Lactoperoxidase-catalyzed Radioiodination Research Articles

Host cellular annexin II is associated with cytomegalovirus particles isolated from cultured human fibroblasts.

A significant amount of host cellular annexin II was found to be associated with human cytomegalovirus isolated from cultured human fibroblasts (approximately 1,160 molecules per virion). This composition was established by four different analytical approaches that included (i) Western blot (immunoblot) analysis of gradient-purified virions with a monoclonal antibody specific for annexin II, (ii) peptide mapping and sequence analysis of virus-associated proteins and proteins dissociated from virus following EDTA treatment, (iii) electron microscopic immunocytochemistry of gradient-purified virions, and (iv) labeling of virus-associated proteins by lactoperoxidase-catalyzed radioiodination. These results indicated that annexin II was primarily localized to the viral surface, where it bound in a divalent cation-dependent manner. In functional experiments, a rabbit antiserum raised against annexin II inhibited cytomegalovirus plaque formation in human foreskin fibroblast monolayers in a concentration-dependent manner. Cumulatively, these studies demonstrate an association of host annexin II with cytomegalovirus particles and provide evidence for the involvement of this cellular protein in virus infectivity.

Determination of the sidedness of the carboxy-terminus of the Na +/K +-ATPase α-subunit using lactoperoxidase iodination

The orientation of the carboxy-terminal pair of tyrosines of the Na +/K +-ATPase α-subunit with respect to the plane of the plasma membrane was determined. The approach was based on lactoperoxidase-catalysed radioiodination of the tyrosine residues accessible on the surface of the enzyme molecule in intact cells of a pig kidney embryonic cell line and those accessible in a broken plasma membrane fraction and in isolated membrane-bound Na +/K +-ATPase. The labeled α-subunit was isolated by SDS gel electrophoresis followed by electroblotting. Then the COOH-terminal amino acids were hydrolyzed by carboxypeptidases B and Y. Radioactivity and quantitative analysis of the protein and released amino acids showed that the COOH-terminal tyrosine residues of the α-subunit were only accessible to modification only when lactoperoxidase had access to the inner side of the plasma membrane. Therefore, the COOH-terminus of the Na +/K +-ATPase α-subunit is located on the cytoplasmic surface of the pump molecule and its polypeptide chain must have an even number of transmembrane segments.

Evidence for the selective association of a subpopulation of GPIIb-IIIa with the actin cytoskeletons of thrombin-activated platelets.

Activation of blood platelets triggers a series of responses leading to the formation and retraction of blood clots. Among these responses is the establishment of integrin-mediated transmembrane connections between extracellular matrix components and the actin cytoskeleton of the platelet. Here we report that a specific subpopulation of the major platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) (also referred to as alpha IIb beta 3 integrin), becomes incorporated into the detergent-insoluble actin cytoskeleton of platelets during the platelet activation response. The cytoskeletal association of GPIIb-IIIa is independent of platelet aggregation and fibrin sedimentation and is sensitive to cytochalasin D treatment. As determined by Western immunoblot analysis, approximately 22% of the total cellular GPIIb-IIIa becomes associated with the actin cytoskeleton upon thrombin activation in a manner that is independent of the detection of talin, alpha-actinin, or vinculin in the complex. We found that the cytoskeleton-associated GPIIb-IIIa is derived from an intracellular source since it is not available for lactoperoxidase-catalyzed radioiodination before platelet activation. Two intracellular sources of GPIIb-IIIa are present in resting platelets: GPIIb-IIIa associated with the alpha-granule secretory compartment as well as surface-inaccessible domains of the surface-connected canalicular system. Interestingly, alpha-granule secretion, which occurs in thrombin-activated platelets and results in the translocation of intracellular GPIIb-IIIa to the plasma membrane, appears to be required for the cytoskeleton incorporation of GPIIb-IIIa that we observe. Collectively, our data provide evidence that a subpopulation of GPIIb-IIIa derived from an intracellular source is selectively linked to the actin cytoskeleton of platelets upon thrombin activation in the absence of platelet aggregation.

WGA-binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells.

