Lentil Lectin Affinity Chromatography Research Articles

Evaluation of two Taenia solium cysticercal antigenic preparations (vesicular fluid and a glycoprotein fraction with affinity for lentil lectin) for the immunodiagnosis of neurocysticercosis by enzyme-linked immunosorbent assay (ELISA)

To evaluate the performance of two antigenic preparations (vesicular fluid - VF and a glycoprotein fraction, LLa-Gp fraction, purified from a whole parasite extract by lentil lectin affinity chromatography) from Taenia solium cysticerci for the immunodiagnosis of neurocysticercosis. Fifty-six cerebrospinal fluid (CSF) samples (22 from patients with neurocysticercosis and 34 from patients with other neurological disorders) and 57 serum samples (22 from patients with neurocysticercosis, 18 from patients with other infections and 17 from presumably healthy persons) were assayed for anticysticercal IgG antibodies with an enzyme-linked immunosorbent assay (ELISA). The VF ELISA showed 100% sensitivity and specificity in CSF and serum samples, whereas the sensitivity and specificity of the LLa-Gp ELISA were, respectively, 90.9% and 97.1%, with the CSF samples and 95.5% and 100% with serum samples. There was no significant difference in the sensitivity and specificity of the two antigenic preparations used to screen CSF and serum samples. Considering the complexity and high cost of obtaining the LLa-Gp fraction, VF could be more suitable for screening specific antibodies by ELISA in CSF and serum samples from patients with neurocysticercosis.

Schistosoma mansoni gene GP22 encodes the tegumental antigen sm25: (1) antibodies to a predicted B-cell epitope of Sm25 cross-react with other candidate vaccine worm antigens; (2) characterization of a recombinant product containing tandem-repeats of this peptide as a vaccine.

Monospecific antibodies against two putative epitopes of schistosome protein encoded by gene GP22 (182 codons, no introns) were used to probe worm extracts fractionated by lentil-lectin affinity chromatography or by electrophoresis. Anti-peptide-alpha (codons 70-84) exclusively identifies the N-glycanase-sensitive, 25 kDa tegumental glycoprotein Sm25 in the lectin-bound fraction of detergent-solubilized adult worm extract S3. In contrast, antipeptide-delta (codons 151-162) does not react with Sm25 but cross-reacts with other schistosome proteins, including candidate vaccine antigens paramyosin (Sm97) and glutathione-S-transferases (Sm26, Sm28, Sj26). Recombinant protein r4 x 47, constructed to express multiple copies of codon sequence 117-163 (containing delta), reacts with anti-delta and is uniquely recognized by protective Fischer twice-infected (F-2x) rat antiserum. Immunization with r4 x 47 induces antibodies with cross reactivities similar to anti-delta, but which also recognize Sm25. Despite these cross-reactivities with protective antigens, rodents vaccinated with r4 x 47 were not protected against cercarial infection. On the basis of these data, two hypotheses are proposed: (1) antigenic epitopes other than delta are present within the r4 x 47 sequence which induce antibodies reactive with Sm25 and/or (2) peptide-delta assumes alternative antigenic conformations, dependent upon the context of neighbouring sequences, some of which mimic epitopes of proteins encoded by other schistosome genes. These mimotopes are not targets of protective antibodies.

Molecular and immunological characterization of soluble aggregated A/Victoria/3/75 (H3N2) influenza haemagglutinin expressed in insect cells.

A/Victoria/3/75 (H3N2)-derived cDNA coding for a secreted haemagglutinin (HA0s) was cloned into the polyhedrin promoter-based pVL1392 transfer vector, and a recombinant baculovirus was isolated. 5 to 10 micrograms/ml of secreted HA were obtained following infection of Spodoptera frugiperda-9 cells. Gel filtration revealed the presence in the cell supernatant of immunoreactive HA molecules with varying M(r). The high M(r) fraction (aHA0s) could be purified by Matrex Cellufine Sulphate and Lentil-lectin affinity chromatography, followed by Sephacryl S300 HR gel filtration. aHA0s consisted of aggregated, non-covalently linked subunits which were not cleaved into HA1 and HA2 polypeptides; aHA0s was highly susceptible to trypsin treatment and reacted with two low pH-specific monoclonal antibodies, suggesting that a HA0s consists of monomeric subunits. When the expression medium was adjusted to pH 8.5, no aHA0s was observed, suggesting that aggregation occurred in the cells due to a low intracellular pH. Balb/c mice immunized with purified aHA0s developed high, aHA0s-specific antibody titres. Despite these high titres, almost no binding to trimeric viral HA was observed, and immunized mice were not protected against a challenge with homologous mouse-adapted X47 virus. However, when virus was subjected to low pH, resulting in a profound conformational rearrangement, strong binding was observed. Moreover, binding to the low pH-treated HA of different drift variants, isolated between 1968 and 1989, occurred.

