Differences In Sialic Acid Content Research Articles

Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations.

Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purified PMSG. To gain insight into the structural and functional characteristics of reCG, a bioinformatics analysis was performed. An exhaustive characterization comprising the determination of the purity degree, aggregates and nicked forms through SDS-PAGE, RP-HPLC and SEC-HPLC was performed. Higher order structures were studied by fluorescence spectroscopy and SEC-HPLC. Isoforms profile were analyzed by isoelectric focusing. Glycosylation analysis was performed through pulsed amperometric detection and PNGase F treatment following SDS-PAGE and weak anion exchange-HPLC. Slight differences between the purified recombinant hormones were found. However, recombinant molecules and PMSG exhibited variations in the glycosylation pattern. In fact, differences in sialic acid content between two commercial preparations of PMSG were also obtained, which could lead to differences in their biological potency. These results show the importance of having a standardized production process, as occurs in a recombinant protein bioprocess. Besides, our results reflect the importance of the glycan moieties on eCG conformation and hence in its biological activity, preventing denaturing processes such as aggregation.

Comparison of clot lysis activity and biochemical properties of originator tenecteplase (Metalyse®) with those of an alleged biosimilar

The bioengineered tissue plasminogen activator tenecteplase is an important treatment modality of acute myocardial infarction recommended by international guidelines. Following introduction of originator tenecteplase (brand names Metalyse® and TNKase®), a “biosimilar” tenecteplase became available for commercial use in India under the brand name Elaxim® in the absence of Indian biosimilar guidelines which came into force from September 15th, 2012. Based on a report of biochemical and fibrinolytical differences between Metalyse and Elaxim, we have systematically compared them in a range of routine quality testing assays. As compared to Metalyse, Elaxim exhibited less clot lysis activity and contained less of the two-chain form of tenecteplase. Even upon full in vitro conversion to the two-chain form Elaxim exhibited less clot lysis activity. This was linked to differences in sialic acid content and glycosylation pattern with Elaxim exhibiting less bi- and more tetra-antennary glycosylation, leading to a different charge heterogeneity profile. Regarding purity, Elaxim contained more tenecteplase aggregates and, in contrast to Metalyse, considerable amounts of Chinese hamster ovary cell protein. Taken together these data demonstrate that Metalyse and Elaxim differ considerably in clot lysis activity and biochemical properties. These data question whether Elaxim indeed can be considered a “biosimilar” of Metalyse, i.e., whether and to which extent the clinical efficacy and safety properties of Metalyse can be extrapolated to Elaxim in the absence of comparative clinical data.

Sialic acid content of tissue-specific gp96 and its potential role in modulating gp96-macrophage interactions

Cancer-derived heat shock protein gp96 induces a tumor-specific protective immune response primarily mediated by cytotoxic T lymphocytes (CTL) directed toward cancer-associated peptides associated with gp96. Both innate and adaptive immune responses have been demonstrated using a cell culture-based signaling mechanism. When used as an extraneous vaccine, one critical interaction which must occur for an immune response to be generated is the interaction between gp96 and the antigen presenting cell (APC) surface receptors (CD91, SR-A, TLR-2, and TLR-4). Our previous study concluded that gp96 purified from various rat and human prostate cancers is differentially glycosylated based on the amino and neutral monosaccharide content, and it was postulated that the monosaccharides may play a role in its biological activity. In this report, we report differences in the cancer-specific sialic acid content of gp96 purified from normal rat prostate compared to two rat prostate cancers, MAT-LyLu and Dunning G, as well as between two human prostate cancer cells, LnCaP and DU145. We also examined the modulatory effect of sialic acid residues on the binding of gp96 to APCs and its subsequent activation. Our results supported the contention that significant differences in the sialic acid content exist between Dunning G, MAT-LyLu, and normal rat prostate gp96, which affected its binding and biochemical activity to APCs. We therefore postulate that varied glycans of HPS96, a hitherto neglected structural component, may play a pivotal role in its anticancer activity. We suggest that construction of the glycan tree is a key to identification of the necessary and sufficient elements in the structure-function activity of HSP96.

