kDa Doublet Research Articles

Microsomal dexamethasone binding sites identified by affinity labelling

Binding studies with [3H]dexamethasone† identified a class of binding sites on male rat liver microsomes. The binding sites were glucocorticoid-dependent and specific for glucocorticoids and progestins. Scatchard binding parameters, competition studies with triamcinolone acetonide, a synthetic glucocorticoid which competes well for the glucocorticoid receptor, and immunoblotting with an antiglucocorticoid receptor antibody indicated that these sites are distinct from the cytosolic glucocorticoid receptor. Affinity labelling experiments with [3H]dexamethasone 21-mesylate revealed two specifically labelled peptides, one at approx. 66 kDa and a doublet at 45 kDa. The 66 kDa peptide had been previously identified in serum and may be present as a result of serum contamination of the microsomal preparation. The 45 kDa doublet, on the other hand, had been shown to be absent from rat serum. The characteristics of the 45 kDa peptide(s) were identical to those of the dexamethasone binding site identified in the binding studies. [3H]Dexamethasone binding characteristics and affinity labelling of microsomal subfractions, separated by isopycnic centrifugation, showed that the binding sites are located in the endoplasmic reticulum. The identification and role of the 45 kDa peptide doublet remain to be determined.

Histamine and bradykinin stimulate the phosphoinositide turnover in human umbilical vein endothelial cells via different G-proteins

The G-proteins which regulate hormonal turnover of phosphoinositide (PI) in human umbilical vein endothelial cells have been investigated. A 40–41 kDa doublet present in the membranes of these cells was selectively ADP ribosylated by pertussis toxin (PTx), and this doublet was G α 2 and G α 3 according to immunoblotting with specific antisera. By contrast, a doublet of 24–26 kDa proteins in the same membrane preparations was ADP ribosylated by the C 3 component of botulinum toxin (BoTx). PTx-dependent ADP ribosylation blocked stimulation of PI turnover by histamine, but did not affect stimulation by bradykinin, whereas BoTx (C 2 + C 3 components) had the opposite effect. Thus two different groups of G-proteins may be involved in hormone-dependent stimulation of PI turnover in human umbilical vein endothelial cells.

Identification of a novel binding protein for insulin-like growth factors in adult rat serum

Using gel filtration, ligand-affinity chromatography and reversed phase HPLC, four insulin-like growth factor-binding proteins (IGF-BPs) have been purified from adult rat serum. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis revealed that all four proteins migrated as doublets at 45 37 (peak 1 in Fig. 4), 36 32 (peaks 2 and 5 in Fig. 4), 35 33 (peak 3 in Fig. 4) and 22 28 kDa (peaks 4a, 4b in Fig. 4), respectively under reducing conditions. N-terminal sequence analysis of the 45 37 kDa doublet showed that it is identical to the N-terminal of the 45 kDa rat IGF-BP whereas the 22 28 kDa doublet is the C-terminal truncated form of the 45 kDa species. The 35 33 kDa doublet has the same N-terminal sequence as that of the rat IGF-BP isolated from a BRL-3A cell line. However, the N-terminal sequence of the 36 32 kDa doublet is unique, although it may be related to the BRL-3A protein. The most abundant IGF-BP in adult rat serum corresponds to the 45 kDa species plus its C-terminal truncated forms, whereas the second most abundant IGF-BP is the novel protein detected in this study, while the least abundant species is the BRL-3A IGF-BP.

Identification of genus-specific epitopes on the outer membrane complexes of Chlamydia trachomatis and Chlamydia psittaci immunotypes 1 and 2

Polyclonal and monoclonal antibodies were used to study the immunogenic and antigenic characteristics of chlamydiae. We focused on the most predominant proteins in the outer membrane complex, the major outer membrane protein (MOMP) and the doublet consisting of proteins of 57 and 62 kilodaltons (57-62 kDa doublet). Immunoblot analyses were performed with chlamydial elementary bodies by using (i) immune sera from sheep which had undergone a recent episode of abortion due to the ovine abortion (OA) strain of C. psittaci, (ii) rabbit hyperimmune anti-C. psittaci (OA) and -C. trachomatis sera, and (iii) monoclonal antibodies to the MOMP of C. trachomatis. The typical pattern of response with polyclonal antisera against heterologous elementary bodies was reactivity with the 57-62 kDa doublet and lipopolysaccharide with weak and sometimes no anti-MOMP activity. Three distinct genus-specific anti-C. trachomatis MOMP monoclonal antibodies showed different patterns of reactivity with the MOMPs of the two immunotypes of C. psittaci and C. trachomatis serovars. Our data confirm the predominance of a genus-specific 57-62 kDa doublet response despite the presence of genus-specific epitopes on the MOMP.