UDP galactose:ceramide galactosyltransferase and glutamate/aspartate transporter. Copurification, separation and characterization of the two glycoproteins.

The oligodendrocyte-specific UDP-galactose:ceramide galactosyltransferase (CGT) is the key enzyme involved in the biosynthesis of the oligodendrocyte- and myelin-specific cerebrosides. The galactosyltransferase was isolated and purified to homogeneity from Triton-X-100-solubilized rat brain microsomes by ion exchange, dye ligand and lectin affinity chromatography as a 64-kDa protein homogenous in SDS/PAGE. It copurified with the brain-specific Na(+)-dependent high-affinity L-glutamate/aspartate neurotransmitter transporter (GLAST-1) of the central nervous system. Differential lentil lectin affinity chromatography led to the separation of two glycoproteins with very similar physical properties. CGT was identified as a high-mannose glycoprotein and GLAST-1 as a hybrid glycoprotein, both with a molecular mass of 64 kDa. Deglycosylation reduced the molecular mass of the two proteins to 59 kDa. A 70-kDa isoform of GLAST-1 was isolated from whole brain by wheat germ lectin affinity chromatography. Deglycosylation again reduced the molecular mass to 59 kDa. Therefore the 70-kDa isoform differs only in the degree of glycosylation from the 64-kDa GLAST-1 isoform. The two isoproteins form homodimers of 130 and 140 kDa, respectively. They were isolated and characterized with protein-chemical and immunological methods. Oligonucleotides derived from respective peptide sequences of CGT and GLAST-1 were successfully applied to the cloning of CGT and the first high-affinity glutamate neurotransmitter transporter (GLAST-1) in glia of the central nervous system as well.

Characterization of the ATPase activity of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells

P-Glycoprotein (Pgp) was isolated from CHRC5 membranes by selective detergent extraction and further purified by lentil lectin affinity chromatography. The purified product displayed a very high basal ATPase activity (1.65 mumol/min per mg protein in the absence of added drugs or lipids) with an apparent Km for ATP of 0.4 mM. There was no evidence of cooperativity, suggesting that the two ATP sites operate independently of each other. Pgp ATPase activity was stimulated by verapamil, trifluoperazine and colchicine, and inhibited by daunomycin and vinblastine. All drugs and chemosensitizers acted as mixed activators or inhibitors, producing changes in both the Vmax of the ATPase and the Km for ATP. ADP competitively inhibited Pgp ATPase, with a Ki of 0.2 mM. The macrolide antibiotics bafilomycin A1, concanamycin A and concanamycin B, inhibited Pgp ATPase at concentrations of 0.1-10 microM, and at an inhibitor:protein stoichiometry of 0.65-1.0 mumol/mg protein, which is at the low end of the range characteristic of P-type ATPases. Pgp ATPase was relatively selective for adenine nucleotides. Several phospholipids stimulated Pgp ATPase activity in a dose-dependent manner, whereas others produced inhibition. Metabolic labelling showed that the endogenous phospholipids associated with purified Pgp consisted largely of phosphatidylethanolamine and phosphatidylserine, with only a small amount of phosphatidylcholine. 32P-Labelling studies indicated that purified Pgp was partially phosphorylated. It can be concluded that Pgp is a constitutively active, adenine nucleotide-specific ATPase whose catalytic activity can be modulated by both drugs and phospholipids.