Expression of apical cell surface proteins and glycoproteins was examined in polarized primary cultures of mouse uterine epithelial cells (UEC). Lectin-gold cytochemistry revealed that wheat germ agglutinin (WGA) bound specifically to the components of the apical glycocalyx as well as intracellular vesicles. Double labeling with the pH sensitive dye 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP) demonstrated the acidic nature of the WGA-staining intracellular vesicles. The enzymatic and chemical sensitivities of the WGA binding sites on the apical cell surface were monitored both by WGA-gold staining as well as by 125I-WGA binding assays. In thin sections, a large fraction of these sites were removed by pronase; however, application of a wide variety of proteases, glycosidases, or chemical treatments to the apical surface of intact UEC failed to reduce WGA binding. In no case did treatments designed to remove sialic acids reduce 125I-WGA binding more than 12%. In contrast, endo-beta-galactosidase as well as a combination of beta-galactosidase with beta-hexosaminidase succeeded in removing 28% and 77% of these sites, respectively. These studies suggested that the majority of the apically disposed WGA binding sites involved N-acetylglucosamine residues rather than sialic acids and included lactosaminoglycans. Many of the proteins detected at the apical cell surface by lactoperoxidase-catalyzed radioiodination were WGA-binding glycoproteins. A major class of these glycoproteins displayed Mr > 200 kDa by SDS-PAGE and was heavily labeled metabolically by 3H-glucosamine or by vectorial labeling at the apical cell surface with galactosyl transferase and UDP-3H-galactose. Analyses of the 3H-labeled oligosaccharides labeled by either procedure indicated that a large fraction of the apically disposed WGA-binding oligosaccharides consisted of neutral, O-linked mucin-type structures with median MW of approximately 1,500. Oligosaccharides in this fraction were partially (15%) sensitive to endo-beta-galactosidase digestion and bound to Datura stramonium agglutinin (68%), demonstrating the presence of lactosaminoglycan sequences. UEC were an extremely effective barrier to attachment or invasion by either a highly invasive melanoma cell line, B16-BL6, or implantation-competent mouse blastocysts. In contrast, neither uterine stromal cells nor a non-polarizing UEC cell line, RL95, prevented B16-BL6 attachment.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of Plasma Membrane and Cell Surface Phospholipids and Gangliosides of Chick Embryo Neurons Grown in Primary Cultures: Developmental Studies

Plasma membranes isolated from chick embryo neurons after 2 days (division phase) and 5 days (maturation phase) in culture showed no differences in the phospholipid (PL) pattern or in the plasma membrane average fluidity. Significant ganglioside (Ggl) variations were only found in GM1 and GD3 which represent 35% of the total plasma membrane N-acetylneuraminic acid (NeuNac) content (1.8-fold GM1 NeuNac increase, 1.4-fold GD3 NeuNac reduction). Lactoperoxidase-catalyzed radioiodination (125I) under saturating conditions indicated that the hydrophobic core of cell surface Ggl was more exposed to the hydrophilic extracellular environment than that of PL. Nevertheless, when interneuronal contacts start to form, the 125I-labelling of both PL and Ggl dropped progressively throughout the maturation phase. Further analysis revealed that (1) plasma membrane GD3 was not accessible to the enzymatic labelling, and (2) surface phosphatidylethanolamine (PE) and GT1b became more accessible to the external medium during development, whereas the opposite was found for phosphatidylcholine (PC) and GM1. The overall profile of radioiodinated PL and Ggl was unaltered when poly-L-lysine was substituted for polyethyleneimine as culture substratum, except for an increase in GT1b labelling 24 h after plating. These results suggest the existence of a 'buffering' mechanism protecting the neuronal plasma membrane during cellular growth from marked changes in fluidity, polar lipid composition and relative localization of polar lipids with regard to the extracellular environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the pattern of cell surface proteins during transformation of bloodstream forms of Trypanosoma brucei in vitro

Transformation of coated bloodstream forms of Trypunosomu hrircei t o procyclic forms in vitro occurs over a period of 4-5 days 111, during which time the parasite changes its morphology and adapts its metabolism to life within the insect mid-gut [2,3]. One of the best characterized events during transformation is the loss o f the variable surface glycoprotein (VSG) that constitutes the dense surface coat of bloodstream forms of the parasite [4]. However, very little is known about the membrane proteins that are located beneath the VSG; for example, it is not clear whether these other 'masked' antigens are also shed during transformation or whether they remain on the surface of the procyclic forms. In this present report, we have used lactoperoxidasecatalysed radioiodination of the cell surface to label proteins that are located underneath the VSG of bloodstream forms and to follow the change in composition of the surface antigen population at various stages during transit through the transformation pathway.

