Radioactive human lymphoblastoid interferon. One-step purification, regulation of heterogeneous species production and its use for radioimmunoassay

Human lymphoblastoid interferon (alpha type), labeled with [3H]leucine added to virus-induced Namalwa cells, was purified quantitatively and in one step from the culture fluid by immune precipitation. The material showed, upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only four radioactive bands with molecular weights ranging from 17000 to 21000, which coincided well with interferon activity. They coincided also with the four interferon protein bands in the electropherogram of unlabeled interferon purified by a different method. The purity of the labeled interferon was ascertained also by polyacrylamide gel electrophoresis in the absence of dodecyl sulfate. Pulse-labeling of interferon with [3H]leucine for 1 h at various times after induction indicated that the cells always synthesized and secreted the four interferon species in parallel during the interferon production period. Competitive radioimmunoassay for human interferon alpha was achieved by the use of purified radioactive interferon, anti-(interferon alpha) serum, and bacterial adsorbent. The immune precipitation of the labeled interferon was inhibited by unlabeled interferon alpha, and 100 international reference units of interferon alpha could be measured in this way.     

Mitochondrial phosphate transport. Large scale isolation and characterization of the phosphate transport protein from beef heart mitochondria

The phosphate transport protein from beef heart mitochondria has been purified on a large scale by hydroxylapatite chromatography in the presence of sodium dodecyl sulfate and urea. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain), the pure phosphate transport protein preparation consists of two protein bands (alpha and beta, ratio 1:1) with similar mobilities (34 kDa) which display identical peptide maps if fragmented with either CNBr or HCl/dimethyl sulfoxide/HBr. The complete amino acid composition of phosphate transport protein is presented. Quantitative determination of N-terminal amino acids underlines the purity of the preparation and shows for alpha and beta the identical amino-terminals H2N-Ala-Val-Glu-Glu-Glx-Tyr-. Qualitative digestion shows that carboxypeptidase A is able to release at least three amino acids from the C termini of the alpha as well as the beta band of phosphate transport protein. The nature of these two protein bands is discussed. The sum of phosphate transport protein (alpha + beta) per total mitochondrial protein amounts to 2.3% or 1.4 nmol of phosphate transport protein (34 kDa) per nmol of cytochrome b.     

Isolation of a β2-glycoprotein from human serum after precipitation with dextran sulfate and manganese chloride

A new procedure was developed which affords isolation from among the euglobulins of human serum a beta 2-glycoprotein with a high degree of immunological and electrophoretic homogeneity. The isolated protein displays specific binding affinity for the activated form of the C4 component (C4b), and was identified by immunological and physico-chemical criteria as the C4 binding protein. The isolation procedure comprises the following steps: precipitation of euglobulins from serum at pH 5.5 and low ionic strength; precipitation of beta-lipoproteins from the redissolved precipitate with dextran sulfate and CaCl2; precipitation of a fraction of the lipoprotein-free euglobulins with dextran sulfate and MnCl2; redissolution of the precipitate and eventually chromatography on Sepharose 4B. The overall yield was between 15 and 20 per cent. The final product, devoid of immunologically detectable protein contaminants, was a homogeneous proline-rich monomeric beta 2-glycoprotein made up of eight disulfide-bonded polypeptide chains of the same molecular weight 63,000 +/- 3,000. Under non-reducing conditions, the molecular weight of both the native and the SDS-treated protein was 490,000 +/- 25,000. A monospecific antiserum to the isolated protein was raised in rabbits and used for the quantitation of the protein in sera of normal fasting donors; a mean concentration of 25 mg per 100 ml of serum (1 SD: 5) was established.     