Serum thyrotropin (TSH) heterogeneity in euthyroid subjects and patients with subclinical hypothyroidism: the core fucose content of TSH-releasing hormone-released TSH is altered, but not the net charge of TSH

The aims of the present study were to determine the influence of brief subclinical hypothyroidism on the isoforms of serum thyrotropin (TSH) and to examine the net charge of TSH in different metabolic states. Sera were obtained from euthyroid subjects (n = 7) and from patients with subclinical hypothyroidism (n = 8) before and 30 min after the intravenous administration of 200 micrograms thyrotropin-releasing hormone (TRH). The TSH from human pituitary extracts (IRP 68/38), basal and TRH-stimulated serum TSH was immunoconcentrated and further analysed by isoelectric focusing (IEF) and lentil lectin affinity chromatography. TSH immunoreactivity was determined in each specimen or fraction with an automated highly sensitive chemiluminometric TSH assay. We found that basal TSH in subclinical hypothyroidism, and TRH-released TSH in euthyroidism and in subclinical hypothyroidism is distributed in a similar neutral to acidic pattern, which significantly differs from the more alkaline to neutral isoform pattern of intrapituitary TSH (P < 0.05). IEF analysis of pituitary standard TSH revealed 3 major peaks (pI values 7.5; 6.6; 5.8) whereas in most euthyroid or subclinically hypothyroid subjects 5 peaks were found. Lentil lectin affinity chromatography revealed that TRH-released TSH in euthyroid subjects has more core fucose residues than TSH from patients with subclinical hypothyroidism (64.6 +/- 6.7 vs 12.5 +/- 2.7%, P < 0.0001). Thus pituitary standard TSH seems to be less mature material than circulating TSH. Perhaps no alteration in the IEF pattern of TSH was detected during early hypothyroidism because sialylation of TSH was increasing as sulfatation was decreasing. Nevertheless, a change in the core fucose content of TSH was detectable by lentil analysis.

Loss of biological activity of arginine vasopressin during its degradation by vasopressinase from pregnancy serum

Degradation of AVP by placental vasopressinase may precipitate gestational diabetes insipidus, which in some cases is accompanied by pre-eclampsia. Abnormally elevated vasopressinase has also been reported in pre-eclampsia without diabetes insipidus. This association between excessive vasopressinase production and pre-eclampsia might be explained if the products of AVP degradation by vasopressinase retained pressor activity even after anti-diuretic activity had been destroyed. Recent evidence indicates that such products may raise blood pressure in rats. The objective of this study was, therefore, to purify vasopressinase and investigate its action on both the V1 and V2 receptor-stimulating activity of AVP. Vasopressinase was purified from pooled pregnancy serum by ammonium sulphate precipitation, followed by sequential ion exchange, lentil lectin affinity and gel filtration chromatography. Purified enzyme was then used to degrade AVP and the loss of both immunoreactivity and biological activity monitored. Loss of V1 receptor-stimulating activity and V2 receptor-stimulating activity was compared by two-way ANOVA. Blood was obtained from healthy women between week 34 and the end of pregnancy. Pooled serum from 20-30 patients was used as starting material for the purification of vasopressinase. AVP immunoreactivity was measured by radioimmunoassay, V1 receptor-stimulating activity by a platelet aggregation bioassay, and V2 receptor-stimulating activity by adenylate cyclase stimulation in LLC-PK1 target cells. Purified vasopressinase was a dimeric protein of molecular weight 330 kDa, which cleaved the synthetic substrate S-benzyl-L-cysteine-4-nitroanilide with a Km of 0.33 mM. Incubation of AVP (0.1 mM) with vasopressinase (0.66 milligrams) at 37 degrees C led to a parallel loss of both AVP immunoreactivity and biological activity. The rates of loss of V1 and V2 receptor mediated activities were not significantly different. We report the first direct comparison between the loss of V1 and V2 receptor mediated activities of vasopressinase degraded AVP. There was no significant retention of V1, relative to V2, receptor mediated activity. AVP degradation products are unlikely to be pathogenic in hypertensive pregnancy.

