded by the two rabies mRNA's.     

Red cell uridine monophosphate kinase: a comparative study in various vertebrate species.

1. Starch gel electrophoretic patterns and activity of red cell UMP-kinase were compared in different species of vertebrates. 2. A characteristic electrophoretic pattern for each species of animal studied was found. 3. The mammals differed markedly from the rest of the vertebrate compared in having a faster electrophoretic migration. 4. Significant species differences in total UMPkinase activity could be observed.     

Heterogeneity of human cell membrane sialoglycoproteins.

Discontinuous sodium dodecysulfate polyacrylamide gel electrophoresis (disc SDS-PAGE) followed by periodic acid/Schiff staining reveals the presence of six sialoglycoprotein bands in human red cell membranes or glycoprotein preparations therefrom. In agreement with previous investigations it is shown that PAS-1 and PAS-2 (mol. weight 37 000) are different forms of the same molecule (MN glycoprotein). Using separation of glycoproteins by the system of Weber and Osborn and reelectrophoresis of gel slices by disc SDS-PAGE it is demonstrated that the minor component C (mol. weight 41 000) represents the dimeric form of PAS-3 (Ss glycoprotein). Band B corresponds to an aggregate of PAS-3 and PAS-2 and/or the trimer of PAS-3 with possible differences between extracted glycoproteins and those present in the membrane. The minor component D (mol. weight 35 000) is, as far as could be elucidated, not involved in aggregation phenomena. Some technical problems of glycoprotein fractionation by SDS-PAGE and the remarkable effect of phosphate buffers on the glycoprotein pattern are discussed.     

Relationship of Reticuloendotheliosis Virus to the Avian Tumor Viruses: Nucleic Acid and Polypeptide Composition

The RNA content and polypeptide composition of reticuloendotheliosis virus (REV) was compared to that of C-type RNA tumor viruses. Two RNA species with approximate sedimentation values of 64S and 4S were observed after sucrose gradient centrifugation of RNA extracted from purified REV. The high-molecular-weight RNA species of REV sedimented slightly faster than that of the Bryan strain of Rous sarcoma virus (RSV). Although these characteristics were consistent with those of other C-type RNA tumor viruses, significant differences were observed when the polypeptide composition of REV was compared with that of RSV possessing envelope determinants of Rous-associated virus RAV-2 and RAV-3. Five polypeptides of which two were glycosylated were resolved by polyacrylamide gel electrophoresis. The major nonglycosylated polypeptide of REV did not comigrate with that of RSV (RAV-2)-RSV(RAV-3). The majority of the group-specific antigen reactivity resides in this major nonglycosylated polypeptide of avian tumor viruses and comigrates when proteins of several avian tumor viruses are subjected to coelectrophoresis. This difference in the migration of the major polypeptide of REV and RSV(RAV-2)-RSV(RAV-3) may explain the absence of avian tumor virus group-specific antigen in REV.     

Gene protein products of SA11 simian rotavirus genome

When MA104 cells were infected with SA11 rotavirus, 12 protein classes, absent in mock-infected cells, could be distinguished by polyacrylamide gel electrophoresis. At least two of these proteins were glycosylated, and their synthesis could be blocked with tunicamycin. The oligosaccharides of both glycoproteins were cleaved by endo-beta-N-acetylglucosaminidase H, suggesting that they were residues of the "high-mannose" type. Of the 12 viral polypeptides observed in infected cells, 1 was probably the apoprotein of one of these glycoproteins; 5, including 1 glycoprotein, were structural components of the virions, whereas the other 6, including a second and possibly third glycoprotein, were nonstructural viral proteins. When the 11 double-stranded RNA genome segments of SA11 were translated, after denaturation, in an RNA-dependent cell-free translation system, at least 11 different polypeptides were synthesized. Ten of these polypeptides had electrophoretic migration patterns equal to those of viral proteins observed in tunicamycin-treated infected cells. Nine of the 11 double-stranded RNA genome segments were resolved by polyacrylamide gel electrophoresis and were translated individually. Two were not resolved from each other and therefore were translated together. Correlation of each synthesized polypeptide with an individual RNA segment allowed us to make a probable gene-coding assignment for the different SA11 genome segments.     

Purification and characterization of an estrogen-inducible membrane glycoprotein. Evidence that it is a transferrin receptor

A number of N-linked membrane glycoproteins are induced during chick oviduct differentiation. We have purified a major estrogen-inducible glycoprotein (Mr = 91,000) to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of partial NH2-terminal sequence data with membrane glycoproteins having similar Mr showed a limited homology with human and murine transferrin receptors. We observed that oviduct membranes contain estrogen-inducible transferrin receptor activity (Kd = 2-8 x 10(-8) M). Analytical purification of the putative receptor on an ovotransferrin-Affi-Gel affinity column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a protein of Mr, 180,000, which contains two disulfide-linked subunits of Mr 91,000. The receptor reacts very strongly with antibodies prepared against the 91-kDa glycoprotein on Western blots. Western blot analysis confirms that the 91-kDa glycoprotein is induced by estrogen. The protein has 2% total carbohydrate with Man, GlcNAc, Gal, GalNAc, and NeuAc in a molar ratio of 6:4:2:1:1. The protein contains at least one O-linked moiety. Analysis of the O-linked moiety by glycosidase digestions and gel filtration indicates there are sialo tetra- and trisaccharides and a neutral disaccharide(s). Labeled N-linked glycopeptides were prepared by pronase digestion, beta-elimination, and 3H-acetylation. The N-linked oligosaccharides include high mannose and complex neutral nonbisected biantennary types in an approximate ratio of 3:1 as determined by serial lectin affinity chromatography.     