Agonist-enhanced palmitoylation of platelet proteins

When washed human platelets were incubated with [ 3H]palmitic acid, radioactivity was incorporated into a major 38 kDa doublet and several minor proteins that were resolved on polyacrylamide gels. The radioactivity associated with the proteins remained after extractions with organic solvents, but it was lost after hydroxylamine treatment or mild alkali methanolysis. The products of these reactions were analyzed by thin-layer chromatography and HPLC. They were identified as palmitohydroxamate and methyl palmitate, respectively, indicating that the palmitic acid was covalently linked to the proteins via oxygenester or thioester bonds. In resting platelets, radioactivity was detected in the 38 kDa proteins 2 min after the addition of [ 3H]palmitic acid. A plateau was reached between 5 and 11 min, at which time radioactivity was also detected in a 23 kDa protein. Thrombin elicited faster and greater incorporation of label into both proteins. Phorbol 12-myristate 13-acetate (PMA) led to a similar, but slower increase of radioactivity in the 38 kDa proteins, while collagen and A23187 were less effective. Enhanced palmitoylation may be closely linked to platelet activation, as suggested by the following observations: (1) in thrombin- or PMA-activated platelets, the time-course of aggregation correlated with the time-course of enhanced palmitoylation of the 38 kDa proteins; (2) in platelets activated by various concentrations of thrombin with or without prostacyclin, aggregation was correlated with the enhanced incorporation of radioactivity into the 38 kDa proteins.

Antibodies against specific membrane proteins of neurosecretory granules isolated from bovine neural lobes

Membrane proteins from bovine neurosecretory granules isolated by density gradient centrifugation were separated by polyacrylamide gel electrophoresis. A doublet of 120 kDa and 67 kDa bands were identified as specific proteins of the neurosecretory granule membrane. Antibodies against the 120 kDa doublet were raised in rabbits and characterized by western blotting and immunocytochemistry. Analysis of the antiserum by western blotting showed that this recognizes mainly the 120 kDa doublet and some other minor components which seem to be degradation products. The antiserum against the 120 kDa proteins stained, by immunocytochemistry, specifically the supraoptic and paraventricular neurons of the rat hypothalamo-neurohypophysial system. In the neural lobe the immunoreaction was found around blood vessels on structures which appear to be nerve endings and on Herring bodies. Immunoelectron microscopy using protein A-gold showed that the 120 kDa antigens are located on the membrane of neurosecretory granules in sections of rat neural lobes. The presence of the 120 kDa antigens exclusively in the hypothalamo-neurohypophysial system suggests that these proteins are probably not involved in a general secretory mechanism and that they might be a result of the tissue-specific expression of proteins.

Membrane proteins of the nerve growth cone and their developmental regulation

The membrane polypeptides of growth cone fragments ("growth cone particles," GCPs) isolated from fetal rat brain by subcellular fractionation have been analyzed in further detail. The major polypeptides of salt-washed GCP membranes detected by 1-dimensional gel electrophoresis (Ellis et al., 1985b) resolve in 2-dimensional gels as a spot of 52 kDa that comigrates with beta-tubulin and reacts with anti-beta-tubulin; a 46 kDa, pl 4.3, polypeptide (pp46) that has no equivalent in the soluble fraction and is identical to one of the GCP's major phosphoproteins (Katz et al., 1985) and to GAP43 (Willard et al., 1985); a spot of 42 kDa that comigrates with actin; and a species of 34 kDa (p34) without soluble equivalent. The prominent 38 kDa doublet identified in 1-dimensional gels is difficult to resolve in 2-dimensional gels. The major phosphoproteins pp80ac, pp46, and pp40 (Katz et al., 1985), as well as p34 partition into the oil phase of Triton X-114 extracts, suggesting that they are integral membrane proteins, at least in our experimental conditions. The properties of pp46 reported here are in conflict with the highly hydrophilic amino acid sequence predicted for GAP43/B50/F1 (Basi et al., 1987; Karns et al., 1987). Growth-cone and presynaptic membrane proteins are compared as follows. After eye injection of 35S-methionine, GCPs and synaptosomes are isolated from the target areas of optic nerve of fetal and adult rats, respectively. Polypeptides are separated by 1- and 2-dimensional gel electrophoresis and the radiolabeled species identified fluorographically. The comparison of labeled GCP and synaptosome polypeptides shows that all 5 major Coomassie blue-stained polypeptides of GCP membranes (52, 46, 42, 38, 34 kDa) are intensely labeled after eye injection. However, in synaptosomes, these polypeptides are weakly labeled if at all; instead, an intensely labeled polypeptide of 28 kDa, and several additional species not seen in GCPs, have appeared. Therefore, the major growth cone membrane proteins are developmentally regulated, and the rates of synthesis and transport into the axonal ending of neuronal polypeptides change dramatically at the time of synaptogenesis.