Different forms of 130 kD connective tissue protein are specific for boundaries in the nervous system and basement membrane of muscle cells in leech

The nervous system and muscle tissue of the leech express two different organ-specific forms of connective tissue protein. The nervous system-specific form appears in regional boundaries separating cell bodies, axonal tracts and areas of the neuropile during late embryogenesis. In contrast, the muscle-specific form appears earlier during development in the basement membrane of muscle cells. In extraction experiments both forms behave like extracellular matrix proteins and because of their molecular weight, are considered members of a group of cell type-specific 130 kD proteins (leech gp130s). However, the two forms differ in their posttranslational modification. As determined by Con A and lentil lectin affinity chromatography, only the nervous system-specific, but not the muscle-specific form, has fucosylated and high mannose N-linked carbohydrates. These differences in the developmental onset and glycosylation suggest that nervous system-specific and muscle-specific connective tissue proteins are regulated differently and participate in different molecular interactions.

Ricin and lentil lectin-affinity chromatography reveals oligosaccharide heterogeneity of thyrotropin secreted by 12 human pituitary tumors

Some patients with thyrotropin (TSH)-producing pituitary tumors are more hyperthyroid than others despite similar TSH levels in serum, suggesting that qualitatively different TSH molecules with differing bioactivities may be secreted by different tumors. We used ricin and lentil lectin-affinity chromatography to test whether the TSH oligosaccharides varied among 12 patients with TSH-producing tumors. We found that each tumor secreted heterogeneous isoforms of TSH that differed in their extents of exposed galactose (Gal) residues, and their degrees of sialylation and core fucosylation. These biochemical parameters also varied markedly for TSH secreted by different tumors. Isoforms appeared to reflect poor sialyltransferase activity in two tumors and efficient sialyltransferase in the remainder. TSH secreted by tumors was more fucosylated than TSH secreted by control euthyroid persons. There was an inverse relationship between the sialylation and fucosylation of tumor TSH. No simple relationship between TSH oligosaccharide structures and bioactivity was evident, although mixtures of isoforms having the least and most sialylated TSH seemed to be the most bioactive clinically. In three patients from whom serum and medium TSH were both available, TSH in serum was more sialylated than TSH secreted by the tumor in vitro, perhaps reflecting slow clearance of sialylated isoforms form the circulation. Core fucosylation of serum TSH was less than that of medium TSH. These data prove that human tumors secrete TSH with heterogeneous oligosaccharide structures.

Cross-Neutralizing Antibodies in Rabbits Immunized with HIV-1 gp160 Purified from Simian Cells Infected with a Recombinant Vaccinia Virus

A recombinant vaccinia virus in which the transcription of the human immunodeficiency virus type 1 (BRU isolate) env gene is driven by the 11K late vaccinia promoter yields about 10-fold higher amounts of gp160 env protein upon infection of monkey cells than does a recombinant in which gp160 is expressed using the 7.5K early-late promoter. The gp160 was purified from detergent lysates of infected cells by lentil lectin affinity chromatography followed by immunoaffinity chromatography, and was obtained in yields of 1-2 mg/10(9) cells of material estimated to be about 70% pure. Pairs of rabbits were immunized with purified gp160 using either one of five different adjuvants or an immunostimulating complex. In all cases a substantial humoral immune response was obtained after boosting, including an activity that neutralized the homologous (BRU) isolate of HIV-1. In some cases, this activity also neutralized two distantly related isolates, SF2 and MN.

Isolation and characterization of a thermolabile β-2 macroglycoprotein (‘thermolabile substance’ or ‘Hakata antigen’) detected by precipitating (auto) antibody in sera of patients with systemic lupus erythematosus

A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain.

Structure of a new nervous system glycoprotein, Nr-CAM, and its relationship to subgroups of neural cell adhesion molecules.