Plasma testosterone transport in primates

All primate species, including Old and New World primates and prosimians have a plasma testosterone-estradiol binding globulin (TeBG), which is a glycoprotein and has a similar mobility in polyacrylamide gel electrophoresis. In New World primates the TeBG binding capacity for [3H]testosterone was higher and its affinity lower than in Old World primates. These changes were associated with high unbound plasma testosterone concentrations in these species. Binding parameters of TeBG in prosimian species varied markedly. Thus, in primate evolution TeBG was conserved despite marked differences in binding characteristics. In New World primates changes are associated with high total and unbound testosterone, a finding concordant with alterations of other steroid hormones concentration in these species with "generalized steroid hormone resistance".     

Renin substrate in plasma of various mammalian species: electrophoresis on polyacrylamide gel.

The plasma of eight different species was subjected to electrophoresis on polyacrylamide gel, and the position of renin substrate was determined. There are considerable differences in the electrophoretic mobility of the renin substrates tested. Sheep substrate shows the slowest migration and mouse substrate the most rapid. The species tested appear to fall into two groups: slow-moving substrates occuring in the plasmas of sheep, cow, pig, and rabbit and fast moving substrates in man, dog, rat, and mouse. In most species only a single peak of renin substrate appeared, but in man and dog a minor peak was often observed in addition to the prominent major one. The classification of human renin substrate as an alpha-2-globulin is questioned.     

Glycoprotein Ib beta is the only phosphorylated major membrane glycoprotein in human platelets.

Platelets were metabolically labelled with 32P and the phosphoproteins examined by two-dimensional non-reduced/reduced gel electrophoresis and isoelectric-focusing/gel electrophoresis. Comparison with similar separations of surface-labelled platelets showed that the only major glycoprotein which is phosphorylated is the beta-subunit of glycoprotein Ib, indicating that this subunit contains a cytoplasmic segment. The identification was confirmed using immunoblotting with an antibody to the beta-subunit. Phosphoserine was the principal phosphorylation site, with some phosphothreonine, but phosphotyrosine was absent. No quantitative or qualitative differences could be detected in the phosphorylation of glycoprotein Ib beta from resting or activated platelets. These results exclude changes in phosphorylation of the major platelet membrane glycoproteins as a method of signal transmission by these receptors.     

Molecular weight determination and partial characterization of Klebsiella pneumoniae hemolysins

Two thiol-activated Klebsiella pneumoniae hemolysins were purified from growth media by means of salt precipitation, gel filtration, ion-exchange chromatography, and polyacrylamide gel electrophoresis. The hemolysins peaks coincided with the protein and glycoprotein peaks as determined by chromatography and electrophoresis. The molecular weights, estimated by gel filtration, were 8400 and 19,000; by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, the values were calculated as 15,500 and 27,000. The electrophoretic bands were best detected by the periodic acid--Schiff method. Reduction of the disulfide linkages did not cause the originally larger molecule to break into 8400 and 19,000 hemolysins. However, trypsin treatment cleaved the 19,000 hemolysin into an active moiety, with an electrophoretic migration similar to the 8400 hemolysin. A naturally occurring proteolytic activity was investigated using pepstatin and antipain. When the trypsin inhibitor was added to the system, the hemolytic activity was detected only in the 19,000 hemolysin and the smaller hemolysin was absent.     

SDS gel electrophoresis of proteins and glycoproteins from peritrophic membranes of some Diptera

Peritrophic membranes (PM) of larval and adult Calliphora erythrocephala and Sarcophaga barbata contain proteins and glycoproteins which were extracted by 2.5% SDS and 8 M urea from the matrix. The acid mucopolysaccharide moiety of PM which was demonstrated by the carbazole method remained in the insoluble resudues. After SDS electrophoresis the gels were counterstained with PAS and Coomassie blue; the carbohydrate and protein content of the bands were recorded by dual scanning. Besides molecular weight (MW) determination from the migration rate, the MW of some glycoprotein bands of PM were evaluated also from their retardation coefficients. The methods revealed different results indicating anomalous SDS binding and mobility of these glycoproteins in SDS electrophoresis.The glycoprotein patterns of larvae and of adults of Calliphora as well as of Sarcophaga differed markedly. PM of adults of both species contained only one carbohydrate fraction which migrated in the gel according to an apparent MW of about 200.000 daltons. PM of the larvae, however, showed a variety of bands in the range between 30.000 and 80.000 daltons which had binding capacities for the protein as well as for the carbohydrate stain. On the other hand, the patterns of pure protein bands were similar in the larval and in the adult stage. Obviously, the glycoprotein pattern of PM is altered during development according to special requirements. Also the similarities between both species in the larval and in the adult stage point to a special physiological function of the glycoprotein moiety.     