Characterizing an engineered chimeric anti‐ricin A chain antibody

Ricin toxin, derived from the castor bean, is a potent toxin that inhibits protein synthesis. It has been considered a possible agent of bioterrorism for many years. In experimental studies antibodies have been shown to be an effective treatment against ricin poisoning. Maddaloni et al, described a murine monoclonal anti‐ricin A chain antibody, mRAC 18, that was shown to neutralize in vitro and protect in vivo. This antibody has been found effective, even when administered 12 hours post exposure. The use of monoclonal antibodies in therapy has steadily increased since their development. A limitation to the utility of mAb therapy is their immunogenicity. In an effort to create an antibody for use in post‐exposure human therapy of ricin intoxication, we have created a chimeric ricin A chain antibody. We combined the antigen‐specific variable region genes of the mouse antibody RAC 18 to human gamma‐1 and kappa constant region genes. The resultant chimeric antibody retained the same specificity for ricin as the parental antibody, offered passive protection in vivo, and had enhanced in vitro neutralizing ability. A lectin binding assay showed the chimeric antibody had significantly less sialic acid than the parental antibody. We conclude that differences in sialic acid content may be responsible for the increased in vitro neutralizing ability, and that this effect is independent of Fc receptor binding.

Carbohydrate complexity and proportions of serum FSH isoforms in the male: Lectin-based studies

FSH is synthesized and secreted in multiple molecular forms with different oligosaccharide structures which are needed for full expression of biological activity. GnRH and sex steroids modulate oligosaccharide structure and composition. In the present study we have assessed the carbohydrate complexity and proportion of circulating FSH isoforms during puberty, aging and after androgen administration to pubertal anorchid boys. Preparative isoelectrofocusing and lectin chromatography were used to isolate FSH isoforms on the basis of charge and internal carbohydrate complexity. Differences in sialic acid content and a progressive increase of isoforms bearing highly branched oligosaccharides were found during puberty. Less acidic, more bioactive FSH isoforms, secreted at mid-puberty may modulate important maturational events in the Sertoli cell population. Androgen administration to pubertal anorchid boys favoured the secretion of this type of isoforms. In adult men, the predominance of FSH isoforms bearing complex type oligosaccharides remained unchanged until very advanced age. These results show that the predominance of FSH isoforms bearing fully processed oligosaccharides in circulation may contribute to the development and maintenance of seminiferous epithelium function in men.

Human FSH isoforms: carbohydrate complexity as determinant of in-vitro bioactivity

Differences in sialic acid content of the hormone have been considered the main determinant of FSH polymorphism. The aim of the present study was to investigate the effect of variations in the oligosaccharide structure of the intrapituitary human FSH (hFSH) glycosylation variants on their intrinsic biological activity. FSH charge isoforms obtained after chromatofocusing were further separated by lectin affinity chromatography [Concanavalin A (ConA), Wheat germ agglutinin (WGA), Lentil lectin (LcH)]. Isolated isoforms were separately tested for in-vitro bioactivity in a rat Sertoli cell aromatization bioassay. Our results show that: (1) FSH microheterogeneity is due not only to variations in the sialic acid content of the hormone but also to differences in the internal structure of the carbohydrate chains, and (2) variations in the sialic acid content as well as differences in the complexity of the glycans determine the full biological expression of FSH glycosylation variants.

Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells.

We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection.

Tamm-Horsfall glycoprotein: role in inhibition and promotion of renal calcium oxalate stone formation studied with Fourier-transform infrared spectroscopy

Tamm-Horsfall glycoprotein (THP) from healthy probands inhibits the precipitation of calcium oxalate, whereas THP from individuals who repeatedly develop calcium oxalate stones has no effect or even promotes precipitation. Using Fourier-transform infrared spectroscopy, we found a structural differentiation between these functionally different THPs: a decisive difference in sialic acid content. Quantitative analysis for sialic acid showed the same results. THP from healthy probands had a high sialic acid content (51 +/- 9 g/kg), whereas THP from recurrent stone formers had a decreased sialic acid content (21 +/- 4 g/kg). This explains the dual role of THP in the precipitation of calcium oxalate and the formation of renal stones and shows the importance of glycosylation in the function of this glycoprotein.