Topography of a vacuolar-type H+-translocating ATPase: chromaffin-granule membrane ATPase I

Proteins exposed on the cytoplasmic face of isolated chromaffin granules were labelled by lactoperoxidase-catalysed radioiodination and by non-enzymic biotinylation. Granule membranes were then prepared, and the H+-translocating ATPase isolated by fractionation with Triton X-114. The labelling of individual ATPase subunits was assessed by polyacrylamide-gel electrophoresis, followed by autoradiography or by blotting and decoration with 125I-labelled streptavidin. Subunits of 72, 57 and kDa were strongly labelled, and could be removed from the membrane at pH 11: they are therefore extrinsic proteins. The 120 kDa subunit was also labelled, but it was not solubilized at pH 11. Photolabelling with a hydrophobic probe indicated that this subunit penetrates the bilayer, and enzymic degradation studies showed the presence of N-linked oligosaccharides; this subunit therefore spans the chromaffin-granule membrane. Labelling of the 17 kDa subunit occurred predominantly on the extracytoplasmic (matrix) face of the granule membrane. These results are consistent with this V-type ATPase having a structure that is generally similar to that of mitochondrial (F-type) ATPases, although the attachment of the 120 kDa subunit may be asymmetrical.

Identification of Dictyostelium discoideum plasma membrane proteins by cell surface labeling and quantitative two-dimensional gel electrophoresis

Plasma membrane proteins of the cellular slime mold Dictyostelium discoideum were characterized by two-dimensional polyacrylamide gel electrophoresis using a variety of labeling techniques and a microcomputer-based videodensitometer. Algorithms for the determination of molecular weights and isoelectric points were developed to aid in the comparison of polypeptides from different autoradiographs, Coomassie blue-stained gels, and Western blots. Cell homogenates were compared to plasma membranes isolated by a silica density perturbation technique and to cytoskeletons obtained by nonionic detergent extraction. Plasma membrane proteins were distinguished from subcellular contaminants by lactoperoxidase-catalyzed radioiodination, by selective labeling with N-hydroxysuccinimidyl-2-iminobiotin, and by quantitatively determining the enrichments of individual polypeptides from gels of plasma membrane proteins relative to their counterparts in gels of total cell lysate proteins. In contrast to defining plasma membrane purity by measuring a representative marker enzyme activity, the quantitative two-dimensional gel analysis strategy presented allowed for a rigorous evaluation of the enrichments of all detectable polypeptides in the subcellular fraction. Quantitative two-dimensional gel analysis avoided problems encountered with marker enzyme activation or inhibition during subcellular fractionation as enrichments were based solely on polypeptide amounts. It was also capable of identifying a wider spectrum of plasma membrane proteins than any of the labeling techniques employed in this study. A high resolution two-dimensional gel catalog was generated containing information about plasma membrane protein orientation in the bilayer, association with the cytoskeleton, phosphoryalation state, glycosylation state, copy number, isoelectric point, and molecular weight.

Insulin-induced phosphorylation of the beta-4 integrin subunit expressed on murine metastatic carcinoma cells.

A tumor surface protein (TSP-180) that is highly expressed on highly malignant metastatic cells has been identified on murine lung carcinomas. On SDS-PAGE under reducing conditions, TSP-180 shows a complex banding pattern corresponding to 204, 183, 150, 135, and 116 kDa. All bands of the TSP-180 complex are glycosylated and are labeled by lactoperoxidase-catalyzed radioiodination of viable cells. The mouse TSP-180 complex described here is homologous to the human integrin alpha 6 beta 4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the beta 4 subunit of the integrin complex. It has been shown recently that monoclonal antibody to TSP-180 (MoAb 135-13C) stimulates cell growth in vitro and induces phosphorylation of the 204-kDa protein. We now report that insulin increases the phosphorylation of the 204-kDa protein 30-fold in intact carcinoma cells and epidermal growth factor (EGF) causes a threefold increase. Insulin-like growth factor (IGF-I) and platelet-derived growth factor have no effect. The effect of insulin and of IGF-I on phosphorylation of their own receptors was studied using solubilized cell membranes. Insulin and IGF-I each induced a fivefold increase in the phosphorylation of their respective receptor beta subunits. In order to test if phosphorylation of the 204-kDa protein was induced by direct binding of growth factors to TSP-180 and to identify growth factor receptors on line 1 cells, affinity cross-linking studies were performed. Affinity labeling of receptors demonstrated that insulin and IGF-I both bind to a 135-kDa protein that corresponds to the insulin and IGF-I receptor alpha subunits. Affinity labeling of EGF receptors failed to demonstrate EGF receptor molecules (175-kDa protein) on line 1 cells. Further investigations by using a different approach confirmed the very low amount of EGF receptors on line 1 cells. Direct phosphoamino acid analysis of the 204-kDa protein purified from insulin-stimulated cells demonstrated that this beta 4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that beta 4 integrin molecule is a target for phosphorylation through an indirect receptor-mediated mechanism.