Prothrombinase activity of human platelets is inhibited by beta 2-glycoprotein-I

In the present paper the influence of beta 2-glycoprotein-I, also known as apolipoprotein H, upon the prothrombinase activity of platelets and phospholipid vesicles was investigated. The results can be summarized as follows. 1. The prothrombinase activity of resting, non-activated platelets, lysed platelets and vesicles composed of phosphatidylserine and phosphatidylcholine at different molar ratios is inhibited by beta 2-glycoprotein-I in a dose-dependent manner. The concentration of glycoprotein which produces marked inhibition is within the physiological plasma concentration range of beta 2-glycoprotein-I. 2. The time dependence of this inhibition is a relatively slow process, which is not fully expressed before 1 h of incubation. 3. The effect of the glycoprotein is not due to a direct interaction with the components of the prothrombinase complex, i.e. factors Xa, Va, Ca2+ or prothrombin, nor is the inhibitory action abolished by increasing concentrations of coagulation factors Xa and Va. This suggests that beta 2-glycoprotein-I causes a reduction of the prothrombinase binding sites of these coagulation factors to platelets or phospholipid vesicles. 4. The prothrombinase activity of platelets stimulated with ionophore A23187 or with collagen plus thrombin is also inhibited by beta 2-glycoprotein-I in a manner similar to that observed for phospholipid vesicles or for lysed platelets. These findings suggest a regulatory role for beta 2-glycoprotein-I in the pathway of blood coagulation.     

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.     

Resolution of two subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase

The Mo-Fe protein of Azotobacter vinelandii nitrogenase was fractionated on 9.5 M urea isoelectric focusing gels and gave three distinct bands (alpha', alpha", beta'). Protein focused on nondenaturing gels gave a single brown band, which when excised and refocused on a denaturing gel gave the three-band pattern. Partial trypsin digestion of the subunits and fractionation of the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the alpha' and alpha" polypeptide moieties were the same. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha' and beta' proteins with appropriate molecular weight standards indicated Mr = 61,000 and 57,000, respectively. This is consistent with an overall alpha 2 beta 2 mass of 236,000 daltons.     

Rapid, simplified method for production and purification of tetanus toxin

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 ± 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility.     

Isolation of the terminal complement complex from target sheep erythrocyte membranes.

(1) Membranes from sheep erythrocytes lysed with antibody and human complement were solubilized in Triton X-100 and subjected to isoelectric focusing in polyacrylamide gels containing 1% Triton X-100. Membrane-bound serum proteins were located in the gels by subsequent immunoelectrophoresis against antisera to human serum proteins. Monospecific antisera against C9 and C5 were used to locate the terminal complement complex, which is not dissociated by Triton X-100. The complex focused between pH 5.8 and pH 6.5 and was separated from the bulk of other membrane-bound serum proteins, which focused at pH ranges below than 6.0. (2) In a second step, proteins electrophoretically eluted from the gel sections containing the terminal complement complex were chromatographed on Sepharose 6B equilibrated with 0.05% Triton X-100. Fused rocket immunoelectrophoresis was used to monitor separations. This step separated the terminal complement complex from the remaining contaminating proteins. The complex eluted in a broad peak corresponding to a molecular weight range of 800000-4000000. (3) The terminal complement complex thus obtained migrated with alpha-mobility and yielded a single precipitation arc in crossed immunoelectrophoresis using polyvalent antisera to human serum proteins. A distinct precipitate was obtained with monospecific anti-C9. The presence of C5 and C6, in complex with one another and with C9 was demonstrable by immuno-double-diffusion. No immunoprecipitate was obtained with antisera to sheep erythrocyte membrane proteins. (4) Dodecyl sulfate gel electrophoresis of the complex revealed seven protein bands of 190000, 160000, 115000, 93000, 85000, 68000 and 60000 daltons. Planimetric quantitation of densitometric scans gave a molar ratio of approx. 0.7:0.3:1:1:1:2:1 for these bands, respectively. All bands stained faintly with periodate-Schiff. Two-dimensional dodecyl sulfate gel electrophoresis showed that the first two bands (190000 and 160000 daltons, probably C5b and C5c) represented proteins possessing more than one peptide chain linked by disulfide bonds. The main subunit for both bands was a protein of approximately 68000 daltons. Band 5 (83000 daltons, probably C8alpha) was split into two peptide chains of approximately 68000 and 15000 daltons. The other components were not affected by dithiothreitol treatment. (5) The dodecyl sulfate gel electrophoretograms obtained were very similar to that described by Kolb and Müller-Eberhard (Kolb, W.P. and Müller-Eberhard, H.J. (1975) J. Exp. Med. 141, 724-735) for the terminal complement complex isolated from inulin-activated serum. However, certain minor but consistent deviations were observed. A preliminary correction of the electrophoretograms is presented.     