Assessment of DNA strand breakage by comet assay in diabetic patients and the role of antioxidant supplementation

Diabetes patients often show increased production of reactive oxidative species (ROS) together with vascular complications. The presence of these ROS may lead to increased DNA damage in peripheral blood lymphocytes that may be revealed by the comet assay. To test whether DNA is damaged in diabetes, peripheral blood samples were taken from 30 control individuals and 63 diabetic patients (15 insulin dependent (IDDM) and 48 non-insulin dependent (NIDDM)) and the alkaline comet assay was used to evaluate background levels of DNA damage. Significant differences were detected between control and diabetic patients in terms of frequencies of damaged cells. The extend of DNA migration was greater in NIDDM patients by comparison with IDDM patients which might indicate that IDDM patients are handling more oxidative damage on a regular basis. Smoker individuals had higher frequencies of cells with migration by comparison with the non-smokers in both groups. Also, clear differences between patients on placebo and on Vitamin E supplementation for 12 weeks were observed on the basis of the extend of DNA migration during single cell gel electrophoresis.     

Assessment of DNA strand breakage by comet assay in diabetic patients and the role of antioxidant supplementation

Diabetes patients often show increased production of reactive oxidative species (ROS) together with vascular complications. The presence of these ROS may lead to increased DNA damage in peripheral blood lymphocytes that may be revealed by the comet assay. To test whether DNA is damaged in diabetes, peripheral blood samples were taken from 30 control individuals and 63 diabetic patients (15 insulin dependent (IDDM) and 48 non-insulin dependent (NIDDM)) and the alkaline comet assay was used to evaluate background levels of DNA damage. Significant differences were detected between control and diabetic patients in terms of frequencies of damaged cells. The extend of DNA migration was greater in NIDDM patients by comparison with IDDM patients which might indicate that IDDM patients are handling more oxidative damage on a regular basis. Smoker individuals had higher frequencies of cells with migration by comparison with the non-smokers in both groups. Also, clear differences between patients on placebo and on Vitamin E supplementation for 12 weeks were observed on the basis of the extend of DNA migration during single cell gel electrophoresis.     

Rabbit Tamm–Horsfall urinary glycoprotein. Chemical composition and subunit structure

1. Tamm-Horsfall glycoprotein from rabbit urine has been isolated and characterized. The homogeneity of the preparation has been established by a variety of procedures including disc gel electrophoresis and ultracentrifugation in aqueous solution, sodium dodecyl sulphate and formic acid. 2. The chemical composition has been determined and a carbohydrate content of approx. 31% was obtained. The relative contents of the amino acids were shown to be very similar to those in human Tamm-Horsfall glycoprotein. A trace of lipid was also detected. 3. Leucine was identified as the only N-terminal amino acid. 4. The subunit structure was investigated in the presence of sodium dodecyl sulphate by gel filtration and disc gel electrophoresis. These studies indicated that the subunit possessed a molecular weight of approx. 84000+/-6000. A similar value was obtained after reduction and S-alkylation of the glycoprotein indicating that the disulphide bonds were all intrachain. 5. A minimum value for the chemical molecular weight of 85000+/-6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 6. The immunological properties of the glycoprotein were studied. Cross reactivity was demonstrated between human Tamm-Horsfall glycoprotein and a guinea-pig anti-rabbit Tamm-Horsfall antiserum.     

Characterization of the Fc receptors of the murine leukemia L1210

A glycoprotein extract prepared from the plasma membranes of L1210 cells was passed over columns of Sepharose 4B to which either heat-aggregated human IgG or F(ab′)₂ fragments had been coupled. The intact IgG column bound 35.7% of the applied counts, whereas the F(ab′)₂ columns bound 2.8%. The bound glycoproteins were eluted with citrate buffer (pH 3.2) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three peaks with apparent molecular weights of 65,000, 45,000, and 28,000 daltons were identified and purified by electroelution from polyacrylamide gels. The isolated proteins were able to bind to the same subclasses of mouse IgG myeloma proteins as the intact L1210 cells, indicating that these molecules are related to L1210 surface Fc receptors. Amino acid analyses of the 3 proteins were markedly similar suggesting that the observed molecular heterogeneity might be due to carbohydrate differences. Neuraminidase digestion of the isolated proteins resulted in mobility shifts on polyacrylamide gel electrophoresis which were consistent with the interpretation that either the isolated proteins have considerably different sialic acid contents, or that removal of the sialic acid results in disaggregation of an Fc receptor molecule.