Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.     

Immunofluorescence and electron microscopy of the cytoplasmic surface of the human erythrocyte membrane and its interaction with Sendai virus

A method was developed for directly observing the inner surfaces of plasma membranes by light and electron microscopy. Human erythrocytes were attached to cover slips (glass or mica) treated with aminopropylsilane and glutaraldehyde, and then disrupted by direct application of a jet of buffer, which removed the distal portion of the cells, thus exposing the cytoplasmic surface (PS) of the flattened membranes. Antispectrin antibodies and Sendai virus particles were employed as sensitive markers for, respectively, the PS and the external surface (ES) of the membrane; their localization by immunofluorescence or electron microscopy demonstrated that the major asymmetrical features of the plasma membrane were preserved. The fusion of Sendai virus particles with cells was investigated using double- labeling immunofluorescence techniques. Virus adsorbed to the ES of cells at 4 degrees C was not accessible to fluorescein-labeled antibodies applied from the PS side. After incubation at 37 degrees C, viral antigens could be detected at the PS. These antigens, however, remained localized and did not diffuse from the site of attachment, as is usually seen in viral antigens accessible on the ES. They may therefore represent internal viral antigens not incorporated into the plasma membrane as a result of virus-cell fusion.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes.

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface. In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface.In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

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.     

Arylamidases of rat liver and chemically induced hepatomas. II. Preparation and characterization of monospecific antisera against two distinct arylamidase-active antigens.

Monospecific antisera were prepared against the most prominent arylamidase (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) active antigen in plasma membranes (the plasma membrane arylamidase) and lysomal content (the lysosomal content arylamidase), respectively. Plasma membrane extract and lysosomal content were allowed to react in crossed immunoelectrophoresis against their homologous antisera. The electrophoretic plates were washed extensively, dried and subsequently stained for arylamidase activity.The particular immunoprecipitates were thus identified and could be excised to be used for immunizations. The two resulting antisera precipitated the arylamidase used for immunization, but failed to be monospecific as they precipitated additional antigens. These antisera with restricted specificity against some plasma membrane and lysosomal content antigens, respectively, were used to produce immunoprecipitates intended for new attempts to prepare monospecific antisera by a second cycle of immunizations. A monospecific antiserum against the plasma membrane arylamidase was thus obtained, while a third cycle of immunizations was needed to get a monospecific anti-lysosomal content antiserum. The plasma membrane arylamidase showed ATPase activity also after precipitation with the monospecific antiserum, thus still retaining its characteristics as a multienzyme complex.     

Immunological Properties of Micrococcus lysodeikticus Membranes

Membranes of Micrococcus lysodeikticus possess antigens which are distinct from other cellular components such as cytoplasm, ribosomes, and cell walls. Only a few (two to three) components are found when dissociated membranes are examined by immunodiffusion and immunoelectrophoresis techniques. Membranes treated with 0.3% sodium dodecyl sulfate, 0.3% Triton X-100, trypsin, phospholipase A or C, or by sonic oscillation at pH 9.0, all showed the same pattern (three major bands) when examined against membrane antisera by immunoelectrophoresis. Immunological analysis of fractions isolated by sucrose gradient centrifugation or by polyacrylamide gel electrophoresis suggests that individual components cross-react. Antibodies to adenosine triphosphatase (EC 3.6.1.3) and fast-moving component are not removed by absorption with protoplasts. Removal of antibody to one of the membrane antigens by protoplast absorption indicated a surface location. Glutaraldehyde fixation of protoplasts resulted in the loss of membrane antigens detectable by immunodiffusion.     

Immunochemical analysis of inner and outer membranes of Escherichia coli by crossed immunoelectrophoresis.