Improved fractionation of sialylated glycopeptides by pellicular anion-exchange chromatography

The glycoprotein bovine fetuin was treated with trypsin and the Asn-81 tryptic glycopeptide was purified (90% pure by Edman sequencing) by reversed-phase chromatography (RP-HPLC). The Asn-81 glycopeptide, which eluted as a single peak by RP-HPLC, was separable into five peaks on the NucleoPac PA100 column, a pellicular anion-exchange column. Each of the five Asn-81 glycopeptide peaks was shown to contain N-linked oligosaccharides by treatment of each peak with peptide N 4-(N-acetyl-β- d-glucosaminyl) asparagine amidase F (PNGase F) and subsequent oligosaccharide analysis by high-pH anion-exchange chromatography with pulsed amperometric detection. High-pH anion-exchange chromatography-pulsed amperometric detection oligosaccharide analysis revealed that each peak contained a different population of sialylated N-linked oligosaccharides. Hence each peak contained a different group of glycopeptide glycoforms. It was observed that the longer the retention time of the Asn-81 glycopeptide peak on the anion-exchange column, the greater the oligosaccharide sialylation. Two glycopeptide peaks which differed in their distribution of disialylated oligosaccharides demonstrated that the glycopeptide separation was a result of something more than gross differences in sialic acid content. The two other N-linked tryptic glycopeptides of fetuin were also separated into multiple peaks on the NueleoPac PA100 column and these separations were shown to be due to differences in oligosaccharide sialylation. The separations of the three fetuin N-linked glycopeptides demonstrate that pellicular anion-exchange chromatography offers improved separation speed and resolution for the separation of sialylated glycopeptides.

Identification and structural characterization of Lyt-1 glycoproteins from tunicate hemocytes and mouse thymocytes

1. 1. A panel of monoclonal antibodies specific to murine Lyt-1 allotypic and framework determinants was used to investigate the possible occurrence of a Lyt-1 homolog in tunicate (protochoradte) hemocytes. 2. 2. In immunoprecipitation experiments, antigenic activities were associated with a major 67 kDa component on tunicate hemocytes and C57B1/6 mouse thymocytes. 3. 3. Tunicate and mouse Lyt-1 molecules were compared, in terms of glycosylation, by their sensitivity to glycosidases and analyses on one- and two-dimensional gel electrophoresis. 4. 4. Each of the two molecules appeared to bear two N-linked oligosaccharides, one high-mannose and one complex-type glycan. 5. 5. Both molecules revealed charge microheterogeneity with differences in sialic acid content accounting for the charge difference between each other. 6. 6. However, the difference in the glycans did not account for the microheterogeneity within each molecule, suggesting that other post-translational modifications might be responsible. 7. 7. At the polypeptide level, comparisons of chymotryptic and endoproteinase-Arg-C peptide maps, as well as CNBr-cleavage products, suggested that tunicate and mouse Lyt-1 molecules are structurally similar and that each may contain at least one intra-chain disulfide bridge. 8. 8. The significance of these findings is discussed in terms of the possible biological role of Lyt-1 glycoproteins at different levels of evolution.

Heterogeneity of human luteinizing hormone. Discrimination between acidic and basic preparations.

The charge heterogeneity of an I. H preparation containing relatively acidic components (LH Type 1) was studied. Four biological active components, with pI-values of 5.04, 5.60, 6.06 and 6.57 were detected. A total of four different alpha-subunits, with pI-values of 4.49, 4.79, 5.16 and 6.02 could be detected after incubation at 37 degrees C. With the exception of the most acidic component all these alpha-subunits were also present in earlier studied 1.H Type II preparations. After neuraminidase treatment a strong shift to more basic components was observed, resulting in a population of components similar to the one detected in I.H Type II preparations. The beta-subunits detected were very different from those observed in Type II preparations. All six components detected had pI-values greater than 7.5. Upon incubation at 56 degrees C these subunits appeared to be unstable resulting in a shift to more basic pI-values these pI-values being very similar to those of beta-subunits observed before in Type II preparations. After neuraminidase treatment, the pH values of the population of beta-subunits became identical to those of the population in I.H Type II. From these results it is concluded that the major charge difference between LH Type I and Type II is located in the beta-subunits. This difference cannot be explained completely by differences in sialic acid content, but may also be due to heat labile charged groups such as sulphate.