Boar proacrosin is a single-chain molecule which has the N-terminus of the acrosin A-chain (light chain)

Boar proacrosin was isolated from spermatozoa by a novel procedure under conditions preventing proenzyme activation. The spermatozoal extract was fractionated by gel filtration and reversed-phase FPLC, all in acidic solutions. Isolated proacrosin had a molecular mass of 55/53 kDa (doublet) and was devoid of amidolytic activity. Its single N-terminal sequence corresponded to that of the 23-residue acrosin A-chain and continued with that of the acrosin B-chain. Autoactivation at pH 7.8 did not influence the molecular mass. However, activated material contained two parallel N-terminal sequences, those of the A- and B-chain. Thus, activation of proacrosin is analogous to that of other serine proteinase proenzymes.

Further characterization of calpain-mediated proteolysis of the human erythrocyte plasma membrane Ca 2+-ATPase

The membrane-bound form and a solubilized and purified form of the Ca 2+-ATPase from human erythrocyte have been proteolyzed under controlled conditions by highly purified. Ca 2+-dependent neutral cysteine-protease, calpain I, in the absence and in the presence of the calmodulin-calcium complex. In the absence of calmodulin the 136-kDa enzyme was transformed into a group of fragments of 125-124 kDa, followed by the slower formation of a second group of fragments of 82-80 kDa. These heterogeneous fragments were capable of forming an acylphosphate intermediate. The 125- and 82-kDa minor components of each heterogeneous group of fragments (125-124 and 82-80 kDa) were capable of binding calmodulin, whereas the 124- and the 80-kDa major components did not. In the presence of calmodulin, however, the native enzyme was transformed into a 127-kDa fragment followed by the slower formation of an 85-kDa fragment. Both fragments (127 and 85 kDa) formed an acylphosphate intermediate and were capable of binding calmodulin. The presence of calmodulin during calpain action effectively protected the Ca 2+-ATPase from proteolytic activation (K. K. W. Wang, A. Villalobo, and B. D. Roufogalis (1988) Arch. Biochem. Biophys. 260, 696–704) and prevented the formation of the calmodulin-insensitive 124- and 80-kDa fragments. Smaller fragments not capable of forming the acylphosphate intermediate were also produced, in particular a 39-37 kDa doublet band retaining the capacity to bind calmodulin. In contrast to the membrane-bound form, the purified form of the Ca 2+-ATPase was proteolyzed by calpain at a slower rate.

Monoclonal antibodies to axolotl immunoglobulins specific for different heavy chains isotypes expressed by independent lymphocyte subpopulations

An immunoblotting analysis of purified axolotl immunoglobulins (Ig) separated by SDS—PAGE reveals two heavy (H) chains isotypes: a 76 kDA chain recognized by the monoclonal antibody (mAb) 33.45.1 and a 66–68 kDa doublet recognized by the mAb 33.39.2. The 76 kDa chain is associated to high molecular weight (HMW) Ig molecules and the 66–68 kDa H chains are associated to low molecular weight (LMW) Ig of 172 kDa. Both H chains isotypes are linked to identical light (L) chains, labelled in immunoblotting by the mAb 33.101.2. Two different axolotl lymphocyte subpopulations are characterized by these two distinct H chains isotypes. One population of splenic lymphocytes (∼40%) is labeled by indirect immunofluorescence with mAb 33.45.1, specific for the 76 kDa H chain isotype. Another population (∼20%) is labeled by mAb 33.39.2 specific for the 66–68 kDa H chain isotype. Both populations of splenic lymphocytes are stained by mAb 33.101.2 specific for the axolotl L chains. Therefore, the presence of at least two independent Ig classes is now confirmed in a urodele amphibian species at the humoral and cellular levels.