Isolated membrane fractions of Escherichia coli K-12 yielded complex immunoprecipitate patterns when Triton X-100 and sodium dodecyl sulfate extracts were examined by crossed immunoelectrophoresis with antienvelope immunoglobulins. Twelve of the 46 antigens in the immunoprecipitate patterns of inner (plasma) membranes were identified by zymograms and/or by the use of specific antisera. The following enzyme activities were detected in immunoprecipitates: 6-phosphogluconate dehydrogenase (EC 1.1.1.43); adenosine triphosphatase (EC 3.6.1.3); glutamate dehydrogenase (EC 1.4.1.4), two separate components; malate dehydrogenase (EC 1.1.1.37); dihydroorotate dehydrogenase (EC 1.3.3.1); succinate dehydrogenase (EC 1.3.99.1); lactate dehydrogeanse (EC 1.1.1.27); reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3); protease (EC 3.4.21.1); and glycerol 3-phosphate dehydrogenase (EC 1.1.99.5). The corresponding immunoprecipitate pattern for isolated outer membranes consisted of at least 25 discrete antigens and differed strikingly from that obtained with inner membranes. Two major immunogens were identified as lipopolysaccharide and Braun lipoprotein. A protease-active immunoprecipitate was also detected in this fraction, but attempts to identify the Rosenbusch matrix protein in the crossed immunoelectrophoretic profile were unsuccessful.     

Purification and characterization of Rana pipiens brain Thy-1 glycoprotein

The occurrence of Thy-1 antigens in Rana brain has been studied by the use of heterologous anti-Rana brain antisera raised in rabbit and BALB/c mouse (Thy-1.2) and AKR/J mouse (Thy-1.1) strains and by monoclonal anti-mouse Thy-1.1 and anti-mouse Thy-1.2 antibodies with the use of quantitative absorption assays. Three antigenic determinants were defined on Rana brain and referred to as: 1) the Rana-specific xenoantigen, 2) the Rana-mouse cross-reacting xenoantigen, and 3) the Thy-1.1 antigen. Thy-1 antigenic activities were solubilized from crude brain membranes in deoxycholate and followed by measuring the Rana-specific and the Thy-1.1 antigenic determinants. After solubilization, Rana brain Thy-1 antigens were purified by lentil lectin affinity chromatography and gel filtration on Sephadex G-200. A 605-fold and 400-fold enrichment in the Rana-specific and the Thy-1.1 antigenic activities with a yield of 25% and 17%, respectively, were obtained. Both antigenic activities were associated with a single glycoprotein of molecular size 3.1 nm and m.w. estimated at 27,000 by SDS-polyacrylamide gel electrophoresis. The serologic and biochemical properties of our purified Rana brain Thy-1 glycoprotein were very similar to those of the mammalian Thy-1 molecule, suggesting the conservation of the gene coding for Thy-1 during vertebrate evolution.     

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.     

Expression of the asialo GM1 determinant on murine intestinal epithelia.

Murine intestinal epithelial cells were studied by flow cytometric analyses for the expression of lymphocyte-associated membrane antigens. Three lymphocyte antigens were found to be expressed at high density on most nonhematopoietic intestinal epithelial cells. These included major histocompatibility complex class II antigens, the T cell-associated CT carbohydrate determinant, and the asialo GM1 (aGM1) neutral glycolipid. Examination of aGM1 determinant density on epithelial cells, estimated by fluorescence intensity, indicated that aGM1 was expressed at levels equal to those present on lymphoid cells known to be aGM1+. The potential role for lymphocyte antigenic determinants on nonhematopoietic cells of murine epithelia with respect to local regulation of intestinal lymphocytes is discussed.     

Virus-Specific Antigens in Hamster Cells Transformed by Rous Sarcoma Virus

Virus-specific antigens were studied in hamster cells transformed by Rous sarcoma virus (RSV). Antigens were localized in the cytoplasm, as demonstrated by fluorescent antibody staining of fixed cells as well as by complement fixation (CF) following subcellular fractionation. Cytoplasmic extracts were analyzed by velocity and isopycnic centrifugation. CF antigens were found in a soluble form and in association with membranes and polyribosomes. Isolated plasma membranes had no CF antigen. Both soluble and particulate fractions with CF activity contained the same antigenic determinants by Ouchterlony analysis. These antigenic determinants were identical to those released by ether treatment of RSV.     

Presence of normal human cell surface antigens in plasma of athymic mice bearing a human colon carcinoma and in normal human plasma

Summary The mixed haemadsorption (MHA) method was employed for detection of several normal antigenic components on the surface of human colon carcinoma cells (HT-29). The antigens were expressed by cells in monolayer cultures and in suspensions prepared by monolayer trypsinization, and by cells of tumours growing progressively in athymic mice. The plasma of such animals bearing medium sized and large, non-necrotic tumours contained all the antigens, as determined by the radial diffusion immune haemolysis method (RDIH); the plasma of animals with small or large heavily necrotic tumours did not contain detectable amounts of any of the determinants. The half-life of the determinants in the circulation as extracellular entities was ca. 20 h. The same antigens, and fibronectin, were found to be ubiquitously represented in normal human plasma. It is proposed that the presence of membrane antigens in plasma is the result of physiological shedding of cell surface constituents by living cells.     