Stage-specific proteins of Plasmodium berghei-infected red blood cells detected by antibodies of immune mouse serum.

The asynchronously developing malaria parasite Plasmodium berghei was synchronized using in vitro cultivation techniques (Mons et al. 1985). After the infection of naive mice, preparations of parasitized erythrocytes with a high level of synchronism could be obtained. Immunofluorescence and immunoblotting techniques using serum from immunized mice were applied to determine stage-specific immunogenic molecules in the parasitized erythrocyte preparations. These techniques allowed the detection of not only parasite-derived but also altered-self molecules. Membrane fluorescence of infected erythrocytes was detected only in preparations containing late trophozoites and schizonts. The appearance of this fluorescence pattern coincided with the presence of immunogenic polypeptides of mol. wt. greater than 200 kD, 86 kD, and 56 kD especially, among some other polypeptides. Preliminary experiments using lactoperoxidase-catalyzed radioiodination suggested that the greater than 200 kD and 56 kD molecules were present at the erythrocyte surface. One molecule with mol. wt. 153 kD was associated with the presence of ring-infected erythrocytes. However, membrane fluorescence of ring-stage-infected erythrocytes was not found. Noninfected erythrocytes sometimes showed membrane fluorescence.

A Japanese Family with Two Sisters Apparently Homozygous for M^k

Two Japanese sisters with consanguineous parents have M —N — En(a —) Wr(a — b — ) S — s — U— red cells and are therefore apparently homozygous for M^k; the third reported family with members of this genotype. The serum of the proposita (ORCMK) contained anti-En^aTS, anti-En^a FR and possibly anti-Wr^b, whereas the serum of her M^kM^k sister contained no atypical antibodies. Total absence of sialoglycoproteins α and δ from red cell membranes of an M^k homozygote was demonstrated by lactoperoxidase-catalysed radioiodination of accessible tyrosine residues with subsequent SDS polyacrylamide gel electrophoresis and autoradiography, and by use of a monoclonal antibody directed at the cytoplasmic portion of α-sialoglycoprotein.

Isolation of plasma membranes from neurons grown in primary culture

Plasma membranes from chick embryo neuronal primary cultures were isolated after subjecting 5-day-old cells, previously surface labeled with either lactoperoxidase-catalyzed radioiodination or galactose oxidase/NaB 3H 4, to a freeze-thaw cycle. The cellular material adhering to the culture substratum was washed, and the “wash” fractions were pooled and centrifuged at 37,000 g. The resulting pellet was resuspended in 3 ml of buffer, layered on 33 ml of 33% sucrose, and centrifuged at 105,000 g. Radioactivity was recovered at the top of the gradient. Sedimentation of these fractions and biochemical studies revealed that the pellet was 20-and 12-fold enriched in (Na +,K +)-adenosinetriphosphatase and 5′-nucleotidase, respectively. The preparation was devoid of inner mitochondrial (succinate dehydrogenase), outer mitochondrial (monoamine oxidase), endoplasmic reticulum (glucose-6-phosphatase), and Golgi (UDP galactose: N-acetylglucosamine galactosyltransferase) enzymatic markers. Ultrastructural studies showed that the membrane preparation was homogeneous and lacked mitochondria endoplasmic reticulum and lysosomes. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of 11 protein components with molecular masses ranging from 120 to 300 kDa. This method for the isolation of plasma membranes probably depends on the capacity of the cellular material to adhere to the culture substratum and to entrap intracellular organelles during the freeze-thaw cycle. The membrane preparation seems suitable for studying the function of high-molecular-weight protein components of neuronal plasma membranes.

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane.

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.

Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination.

Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. Several control experiments indicated that the labeled proteins are integral membrane proteins and that label is limited to the plasma membrane. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger.

Resting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins.