We have identified and characterized a new glycoprotein in the chicken nervous system using immunological and molecular biological methods and we have examined its tissue distribution. Analysis revealed that this protein is very similar in structure to the chicken neuron-glia cell adhesion molecule, Ng-CAM, and to mouse L1. cDNA clones encompassing the entire coding sequence of this Ng-CAM related molecule, called Nr-CAM, have been isolated and sequenced. A glycoprotein containing one major component of Mr 145,000 on SDS-PAGE was purified from brain by lentil lectin affinity chromatography and FPLC, and its amino-terminal sequence was identical to that predicted from the Nr-CAM cDNA. The complete cDNA sequence encodes six Ig-like domains, five fibronectin type III repeats, a predicted transmembrane domain, and a short cytoplasmic domain. On Northern blots, nucleic acid probes for Nr-CAM recognized one major RNA species of approximately 7 kb and much lesser amounts of larger RNAs. Most of the same probes hybridized to single bands on genomic Southern blots, suggesting that Nr-CAM is encoded by a single gene that may be alternatively processed to yield several mRNAs. In support of this notion, two Nr-CAM cDNA clones had a 57-bp sequence located between the second and third Ig-like domains that was not found in two other Nr-CAM cDNA clones, and two other clones were isolated that lacked the 279-bp segment encoding the fifth fibronectin-like type III repeat. Antibodies against the purified protein and synthetic peptides in Nr-CAM both recognized a predominant Mr 145,000 species and a much less prevalent species of Mr 170,000 in neural tissues. Levels of Nr-CAM expression increased in the brain until approximately embryonic day (E) 12, followed by slightly lower levels of expression at E18 and after hatching. Immunofluorescent staining with anti-Nr-CAM antibodies showed that most neurons in the retina were positive at E7 and the pattern of expression became restricted to several layers on neuronal cell bodies and fibers during development. Anti-Nr-CAM antibodies labeled specifically cell surfaces on neurons in culture. Although the structure of Nr-CAM resembles that of chicken Ng-CAM and mouse L1, the identity with each of these neural CAMs does not exceed 40%. The differences indicate that Nr-CAM is distinct from Ng-CAM and L1, but there are sufficient similarities to suggest that all of these molecules are members of a subgroup of neural CAMs in the N-CAM superfamily.(ABSTRACT TRUNCATED AT 400 WORDS)

Efficacy of SIV/DeltaB670 glycoprotein-enriched and glycoprotein-depleted subunit vaccines in protecting against infection and disease in rhesus monkeys

Immunization with an inactivated whole-virus vaccine is highly effective in preventing lentivirus infection. The viral protein(s) essential to the induction of protective responses, however, have not been identified. To define the role of virion components in the induction of protective immunity, we evaluated the efficacy of glycoprotein-enriched and glycoprotein-depleted simian immunodeficiency virus (SIV) subunit vaccines prepared by lentil-lectin affinity chromatography of gradient-purified virions using the immunization and challenge regimen previously found successful with an inactivated whole-virus vaccine. Infection was determined by successful recovery of virus, the induction of SIV-specific antibody responses, and infection of naive recipients by inoculation with lymph-node-derived lymphocytes from the vaccinates. Immunization with the glycoprotein-enriched preparation prevented infection in two out of four monkeys, whereas the glycoprotein-depleted vaccine failed to prevent infection in all four vaccinates tested. However, the glycoprotein-depleted vaccine appeared to moderate the progression of SIV-induced disease compared with non-immunized infected control monkeys inoculated with the same challenge dose. These data suggest that subunit vaccines containing sufficient quantities of viral glycoproteins can protect against SIV infection, whereas subunit vaccines composed predominantly of viral core proteins cannot. The development of effective vaccines against HIV infection should include studies on the optimum presentation of the viral envelope glycoproteins to produce long-term broadly protective immune responses.

Improvement of Gelatin Particle Agglutination Test for Detection of Anti‐HTLV‐I Antibody

Partial modifications of antigen components were made to improve the gelatin particle agglutination (PA) test for the detection of antibodies against human T cell leukemia virus type‐I. Envelope glycoproteins prepared by lentil lectin affinity chromatography were further added to the purified viral antigens to be coated on the gelatin particles. Comparative studies with a conventional PA test kit (Serodia ATLA) and indirect immunofluorescence assay showed that the specificity and sensitivity of the new PA test were increased and that abnormal agglutination such as the prozone phenomenon was abolished by this improvement.

Isolation and characterization of a membrane glycoprotein from human brain with sequence similarities to cell adhesion proteins from chicken and mouse

We previously described the production of monoclonal antibodies against a preparation of membrane glycoproteins from human brain [Berglund et al. (1987) J. Neurochem. 48, 809-815]. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135). Gp135 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 115 kDa following N-glycanase digestion. The glycoprotein is anchored in the membrane by a glycosylphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments. Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins.