Analysis of cancer-associated colonic mucin by ion-exchange chromatography: evidence for a mucin species of lower molecular charge and weight in cancer

Cancer-associated mucins in the colon are antigenically distinct and glycosylated differently from their normal counterparts. Mucin-rich glycoconjugate preparations were made from nine non-neoplastic colons, seven colon cancers, and two different xenografts from mucin-producing human colon cancer cell lines, and radiolabeled with 3H. The preparation was applied to a DEAE-cellulose ion-exchange column, and eluted with a discontinuous ascending NaCl gradient resulting in seven discrete fractions or ‘species’. Over half of the 3H-labeled glycoconjugates from specimens of non-neoplastic colonic epithelium eluted in fraction V (eluted with 0.25 NaCl). Significantly less of the 3H-labeled glycoconjugates from specimens of colon cancer eluted in fraction V (34%, P<0.0005), and there were significant increases in glycoconjugates eluted in fractions IV ( P<0.008), III( P<0.0005), and II ( P<0.028). Additional samples were prepared without the radiolabeling procedures, chromatographed on a DEAE-cellulose ion-exchange column, and analyzed for monosaccharide content. Each of the fractions contained the monosaccharides expected in mucin-type glycoproteins, but only sialic acid was differentially expressed in the seven fractions or ‘species’, occurring principally in the more charged species. However, differences in sialic acid content were not sufficient to explain the differences in retention on the ion-exchange column, nor were differences in O-acetylation of the mucins. Mucin-type glycoconjugates from colon cancers are relatively less charged than those from the normal colon, and elute at lower ionic strengths. Of interest, cancer-associated mucins appear to be a lower molecular weight than their normal counterparts. Additional studies of oligosaccharide and apomucin structure will be required to explain the molecular basis of these differences in charge.

Electrophoretic heterogeneity of alkaline phosphatase isozymes in seminoma and normal testis.

Electrophoretic patterns of seminoma- and normal-testis-derived alkaline phosphatase isozymes, the placental alkaline phosphatase (PLAP)-like enzyme and the tissue-nonspecific (liver) alkaline phosphatase (LAP), were studied on starch gel and isoelectric focusing (IEF). Different migration patterns of the PLAP-like enzyme were observed with respect to both seminomas and normal testes on starch gel electrophoresis. On IEF, seminomas showed different staining patterns among different tumors; however, a common main activity was focused at pIs of 4.3-4.6, corresponding to pIs of PLAP. Normal testes showed two enzyme-staining regions, at pIs of 4.1 and 5.0-5.2, which were discriminated from pIs of PLAP and the PLAP-like enzyme in seminoma. The PLAP-like enzyme in seminoma was differentiated from PLAP by digestion with neuraminidase. Neuraminidase treatment simplified the distribution patterns of the PLAP-like enzyme in normal testis, but did not alter the pattern of microheterogeneity in seminoma. Two factors other than sialylation, namely structural modification of the carbohydrate moiety and variation of hydrophobicity, were shown to contribute to the microheterogeneity of the PLAP-like enzyme in seminoma. LAP in seminoma and in normal testis also showed marked electrophoretic heterogeneity and differences in pI distributions from LAP of liver. However, the migration patterns after desialylation were very similar to each other. The findings imply that electrophoretic heterogeneity demonstrated in LAP in seminoma and in normal testis is caused by a difference in sialic acid content in the molecule, and the heterogeneity of the PLAP-like enzyme in seminoma is considerable.

Sialic acid content in mouse myeloma cells and derived B-cell hybridomas with different metastic potentials

In a previous work we have reported on the metastatic capacities of mouse myeloma cells (NSO) and of two hybridomas derived from them. The present study was designed to examine the sialic acid content of these cell types, their attachment to glass and their capping in the presence of wheat germ agglutinin (WGA). The results showed no significant differences in sialic acid content between NSO and Hybridoma A (Hy A) cells which are non and low metastatic, respectively, whereas Hybridoma B (Hy B), which is highly metastatic, contained 1.4–1.6-fold higher sialic acid compared to the other two cell types. NSO cells exhibited a high, Hy A a moderate and Hy B a negligible attachment to glass. Capping experiments showed a low response of NSO cells, a moderate response of Hy A and a high response of Hy B in the presence of WGA. The results are discussed with regard to the metastatic potential of these cell types taking into consideration the properties cell surface sialic acid is known to endow to cells, determining various aspects of their behaviour.