Characterization of a family of rhoptry proteins of Toxoplasma gondii

A monoclonal antibody (McAb 4A7) raised against a rhoptry enriched subcellular fraction of Toxoplasma gondii tachyzoites reacted in immunofluorescence studies with rod-like organelles located in the anterior part of the organisms and gave specific labeling of rhoptries in immunoelectron microscopy. On immunoblots, two major proteins of 55 and 60 kDa were identified by McAb 4A7. Similar results were obtained both by immunodetection and immonoblotting with tachyzoites, bradyzoites and sporozoites. Pulse chase analysis of [ 35S]methionine labeled tachyzoites demonstrated that the 55 and 60 kDa rhoptry proteins derived from a 66–68 kDa doublet which was processed approximately 30 min after biosynthesis. Two other monoclonal antibodies (McAb 2F8, McAb 2H3) respectively specific for rhoptry proteins of 55 kDa having a 66 kDa precursor and 60 kDa having a 68 kDa precursor were also obtained; we suggest that they recognize separately the two components of the 55–60 kDa rhoptry protein family of Toxoplasma.

Characterization of two molecules at 30,33 kDa as major target antigens of natural thymocytotoxic autoantibodies

We have attempted to characterize, by immunoblotting, the cell surface determinants which are recognized by natural thymocytotoxic autoantibodies (NTA). NTA-positive sera from spontaneously autoimmune mice and from mice rendered autoreactive by neonatal induction of tolerance were used as probes on blots of thymocytes. Fifty percent of the sera screened under these conditions reacted with a doublet of 30,33 kDa molecular weight present on membranes, but not detectable on blots of total cell extracts. Additional bands of various molecular weights were detected by NTA-positive sera at a much lower frequency than the 30,33 kDa determinants. Direct evidence of identity between the antibodies inducing thymocytotoxicity and those detecting the doublet in immunoblotting was provided by immunoadsorption tests. Among a panel of immunoadsorbents prepared with thymocyte membrane proteins of various molecular weights, only the one containing the 30,33 kDa molecules efficiently adsorbed cytotoxic antibodies whereas the other preparations containing some of the bands occasionally detected in immunoblotting had practically no effect. In addition we showed that the 30,33 kDa doublet is also detected on membrane preparations of T and B lymphocytes but not on preparations of a non-lymphoid cell line, and is composed of two independent molecules not covalently linked, in their native configuration, by disulfide bonds. Altogether these results strongly suggest that the 30,33 kDa molecules represent major cell surface determinants for thymocytotoxic autoantibodies.

An antigenic complex in the rhoptries of Plasmodium falciparum

A previously identified putative rhoptry antigen of Plasmodium falcipurum is composed of two major components, one of 80 kDa and a doublet at 42/40 kDa. An inhibitory monoclonal antibody immunoprecipitated both the 80 kDa protein and the 42/40 kDa doublet, but immunoblotted only the 80 kDa component. A second monoclonal antibody, raised against the affinity purified complex, immunoblotted only the 42 kDa band under non-reducing conditions. Electron microscopic examination of thin sections of parasites immunolabeled with these monoclonal antibodies and colloidal gold anti-mouse conjugate has confirmed that this antigen is localised in the rhoptry organelles of mature schizonts and free merozoites. The antigen is associated with apparent membranous structures released from free merozoites. Immunoblotting and immunoprecipitation with two different monoclonal antibodies, and protease digestion experiments, have clearly demonstrated that this antigen is a complex composed of two separate and distinct proteins, and does not represent a monomer/dimer pair. The 80 kDa protein is synthesised as an 84 kDa precursor.

Cloning of diagnostic 31/32 kilodalton antigens of Schistosoma mansoni

A cDNA library derived from messenger RNA of adult Schistosoma mansoni was constructed in λgt11 and schistosome antigens were expressed as fusions with the amino terminus of the β-galactosidase of Escherichia coli. Using mouse and human infection sera, recombinant clones specific for a 31/32 kDa doublet having a potential in the immunodiagnosis of schistosomiasis were selected. The specificity of the clones was verified by their reactivity with monoclonal antibodies. The identity of the cloned epitopes with those of the native proteins was confirmed by Western blot analysis of total schistosome proteins, using both antibodies affinity purified from the fusion proteins and antisera raised in rabbits against the fusions. The reactivity of the cloned antigens with human infection sera suggests their usefulness for the immunodiagnosis of human schistosomiasis.