Localization of the Tween 20-soluble membrane proteins of Acholeplasma laidlawii by crossed immunoelectrophoresis

The Tween 20-soluble membrane proteins from Acholeplasma laidlawii have previously been fractionated by preparative agarose-suspension electrophoresis. The fractions obtained have now been characterized by crossed immuno-electrophoresis in the presence of Tween 20 and with antiserum containing antibodies directed against the membrane proteins. This antiserum was also utilized in order to get some information about the location of proteins, i.e. whether they are located on the inside or the outside of the membrane. The method used is based upon crossed immunoelectrophoresis of the Tween 20-soluble membrane proteins as antigens and uses an antiserum that has been depleted of the antibodies that are directed against proteins with antigenic determinants exposed either on the outside of the membrane or on both sides. These two types of antisera (called I and II) can be produced by adding intact cells or washed, lysed cells, respectively, to the original antiserum and then removing the cells with the adsorbed antibodies by centrifugation. If there exists in the intact membrane a protein which has antigenic determinants, e.g. only on the inside of the membrane, a precipitation line corresponding to this protein will appear in crossed immunoelectrophoresis experiments with the original antiserum and antiserum type I, but not with antiserum type II. Using this method we found that probably only one of the Tween 20-soluble proteins is exposed on the outside and three on the inside of the A. laidlawii membrane. These findings, combined with results obtained by digesting and labelling erythrocytes and by immunological investigations of protoplasts of Micrococcus lysodeikticus, may reflect a possible, general feature of the structure of the plasma membrane, namely that most of its proteins are associated with the inner surface of the membrane. There is also some evidence that no protein is buried within the lipid layer, which also has been found for erythrocyte ghosts by a labelling technique, and therefore may be another characteristic architectural feature of plasma membranes.     

Antigenic architecture of membrane vesicles from Escherichia coli.

The antigenic architecture of membrane vesicles prepared from Escherichia coli ML 308--225 has been studied using crossed immunoelectrophoresis. Progressive immunoadsorption experiments conducted with control vesicles and with physically disrupted vesicles were used to monitor and quantitate the expression of 14 different immunogens. Eleven immunogens, including NADH dehydrogenase (EC 1.6.33.3), D-lactate dehydrogenase (EC 1.1.1.27), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), and beta-galactosidase (EC 3.2.1.23), exhibit minimal expression (10% or less) unless the vesicles are disrupted. Three unidentified antigens are expressed to a similar extent in untreated and disrupted vesicles. Consideration of these and other results [Owen, P., & Kaback, H. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3148] in terms of membrane polarity, dislocation of antigens, and possible transmembrane orientation of some immunogens reveals that over 95% of the membrane in the vesicle preparations is in the form of sealed sacculi with the same orientation as the intact cell. Furthermore, antigens are distributed across the membrane in a highly asymmetric manner, indicating that dislocation of components from the inner to the outer surface of the membrane during vesicle preparation does not occur to an extent exceeding 10%.     

Novel monoclonal antibodies identify antigenic determinants unique to cellular senescence.

Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used as a model to study in vivo aging. Monoclonal antibodies were generated against partially purified surface membranes from human diploid fibroblasts at the end of their lifespan (senescent). Three hybridomas were isolated that secreted antibodies reacting to cellular determinants expressed specifically on senescent human fibroblasts of different origin, including neonatal foreskin, embryonic lung, and adult skin punch biopsy, but not expressed on matched young cells. The antibodies did not bind to immortal human cells and normal young cells made reversibly nondividing, indicating the antigens are not expressed in cells that are not senescent. The antibodies identified senescent cells in a mixed cell population and expression of the senescent cell antigens correlated strongly with the cells inability to synthesize DNA at the onset of senescence. The antigens appeared to be cell surface or extracellular matrix associated, and the epitopes were destroyed by mild trypsin treatment. Western analysis indicated all three antibodies reacted with fibronectin. Though the antigenic determinants on the fibronectin molecule were not accessible in the intact young cell, the epitopes were present in fibronectin extracted from both senescent and young cells, as well as purified human plasma fibronectin. These antibodies and the senescent specific expression of the antigens provide powerful tools to investigate the mechanisms leading to in vitro senescence. This may enable us to investigate directly the relationship between cellular aging and aging of the individual.     