A simple novel approach for the purification of pertussis toxin

Abstract Pertussis toxin (PT) was purified from concentrated culture supernatants of Bordetella pertussis by a single step based on affinity chromatography on heparin-Sepharose 6B with a recovery of 36% in two fractions. The fraction of highest specific activity (0.91 mg of toxin per mg protein) constituted 15.3% of toxin content in crude material and appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It exhibited the five bands corresponding to toxin subunits. The purified fraction induced clustering of Chinese hamster ovary cells at as little as 0.06 ng per ml. PT solution gave a single precipitation line with rabbit immune serum raised against crude Bordetella pertussis supernatant.     

Characterization of the sex steroid binding protein of human pregnancy serum. Improvements in the purification procedure

The sex steroid binding protein (SBP) of human pregnancy serum was purified to homogeneity by the sequential use of ammonium sulfate precipitation, affinity chromatography on 5alpha-dihydrotestosterone-17beta-succinyldiaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, and preparative polyacrylamide gel electrophoresis. The yield of pure SBP was improved from 5% as originally reported [Mickelson, K. E., and Petra, P. H. (1975), Biochemistry 14, 957] to 34%. Homogeneity of SBP was shown by equilibrium sedimentation ultracentrifugation in 6 M guanidine hydrochloride containing 0.1 M mercaptoethanol which yields a minimum molecular weight of 36 335 +/- 525. The protein is also homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A value of 52 000 for the molecular weight is obtained by this method. SBP partially purified from Cohn fraction IV has also a molecular weight of 52 000 by gel electrophoresis in the presence of sodium dodecyl sulfate; that fraction is contaminated with another protein of molecular weight 90 000 which must be removed to obtain homogeneous SBP. The amino acid composition of SBP isolated from pregnancy serum is presented.     

Purification and partial characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) was purified from Streptomyces clavuligerus by a combination of salt precipitation, ultrafiltration, and anion-exchange chromatography. The final purified material gave two protein bands with molecular weights of 283,000 and 32,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis in nondenaturing gels gave a single protein band with an estimated molecular weight of 560,000. These results suggest that ACVS is a multimer composed of nonidentical subunits.     

Neurospora crassa mitochondria contain two forms of a 4'-phosphopantetheine-modified protein

When Neurospora crassa was labeled with [14C]pantothenic acid during growth, the mitochondrial fraction contained two bands of radioactivity of Mr 19,000 and 22,000 by sodium dodecyl sulfate gel electrophoresis. The 19-kDa band was converted to the 22-kDa band by four treatments which are characteristic of the cleavage of a thioester bond: dithiothreitol and 2-mercaptoethanol at basic but not neutral pH, alkaline methanolysis, sodium borohydride in tetrahydrofuran, and hydroxylamine at neutral pH. Mitochondrial subfractionation indicated that the 22-kDa form was preferentially associated with the soluble fraction while the 19-kDa form was found in all fractions. Several properties of the mitochondrial protein were similar to the Escherichia coli acyl carrier protein: Mr on sodium dodecyl sulfate gels, decreased electrophoretic mobility under deacylating conditions, isoelectric point, and covalent attachment of 4'-phosphopantetheine. The 19- and 22-kDa bands may therefore represent acylated and deacylated forms of a mitochondrial acyl carrier protein.     

A typical dermatan sulfate isolated from whale intestine

Alkaline extraction of whale intestine, followed by pronase digestion and precipitation of heparin (ω-heparin) with dodecyltrimethylammonium chloride gave a supernatant fraction containing dermatan sulfate. Ethanol at 20% concentration precipitated dermatan sulfate from the supernatant fraction. The crude dermatan sulfate was further fractionated by ion-exchange column chromatography on Dowex-1 (Cl^? form), eluting stepwise with aqueous sodium chloride. The fractions eluted with 1.5M and 1.75M sodium chloride contained a typical dermatan sulfate. Chemical and enzymic studies of these preparations revealed that the sulfate groups were located solely at O-4 of the 2-acetamido-2-deoxy-D-galactose residues. L-Iduronic acid was assumed to be distributed uniformly in the backbone of the polysaccharide chain, with D-glucuronic acid being located in the linkage region to the protein core. A new method for determining the ratio of D-glucuronic acid to L-iduronic acid is also described.     