Immunological evidence for two physiological forms of protein kinase C.

Our recently described purification scheme for rat brain protein kinase C yields an enzyme consisting of a 78/80-kilodalton (kDa) doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (submitted for publication). Antisera against this preparation were raised in two rabbits. One of the antisera detected only the 80-kDa component by immunoblotting of purified protein kinase C and immunoprecipitated an 80-kDa [35S]methionine-labeled protein from a variety of human, rodent, and bovine cells, which was shown to represent protein kinase C by comparative one-dimensional peptide mapping. In contrast, the second antiserum detected both 78- and 80-kDa enzyme forms by immunoblotting and immunoprecipitated a [35S]methionine-labeled 78/80-kDa doublet from mammalian cells. One-dimensional peptide maps of these 78- and 80-kDa proteins were similar to those derived from the 78- and 80-kDa forms of purified protein kinase C, respectively. The two forms were not related by either partial proteolysis or differential phosphorylation, showing that two distinct forms of this enzyme exist in mammalian cells. Treatment of mouse B82 L cells with 2.5 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) per ml for 18 h resulted in complete loss of immunoprecipitable protein kinase C with a half time of disappearance of 48 min. Since the normal half-life of protein kinase C was greater than 24 h and the biosynthetic rate of the protein was not decreased after 18 h by TPA treatment, TPA induces down-regulation by increasing the degradation rate of the enzyme. Treatment of cells with 50 ng of TPA per ml followed by resolution of the membrane and cytosol in the presence of ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) promoted an apparent translocation of both 78- and 80-kDa proteins from the cytosol to the membrane fraction. A similar translocation was effected by cell lysis in the presence of Ca2+, indicating the subcellular localization of protein kinase C to be sensitive to the presence of both activators and micromolar amounts of Ca2+.

Immunological evidence for two physiological forms of protein kinase C.

Our recently described purification scheme for rat brain protein kinase C yields an enzyme consisting of a 78/80-kilodalton (kDa) doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (submitted for publication). Antisera against this preparation were raised in two rabbits. One of the antisera detected only the 80-kDa component by immunoblotting of purified protein kinase C and immunoprecipitated an 80-kDa [35S]methionine-labeled protein from a variety of human, rodent, and bovine cells, which was shown to represent protein kinase C by comparative one-dimensional peptide mapping. In contrast, the second antiserum detected both 78- and 80-kDa enzyme forms by immunoblotting and immunoprecipitated a [35S]methionine-labeled 78/80-kDa doublet from mammalian cells. One-dimensional peptide maps of these 78- and 80-kDa proteins were similar to those derived from the 78- and 80-kDa forms of purified protein kinase C, respectively. The two forms were not related by either partial proteolysis or differential phosphorylation, showing that two distinct forms of this enzyme exist in mammalian cells. Treatment of mouse B82 L cells with 2.5 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) per ml for 18 h resulted in complete loss of immunoprecipitable protein kinase C with a half time of disappearance of 48 min. Since the normal half-life of protein kinase C was greater than 24 h and the biosynthetic rate of the protein was not decreased after 18 h by TPA treatment, TPA induces down-regulation by increasing the degradation rate of the enzyme. Treatment of cells with 50 ng of TPA per ml followed by resolution of the membrane and cytosol in the presence of ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) promoted an apparent translocation of both 78- and 80-kDa proteins from the cytosol to the membrane fraction. A similar translocation was effected by cell lysis in the presence of Ca2+, indicating the subcellular localization of protein kinase C to be sensitive to the presence of both activators and micromolar amounts of Ca2+.