Immunochemical Analysis of the Temporal and Tissue-Specific Expression of an Avena sativa Plasma Membrane Determinant

An immunoglobulin Mk monoclonal (F8IVE9) antibody raised against oat (Avena sativa cv Garry) root homogenate has been produced and characterized. The predominant target bound by this antibody is a 62-kilodalton protein (p62) that is expressed in both oat root and oat shoot cells. Treatment of the oat antigen with periodate, or with recombinant N-glycanase, affects the F8IVE9 binding to the antigen, suggesting that the specific epitope for this monoclonal antibody involves a carbohydrate determinant. Levels of p62 present in cells of the oat root increase approximately twofold as the root tissue matures during the first 11 days postgermination. In contrast, levels of expression in shoot tissue remain relatively constant during the same period. The p62 antigen has been shown to be expressed at the plasma membrane by immunohistochemical means, by immunofluorescent labeling of protoplasts, and by enzyme-linked immunosorbent assay analysis of purified plasma membrane. The F8IVE9 antigenic target appears to be uniformly distributed through root tissue but is differentially expressed in specific sections of the shoot. F8IVE9 antibody also binds to antigens expressed in a number of other species, including clover, corn, pea, broccoli, mustard, and bean, and has been shown to bind to Samanea protoplast plasma membranes. This monoclonal antibody may prove to be useful for a variety of investigations, including an analysis of the specific patterns of cellular differentiation that occur during early morphogenesis, and the characterization of plasma membrane-associated elements in plants.     

Metabolism and functions of phosphatidylserine

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid that is synthesized by prokaryotic and eukaryotic cells. In this review we focus on genes and enzymes that are involved in PS biosynthesis in bacteria, yeast, plants and mammalian cells and discuss the available information on the regulation of PS biosynthesis in these organisms. The enzymes that synthesize PS are restricted to endoplasmic reticulum membranes in yeast and mammalian cells, yet PS is widely distributed throughout other organelle membranes. Thus, mechanisms of inter-organelle movement of PS, particularly the transport of PS from its site of synthesis to the site of PS decarboxylation in mitochondria, are considered. PS is normally asymmetrically distributed across the membrane bilayer, thus the mechanisms of transbilayer translocation of PS, particularly across the plasma membrane, are also discussed. The exposure of PS on the outside surface of cells is widely believed to play a key role in the removal of apoptotic cells and in initiation of the blood clotting cascade. PS is also the precursor of phosphatidylethanolamine that is made by PS decarboxylase in bacteria, yeast and mammalian cells. Furthermore, PS is required as a cofactor for several important enzymes, such as protein kinase C and Raf-1 kinase, that are involved in signaling pathways.     

Epinephrine-binding plasma-membrane antigens in rat liver.

Detergent extracts of isolated rat liver plasma membranes were analysed in two-dimensional immunoelectrophoresis against antiserum to plasma membranes. Enzyme staining of the immunoprecipitates revealed the presence of about ten antigens with nucleoside di- and triphosphatase activity. Most of these were earlier shown also to be NADH-neotetrazolium reductase active. In addition, two of these antigens exhibited L-leucyl-beta-naphthylamidase activity. As judged from autoradiography these plasma membrane antigens earlier characterized as multienzyme complexes bound [14C]epinephrine, and the same antigens were labelled regardless of whether membranes or membrane extracts were incubated with the radioactive hormone. The specificity of this binding was established in displacement experiments with unlabelled hormones or their analogues. Another hormone-binding antigen, also identified in the plasma membrane extract did not exhibit any known enzyme activity while three antigens with different enzyme activities had no epinephrine-binding capacity. [14C]Epinephrine-labelled plasma membrane extracts were chromatographed on Sepharose 4B and the fractions obtained were analysed in two-dimensional immunoelectrophoresis combined with autoradiography. Nucleoside di- and triphosphatases of high molecular weights (5000000) were associated with L-leucyl-beta-naphthylamidase activity, while no such associations were detected in a lower molecular weight region (70000). Further immunological studies on the various fractionated antigens provided evidence that at least two of them occurred in both low and high molecular weight fractions. Hormone-binding membrane components in varying concentrations were found throughout the eluted extract.