Recent evidence suggests the presence in resting platelets of centrally located compartments of glycoprotein (GP) IIb-IIIa. We have employed an experimental procedure which dissociates and antigenically denatures the surface compartment of GP IIb-IIIa and allows internal compartments of GP IIb-IIIa to be studied immunochemically and functionally in intact platelets. When gel-filtered platelets are incubated with 0.25 mM EGTA at 37 degrees C for 30 min, and then supplemented for 30 min with 5 mM calcium, they lose their ability to bind GP IIb-IIIa complex-specific monoclonal antibody Fab fragments. However, when such platelets are subsequently stimulated with thrombin, GP IIb-IIIa-specific Fabs are again able to bind in large amounts to the platelet surface, in concert with the appearance of substantial amounts of receptors for fibrinogen and fibronectin. In immunoprecipitation experiments, we have found that this thrombin-displayed pool of GP IIb-IIIa originates from a pool that is not labeled by lactoperoxidase-catalyzed radioiodination of intact resting platelets. In immunofluorescence experiments, we have found that EGTA-incubated platelets contain a large sequestered internal pool of GP IIb-IIIa which upon thrombin stimulation is translocated to the platelet surface. Additional experiments suggest that this centrally located compartment may be surface connected in resting platelets and that it is accessible to some extracellular proteins, but not others.

Cross-linking of insulin receptors to MHC antigens in human B lymphocytes: evidence for selective molecular interactions.

Molecular interactions between insulin receptors and MHC antigens were investigated in human B cells. Two B lymphoblastoid cell lines, IM-9 and 526, chosen for their high insulin binding capacity, were found to express 15,000 and 25,000 insulin receptors per cell, respectively. Insulin receptors were labeled with a 125I-photoreactive insulin analogue, and all other surface proteins by lactoperoxidase-catalyzed radioiodination. Neighbor proteins were cross-linked with a cleavable homobifunctional reagent dithio-bis-(succinimidyl propionate) (DSP) and solubilized before immunoprecipitation by anti-HLA monoclonal antibodies. Gel analysis of the precipitated proteins showed that 90% of insulin receptors precipitable by anti-insulin receptor antibodies were precipitated by anti-class I antibodies (anti-heavy chain and anti-beta 2-microglobulin) after cross-linking with 2 mM DSP. In neither IM-9- nor 526 cells could HLA antigens be precipitated by anti-insulin receptor antibodies, suggesting that the concentration of class I antigens largely exceeds the concentration of insulin receptors at the cell surface. In 526 lymphocytes, class I MHC antigens were also found to adjoin class II antigens, since both molecules could be coprecipitated with anti-HLA A, B, C and with anti-HLA-DR antibodies after chemical cross-linking. Down-regulation of insulin receptors by chronic exposure of IM-9 cells to insulin did not affect the amount of MHC molecules present on the cell surface, and conversely, class I MHC molecules were internalized in 526 cells irrespective of the presence of insulin. These results thus show that insulin receptors and MHC antigens form multimolecular complexes in the plasma membrane of cultured human B cells. These interactions, which do not appear to influence the regulation of these proteins on the cell surface, may be involved in the mechanism of hormone signaling.

Characterization and Purification of Iodinated Bovine Thyrotropin by High Performance Liquid Chromatography*

Reversed-phase (RP) HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography have been used to study the incorporation of 125I into bovine (b) TSH by the lactoperoxidase-catalyzed radioiodination procedure. Two preparations of [125I]bTSH were studied, being freshly iodinated bTSH and tracer purified from this preparation by the method of receptor adsorption. It is demonstrated with these methods that both the alpha- and beta-subunits of bTSH are labeled with 125I, and that the tracer purified by receptor adsorption retains this incorporation pattern. However, the implied theoretical specific activity of (at least) 2 I atoms per TSH molecule (or approximately 140 muCi/micrograms) suggested by this result was not achieved, with observed tracer specific activity being 30-60 muCi/micrograms, indicating that hormone molecules with varying extents of labeling must exist. Evidence to support this was provided by comparison of the MIT/DIT ratios for the 2 tracer preparations. Receptor adsorption decreased the MIT/DIT ratio from 75:25 in the freshly iodinated bTSH to 93:7, indicating the selection of particular iodinated species. Tryptic mapping by RP-HPLC was used to study both tracer preparations, and it is shown that at least 14 iodine-containing tryptic peptides may be resolved for each preparation, which is greater than the theoretical maximum of 13 peptides if every tyrosine was labeled and tryptic cleavage occurred at all possible lysine and arginine residues. Tracer heterogeneity was also studied by purification using RP-HPLC. Selection of peak fractions demonstrated that intact [125I]bTSH may be recovered from RP-HPLC which in TSH radioreceptor assay exhibit increased assay sensitivity, increased saturable binding, and decreased nonsaturable binding.