Glucocorticoid action on the immune system

Glucocorticoids have profound effects on immune function that are mediated, in part, by steroid-induced cell death. Our studies have been aimed at identifying the mechanism of this lymphocytolytic process using the rat thymocyte as a model system. Administration of glucocorticoids in vivo resulted in internucleosomal cleavage of the lymphocyte genome that was detectable within 2 h of treatment and increased with time after hormone administration. Six h after steroid treatment > 50% of the genome was degraded, yet cell viability remained > 90% indicating that this event preceded cell death. Furthermore, this process appeared to be mediated by the glucocorticoid receptor since the antagonist RU 486 blocked glucocorticoid-mediated DNA degradation. To further characterize this lymphocytolysis we have analyzed glucocorticoid-treated thymocytes for nucleases. Two families of nuclear proteins have been identified, a 30–32 kDa doublet and a series of 3–4 proteins that are 12–19 kDa, both of which are induced by glucocorticoid treatment (137 ± 6% and 342 ± 24%, respectively) and have prominent nuclease activity. These nucleases can also be induced in vitro indicating that glucocorticoids act directly on thymocytes to mediate this response. Moreover, this nuclease induction, like glucocorticoid-mediated DNA degradation, could be blocked by RU 486. Based on these findings we propose a working model of glucocorticoid-mediated lymphocytolysis in which these steroids, acting via a receptor mediated process, induce the expression of a lysis gene product (nuclease) which degrades the genome and results in cell death.

Synthesis and maturation of recombinant human tumor necrosis factor in eukaryotic systems

The biosynthesis of human tumor necrosis factor (hTNF) was studied. The amino-terminal extension of the hTNF precursor (26 kDa polypeptide) was not cleaved off in a cell-free system supplemented with dog pancreas microsomes. Correct maturation of pre-hTNF was nevertheless not restricted to the macrophage system: in the medium of a TNF-producing, transformed CHO cell line, a (weak) ~20 kDa, an ~18.5 kDa (doublet) and a 17 kDa TNF polyeptide, the latter corresponding to mature hTNF, were revealed by specific immunoprecipitation. Similar results were obtained with Xenopus laevis oocytes, injected with hTNF mRNA, except that the 20 kDa band was lacking. The results are discussed in relation to the secretion mechanism of hTNF.

Phase separation in triton X-114 of antigens of transmission blocking immunity in Plasmodium gallinaceum

The distribution of proteins of mosquito midgut forms of Plasmodium gallinaceum in the detergent-free (aqueous) and detergent-enriched phases was studied using a phase separation technique in Triton X-114. Of the three surface proteins on gametes and newly fertilized zygotes (240, 56, and 54 kDa) immunoprecipitated by transmission blocking monoclonal antibodies, 240 kDa protein was recovered in the aqueous phase, whereas 56 and 54 kDa proteins were found preferentially in the detergent phase. The hydrophobic properties of the 56 and 54 kDa proteins were also shown by their strong tendency to interact with the lipid bilayers and a hydrophobic matrix phenyl-Sepharose. Monoclonal antibody IID 3B 3 immunoprecipitated all the three proteins from the whole Triton extract but in the phase-separated extracts reacted only with the 240 kDa protein in the aqueous phase and not with the 56 and 54 kDa doublet in the detergent phase. In Western blot analysis also monoclonal antibody IID 3B 3 reacted only with the 240 kDa protein. The 240 kDa protein in the aqueous phase was retained by monoclonal antibody IID 3B 3 linked to Sepharose 4B beads and could be eluted either with 0.1 M acetic acid or 50 mM diethylamine. The 56 and 54 kDa doublet in the detergent phase could be bound to and eluted from Sepharose 4B beads-linked monoclonal antibody IID 4 or rabbit anti-male P. gallinaceum gamete serum. Two stage-specific glycoproteins of 26 and 28 kDa on the surface of ookinetes of P. gallinaceum were also separated in the detergent phase following Triton X-114 extraction. Phase separation in Triton X-114 offers a simple approach to the separation of a select group of proteins from the bulk of the cellular proteins.

The interaction of human serum with the surface membrane of schistosomula of Schistosoma mansoni

After contact with human serum, a series of proteins become exposed on the surface membranes of schistosomula of Schistosoma mansoni as revealed by radioiodination of the intact parasites. Among the proteins, a doublet ( M r 45 000) is particularly prominent. These doublet proteins, which are believed to be parasite-derived, become apparent after a very short time of incubation with human serum (10 min or less) and are expressed on the surface membranes after contact with a high molecular weight component of human serum ( M r > 80 000). Pretreatment of the parasites with 1.25 mM colchicine or fixation with 2% glutaraldehyde does not prevent the serum-induced expression of the doublet proteins. Extraction of the parasites with chloroform:methanol 2:1 (v/v), however, blocks the human serum effect. Affinity chromatography using immobilized low density lipoproteins (LDL) from human serum shows a tight binding between the 125I-labelled 45 kDa doublet to the LDL. The possible role of the 45 kDa doublet as a receptor for LDL is discussed.