Similarity of the clearance rates of free alpha-subunit and alpha-subunit dissociated from intact human chorionic gonadotropin, despite differences in sialic acid contents.

It is well known that the MCR of proteins can be influenced by their carbohydrate structure; i.e. the presence of terminal galactose on proteins results in uptake by hepatic receptors for galactose-terminated glycoproteins. Thus, a protein with its galactose residues covered or removed exhibits a far longer half life in plasma than one with its galactose residues exposed. The free alpha-subunit of human CG (hCG) has been shown to have a different carbohydrate composition than does the alpha-subunit dissociated from the intact hormone. In our laboratory, analysis of alpha-subunits isolated from pregnancy urine indicated that the alpha-subunit dissociated from hCG (hCG alpha) contains primarily monosialylated oligosaccharides, whereas the free alpha-subunit contains more than one sialic acid per oligosaccharide. This difference in the degree of sialylation prompted us to examine the clearance rates of these two subunits. Accordingly, free alpha and hCG alpha were purified by affinity chromatography and labeled with 125I. The labeled subunits were injected iv into rats and serum samples were removed at various time intervals over a 2-h period. The amount of [125I]alpha-subunit remaining in the serum was determined by immunoprecipitation using an antiserum to hCG alpha. The disappearance curves for the two subunits were indistinguishable and could be analyzed by a biexponential model. The t 1/2 of the faster component was 5 min, while the t 1/2 of the slower component was 74 min. In order to determine whether or not terminal galactose was present on either the hCG alpha or the free alpha-subunit, the labeled molecules were subjected to lectin column chromatography using Ricin or peanut lectin linked to agarose. Both of these lectins bind galactose but have different specificities with respect to the penultimate sugar. Both subunit preparations contained only minor amounts of material which could bind or either lectin. However, after desialylation, both hCG alpha and free alpha bound extensively to Ricin, indicating the presence of penultimate galactose residues in both. We conclude that terminal galactose residues are not present on the oligosaccharides of either hCG alpha or free alpha-subunits, and that the difference observed in the sialic acid contents of the two subunits does not affect their rates of clearance.

Heterogeneity of human luteinizing hormone. Effect of neuraminidase treatment on biologically active hormone and alpha- and beta-subunits.

By preparative isoelectric focussing of a highly purified LH preparation in a sucrose density gradient, four biologically active LH components were isolated. The effect of neuraminidase treatment of each component on the charge heterogeneity was studied by isoelectric focussing followed by in vitro biological and immunochemical techniques. The number of biologically active components with pI-values greater than 7 containing varying amounts of sialic acid is at least six. The pI-value of the most basic (= asialo) LH component was 9.26. The two most basic components were not present in our preparation (NM14) before neuraminidase treatment. It is concluded that the difference between the pI-values of LH components is caused by a difference in sialic acid content. When an intact LH component was incubated with neuraminidase there was detectable dissociation owing to the elevated temperature (37 degrees C) and necessary acidic conditions of the incubation. Under these conditions we found the same subunits as we have described before. The most basic alpha-subunit had a pI-value of 9.29, whereas two beta-subunits with pI greater than 9 were observed at pI 9.26 and pI 9.9. On the other hand, when an LH component was forced to dissociate by incubation at 56 degrees C prior to neuraminidase digestion, two additional alpha-subunits were found. From this it is concluded that in the intact LH molecule, some sialic acid residues are poorer substrates for neuraminidase action than in the free subunits.