Human placental sialidase: further purification and characterization

An acid sialidase [EC 3.2.1.18] has been purified from human placenta by means of successive procedures including extraction, Con A-Sepharose adsorption, ammonium sulfate precipitation, activation, p-aminophenyl thio-beta-D-galactoside-CH-Sepharose (PATG-Sepharose) affinity chromatography and high-performance liquid chromatography on a Shim pack Diol 300 column. The purified enzyme liberated sialic acid residues from sialooligosaccharides, sialoglycoproteins, and gangliosides. In particular, gangliosides GM3, GD1a, and GD1b were hydrolyzed much faster than alpha (2-3) and alpha (2-6)sialyllactoses, and sialoglycoproteins by the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme gave five protein bands with molecular weight of 78,000 (78K), 64,000 (64K), 46,000 (46K), 30,000 (30K), and 20,000 (20K). Rabbit antisera were raised against 78K and 46K proteins, and the two antibodies were specifically reactive with the respective component on immunoblot analysis. Both anti-78K protein and anti-46K protein antisera could precipitate sialidase activity. It is likely that the 78K protein and 46K protein are sub-components which are essential for sialidase activity.     

Conformational transitions of non-helical proteins effected by dodecyl sulfate. Circular dichroism of alpha1-acid glycoprotein, Bence Jones protein, carbonic anhydrase B, deoxyribonuclease, pepsinogen, and plasminogen.

Circular dichroism (CD) of serum alpha1-acid glycoprotein, urinary Bence Jones protein, human carbonic anhydrase B, deoxyribonuclease from bovine pancreas, porcine pepsinogen, and plasminogen from human serum was tested in the absence and presence of 0.005-0.05 M sodium dodecyl sulfate. It was found that in all cases the CD spectra of these proteins were modified by the dodecyl sulfate into spectra indicating the presence of a moderate content of alpha-helix. The transitions were enhanced by addition of acid (pH 2.1-4.4) in all cases tested. Comparison of the various proteins with respect to the amount of reconstruction of the main chain conformation showed that the amount of helix formed depended on the amino acid composition of the protein. Rigidity due to cross-linking by disulfide bridges is the strongest deterrant to the conformational change of the main chain. The CD bands of the native proteins in the 250-350 nm spectral zone were extinguished by sodium dodecyl sulfate, and new weak bands were observed the positions of which corresponded approximately to those of the native proteins. In all cases, except the carbonic anhydrase B, the bands of thus denatured proteins were negative.     

beta 2-Glycoprotein-I (apolipoprotein H) interactions with phospholipid vesicles

The binding characteristics of the human serum protein beta 2-glycoprotein-I, also called apolipoprotein H, with multilamellar phospholipid vesicles has been studied. It was found that beta 2-G-I is not or almost not bound to the "neutral" phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM). The negatively charged compounds phosphatidylserine (PS) and phosphatidylinositol (PI) interact strongly with beta 2-G-I. In terms of phospholipid concentration the binding to PS is about one order of magnitude greater than to PI. The binding capacity is influenced by several parameters such as the molarity of buffer, presence of mono- or divalent cations as well as ethylenediaminotetraacetic acid (EDTA). Proteins like bovine serum albumin (BSA), human serum albumin (HSA) or horse gamma-globulin (HGG) influence the binding also in a concentration dependent manner.     

Dipetalonema viteae: isoelectric focusing and immunochemical studies on somatic extracts of adult worms and microfilariae.

Isoelectric focusing (IEF) of a somatic extract of adult worms (SEAW) yielded nine fractions. Most of the applied protein antigen was recovered in five fractions with pI values of 5.2, 4.4, 4.3, 4.0, and 3.3, respectively. The nine IEF fractions of SEAW gave a total of 37 bands following electrophoresis on polyacrylamide gel (PAGE). IEF fractionation of a somatic extract of microfilariae (SEM) yielded nine fractions. Two fractions with pI values of 4.4 and 3.2, respectively, contained most of the applied protein. The nine IEF fractions of SEM gave a total of 32 bands following PAGE. Crossed immunoelectrophoresis (CI) of SEM and SEAW against serum from hyperinfected hamsters yielded five and eight precipitation peaks, respectively. CI of SEM and SEAW against their homologous rabbit antisera gave 7 and 10 major precipitation peaks, respectively. Immunodiffusion of the nine IEF fractions from both SEM and SEAW against both their homologous and heterologous rabbit antisera indicated three and four precipitin bands peculiar only to SEM and SEAW, respectively. The remaining 23 bands from both preparations showed lines of identity.     

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.