Molecular cloning of a human basic fibroblast growth factor receptor cDNA and expression of a biologically active extracellular domain in a baculovirus system.

A cDNA clone encoding a human fibroblast growth factor (FGF) receptor was isolated from a hepatoma cell line cDNA library. The cDNA encodes a three immunoglobulinlike-domain FGF receptor that is similar to a human placental FGF receptor cDNA but lacks two amino acids. The variation observed at these two amino acids, also seen in the two immunoglobulinlike-domain FGF-receptors, can be explained by an alternate splicing mechanism. We have used a baculovirus expression system to produce high levels of a soluble, extracellular domain form of the FGF receptor (EC-FGF receptor). Spodoptera frugiperda (Sf9) insect cells infected with recombinant EC-FGF receptor viruses synthesized and secreted an EC-FGF receptor of apparent Mr = 58,000. The EC-FGF receptor purified from conditioned media of infected Sf9 cells by lentil lectin affinity chromatography was shown to bind basic FGF with high affinity (Kd = 1-5 nM), to inhibit the binding of radioiodinated basic FGF to its high affinity receptor and to inhibit endothelial cell proliferation. Furthermore, binding of basic FGF to the EC-FGF receptor was shown to be significantly enhanced by heparin. The availability of biologically active FGF receptors will allow an analysis of their interaction with members of the FGF family of proteins and viruses of the herpes family that have been shown to use the FGF receptor system for cell entry.

Inhibition of fibrin(ogen) binding to stimulated platelets by a monoclonal antibody specific for a conformational determinant of GPIIIa

We have purified the integrin GPIIb:IIIa from the membrane fraction of human blood platelets by lentil lectin affinity chromatography followed by gel filtration chromatography. With purified GPIIb:IIIa as an antigen, we have produced monoclonal antibody CS-1, which immunoblotting demonstrates to be specific for native GPIIIa; disulfide bond reduction of GPIIIa resulted in loss of immunoreactivity. Radiolabelled ligand binding studies revealed that CS-1 recognized approximately 55,000 sites per platelet and bound with a K d in the nanomolar range, independent of the state of platelet activation. Binding of CS-1 or its Fab fragment to ADP-and thrombin-stimulated gel filtered platelets caused a 2–3 fold inhibition of binding the soluble ligands fibrinogen and fibrin protofibrils. CS-1 also inhibited aggregation of ADP- and thrombin-stimulated platelets by 2- and 4-fold, respectively. Since CS-1 inhibits fibrin(ogen) interactions with GPIIb:IIIa, we propose that the conformationally dependent epitope on GPIIIa recognized by CS-1 constitutes a region of the receptor which is involved in fibrin(ogen) binding.

Membrane-bound and water-soluble nonclassical class I MHC antigens on rat placenta

We have used mouse monoclonal antibodies to different determinants on rat class I major histocompatibility complex (MHC) antigens in order to identify water-soluble and membrane-bound nonclassical (i. e., non-RT1.A) class I MHC antigens on the spongiotrophoblast and labyrinthine trophoblast of rat placenta. Initial immunohistological studies with monoclonal antibodies reacting with a determinant restricted to classical (RT1.A) rat class I antigens confirmed the presence of these antigens on spongiothrophoblast, but not on labyrinthine trophoblast. Staining with another monoclonal antibody, which reacts with both classical and at least some nonclassical rat class I antigens, gave strong staining of both the labyrinthine and spongiotrophoblast. To distinguish membrane-bound from water-soluble class I molecules, quantitative absorption analyses were carried out using both placental cell membranes and ultracentrifuged aqueous extracts of placenta. The aqueous placental extract had no absorptive capacity for the RT1.A-specific antibodies, but it had very strong absorptive capacity for the more broadly reactive antibody. This strongly suggests the presence of large quantities of a soluble nonclassical class I MHC antigen in rat placenta. The placental cell membranes had four to fivefold greater absorptive capacity for the broadly reactive antibody when compared to the antibodies to classical class I antigens, a result that was consistent with the presence of membrane-bound nonclassical class I antigens on rat placenta. The membrane-bound nonclassical class I antigen was purified from detergent extracts of DA rat placental membranes using monoclonal antibody affinity and lentil lectin affinity chromatography. The putative nonclassical class I antigen had a heavy chain of Mr 43,000, which is 2000 smaller than the classical (RT1.A) class I antigen. N-terminal amino acid sequence analysis demonstrated that the nonclassical placental antigen differed at three amino acid residues from the classical RT1.A class I molecule and also from the Q10-like class I molecule of the DA strain. It differed also from the pAR 1.5 cDNA sequence, the only full-length rat class I DNA sequence available so far.