Purification and characterization of canine urinary kallikrein

The renal kallikrein-kinin system may play a role in the regulation of sodium and water balance. Although the dog is a frequently used experimental animal in the study of the renal kallikrein-kinin system, dog urinary kallikrein (DUKK) has been poorly studied. We have purified DUKK by a series of chromatographic and electrophoretic procedures including anion-exchange chromatography, filtration through p-aminobenz-amidine-Sepharose (to remove contaminating nonkallikrein esterases), gel filtration, isoelectric focusing, and molecular sieve HPLC. This DUKK preparation gave three protein bands on polyacrylamide gel electrophoresis, each having similar esterolytic and kininogenase activities and immunological identity. Preparative isoelectric focusing indicated the presence of multiple forms of kallikrein with p I's of 3.93, 4.05, 4.24, and 4.44, the species with a p I of 4.24 constituting the major component. Neuraminidase treatment converted all of the forms into the component with a p I of 4.44, suggesting the charge heterogeneity was due mainly to differences in sialic acid content. DUKK has a specific activity of 3 mg bradykinin eq/min/mg protein when partially purified dog kininogen is used as a substrate. It is a glycoprotein with a molecular weight of 40,500 (amino acid analysis best fit method) and an alkaline pH optimum (9.0–9.5). DUKK is resistant to soybean trypsin inhibitor and lima bean trypsin inhibitor but is inhibited by several serine protease inhibitors such as antipain, leupeptin, and p-aminobenzamidine. PhePheArgchloromethyl ketone is a very potent inhibitor of DUKK. Contrary to previous reports, DUKK is also inhibited by N-α- p-tosyl- l-lysine chloromethyl ketone and aprotinin, the inhibition by the latter being inversely related to the concentration of NaCl in the medium. The esterolytic and amidolytic activities of DUKK are inhibited by an increase in NaCl concentration of the medium. This inhibition may be related to a NaCl-induced conformational change in the enzyme moiety.

Molecular characteristics of nerve growth factor receptors on PC12 cells.

Cross-linking of 125I-nerve growth factor (NGF) to PC12 cells with the photoreactive heterobifunctional agent N-hydroxysuccinimidyl-4-azidobenzoate results in the labeling of two major bands with Mr 158,000 and 100,000 and a minor band with Mr 225,000 as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions. Binding of 125I-NGF to and cross-linking into all these species is abolished in the presence of excess unlabeled NGF but not in the presence of unlabeled epidermal growth factor, insulin, or bovine pancreatic trypsin inhibitor. When PC12 cells with bound 125I-NGF are incubated in excess unlabeled NGF at 0 degree C prior to cross-linking, only the Mr 158,000 species remains. In addition, binding of 125I-NGF to the Mr 158,000 complex is trypsin-resistant, whereas binding to the Mr 100,000 complex is not. These experiments identify the Mr 158,000 species as the slow NGF-receptor complex (chase stable at 0 degree C) and the smaller Mr 100,000 species as the fast NGF-receptor complex (trypsin sensitive). Furthermore, 125I-NGF bound to the former but not to the latter species is displaced by very-low concentrations of NGF, showing that at least a significant fraction of the high-molecular-weight slow receptor is also a high-affinity receptor. This identification is supported by the finding that chick sensory neurons which possess both high- and low-affinity receptors exhibit two major labeled bands with Mr 145,000 and 105,000 as a result of cross-linking with 125I-NGF, whereas a cell population enriched in non-neuronal cells, which possess only low-affinity receptors, exhibits only the Mr 105,000 component. A shift in molecular weight of both species after pretreatment with neuraminidase indicates that both complexes contain sialoglycoproteins and rules out the possibility that differences in sialic acid content are responsible for the difference in molecular weight of the two complexes. The relative amount of the labeling of these two complexes is not affected by the presence of protease inhibitors nor by a variation of 5000-fold in cross-linker concentration. These results place some limits on possible models for the NGF receptors and their interconversion.

Biochemical and cellular identification of HLA-Bw44 subtypes

Biochemical analysis of HLA-Bw44 antigens by two-dimensional gelelectrophoresis and one-dimensional isoelectric focusing (IEF) from serologically heterozygous and homozygous donors allows the identification of two distinct types of HLA-Bw44 molecules, designated as Bw44-I and Bw44-II. These results are in concordance with the data obtained by CML-typing where at least two types of Bw44 target cells can be distinguished clearly as well. The antigen of type I has a more acidic isoelectric point (IEP) than that of type II. The differences in IEP are not due to differences in sialic acid content.

Iron uptake from sialo transferrins by rat reticulocytes

1. 1. Preparative isoelectric focusing of human diferric transferrin preparations yielded seven bands with different isoelectric points, due to differences in sialic acid content. 2. 2. Incubation of rat reticulocytes at 37 and 4°C with diferric preparations of four of these transferrin forms labeled with 59Fe and 125I show no differences in membrane binding of iron and transferrin and in iron uptake. 3. 3. Hence it is concluded that the carbohydrate chains are not directly involved in the process of iron delivery to reticulocytes.