Membrane orientation and antigenic peptides of an immunoprotective 74 kDa Plasmodium knowlesi glycoprotein

Vaccination trails have shown that a purified, 74 kDa glycoprotein, GP74, isolated from the host cell membrane of Plasmodium knowlesi-infected rhesus erythrocytes, can provide protective immunity against P. knowlesi malaria. We have extended this work by a tryptic peptide analysis of the disposition of GP74 in the host cell membrane. Of the 18 peptides characterized by high-performance liquid chromatography only four were accessible to lactoperoxidase-catalyzed radioiodination of non-leaky, schizont-infected host cells from the extracellular space. Metabolic labeling with radioactive glucosamine indicates that two of the surface exposed peptides are glycopeptides, and one of these, peptide 12, appears to carry a dominant antigenic site, according to its reactivity with immunoglobulin from sera of monkeys protected against P. knowlesis malaria.

Insulin-receptor biosynthesis in cultured lymphocytes from an insulin-resistant patient (Rabson-Mendenhall syndrome). Evidence for defect before insertion of receptor into plasma membrane.

In some patients with genetic forms of extreme insulin resistance, there is a marked decrease in the number of insulin receptors on the cell surface. We studied an insulin-resistant patient (RM-1) with the Rabson-Mendenhall syndrome. As judged by insulin-binding studies, Epstein-Barr virus-transformed lymphocytes from patient RM-1 exhibit a 90% decrease in the number of insulin receptors. Similarly, with either lactoperoxidase-catalyzed radioiodination of cell surface receptors or biosynthetic labeling of receptors with [3H]glucosamine, we demonstrated an 80-90% decrease in the number of insulin receptors in cells from patient RM-1. Previous studies have shown that the marked decrease in insulin receptors of the Rabson-Mendenhall patient is not due to accelerated receptor degradation. Therefore, we investigated the possibility that a slow rate of receptor biosynthesis might account for the 90% reduction of insulin receptors in cells from this patient. Insulin-receptor biosynthesis proceeds through a glycoprotein precursor with an apparent Mr of 190,000. It undergoes endopeptidase cleavage and further posttranslational processing to yield the mature 135,000- and 95,000-Mr glycoprotein subunits. We studied the biosynthesis of the 190,000-Mr precursor and mature receptor subunits by a pulse-chase labeling technique with [2-3H]mannose. The time course of insulin-receptor biosynthesis appeared normal in cells from patient RM-1, despite a 10-fold reduction in the number of receptors on the cell surface. Parallel pulse-chase experiments with either [2-3H]mannose or [35S]methionine yielded the same results regardless of which label was employed. Thus, the receptor precursor in the Rabson-Mendenhall patient seems to be synthesized at a normal rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between diphtheria toxin entry and anion transport in Vero cells. II. Inhibition of anion antiport by diphtheria toxin.

When cells with surface-bound diphtheria toxin were exposed to pH 4.5, the toxin became shielded against lactoperoxidase-catalyzed radioiodination, indicating that the toxin was inserted into the membrane. Cells thus treated had strongly reduced ability to take up 36Cl-, 35SO4(2-), and [14C]SCN-. The reduction of chloride uptake was strongest at neutral pH, whereas that of sulfate was strongest at acidic pH. Lineweaver-Burk plots indicated that the toxin treatment reduced the Jmax but not the Km for the anions. The toxin also inhibited the NaCl-stimulated efflux of 35SO4(2-), indicating that the toxin inhibits the antiporter. No inhibition was found when toxin-treated cells were not exposed to low pH, whereas exposure to pH 4.5 for 20 s induced close to maximal inhibition. Half-maximal inhibition was obtained after exposure to pH 5.4. The concentration of diphtheria toxin required to obtain maximal inhibition (0.3 micrograms/ml) was sufficient to ensure close to maximal toxin binding to the cells. Even in ATP-depleted cells and in the absence of permeant anions, low pH induced inhibition of anion antiport in toxin-treated Vero cells. There was no measurable inhibition of anion antiport in cells with little or no ability to bind the toxin.