Inhibin and activin-like activity in fluids from male and female gonads: different molecular weight forms and bioactivity/immunoactivity ratios

The existence of various molecular weight forms of inhibin in ovarian follicular fluid has been reported earlier, while there is no information on the form of inhibin in testicular tissue. Inhibin bioactivity was therefore estimated in eluates of slices, obtained after SDS-PAGE of rat testicular and ovarian homogenates, rat Sertoli cell-conditioned medium (rSCCM) and bovine ovarian follicular fluid (bFF). The only form of inhibin detected in testes from 22-day-old rats and in rSCCM was a 30 kDa protein. In rat ovarian extracts, larger forms of inhibin were also found as well as the predominant 30 kDa form. An activin-like activity was found in the 25 kDa SDS-PAGE eluates of both rSCCM and ovarian homogenates, which caused a dose-dependent increase of FSH release from cultured pituitary cells. Activin-like activity and several forms of inhibin were found in bFF after SDS-PAGE. After purification of inhibin from bFF using dye affinity, anion-exchange, lentil lectin affinity chromatography and a subsequent reversed phase chromatography step, two pools of inhibin activity were obtained. These were separated by SDS-PAGE revealing 30 and 58 kDa inhibin forms. The immunoactivity of these forms of inhibin was then estimated using antibodies against the 22 N-terminal amino acids of the alpha subunit of 30 kDa bovine inhibin. It appeared that the two molecular weight forms of inhibin had bioactivity/immunoactivity ratios which differed more than five-fold. This indicates that results of radioimmunoassays of inhibin of ovarian origin, using peptide antisera, should be interpreted with caution.

Molecular Cloning and Expression of cDNA Encoding the 53,000-Dalton Glycoprotein of Rabbit Skeletal Muscle Sarcoplasmic Reticulum

The 53-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum was purified by lentil lectin affinity chromatography and preparative polyacrylamide gel electrophoresis and partially sequenced. Polyclonal and monoclonal antibodies were raised against the 53-kDa glycoprotein and found to cross-react with the 160-kDa glycoprotein. A combination of antibody and synthetic oligonucleotide screening was used to isolate a cDNA encoding the 53-kDa glycoprotein of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. The cDNA encodes a protein of 453 amino acids with Mr of 52,421 and a 19-residue amino-terminal signal sequence. The deduced sequence contains two potential glycosylation sites and is largely hydrophilic. The presence of a glycine-rich sequence in the glycoprotein with homology to mononucleotide binding domains supports earlier observations that the glycoprotein binds ATP with high affinity. Although two sequences appear to be hydrophobic on a hydropathy plot, they are not sufficiently long nor sufficiently hydrophobic to qualify unambiguously as transmembrane sequences. The glycoprotein, like calsequestrin, was shown to be inaccessible to trypsin in intact sarcoplasmic reticulum. It can be eluted from the sarcoplasmic reticulum by extraction with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid under hypotonic conditions. Thus, the glycoprotein appears to be localized entirely in the lumen of the sarcoplasmic reticulum and to be associated with the inner membrane surface through Ca2+-dependent mechanisms. Cotransfection of COS-1 cells with cDNAs encoding the glycoprotein and the Ca2+-ATPase led to expression of both proteins with a common localization in the microsomal fraction. The Ca2+ pumping activity of the microsomes isolated from transfected cells was unaltered by the presence of the glycoprotein. Thus the glycoprotein does not appear to modulate Ca2+-ATPase function.