Biomedical subjects

Y Oda

Publications and source records attributed to Y Oda.

At least 199 records · Page 11Linked to original sources

PubMed1999

Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom.

Colominic acid is an alpha2,8-linked sialic acid polymer produced by Escherichia coli. We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC. Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition. SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom. SC did not inhibit phospholipase A2 activity in bee venom. This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively. SC with a higher sulfur content and a larger molecular mass showed more potent activity. The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important. For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity. A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity.

Animals↗

PubMed1999

The AATPAP sequence is a very efficient signal for O-glycosylation in CHO cells.

The peptide signal sequence for protein O-glycosylation is not fully characterized, although a recent in vitro study proposed that the sequence motif, XTPXP, serves as a signal for mucin-type O-glycosylation. Here, we show that the AATPAP sequence acts as an efficient O-glycosylation signal, in vivo. A secreted fibroblast growth factor (secFGF) was used as a model to analyze glycosylation and its effects on the biological activity of FGF. Two constructs encoding [AATPAP]secFGF in which AATPAP was introduced at the N- or C-terminus of secFGF were constructed in an eukaryotic expression vector. [AATPAP]secFGF proteins were then expressed in Chinese hamster ovary (CHO) cells and secreted into the surrounding medium, primarily as modified forms sensitive to sialidase but not to peptide N-glycosidase F. The modifying groups were not seen when the AATPAP sequence was converted to AAAPAP or when [AATPAP]secFGF was expressed in mutant cells incapable of UDP-GalNAc biosynthesis. The results indicate that the modifying groups were mucin-type O-glycans and that the AATPAP served as an efficient O-glycosylation signal sequence. The O-glycosylated forms of [AATPAP]secFGF were as mitogenic toward human vascular endothelial cells as unmodified secFGF, suggesting that introduction of the signal into biologically active polypeptides is a promising approach with which O-glycosylation may be achieved without affecting original activity.

Amino Acid Sequence↗

PubMed1999

Preparation and properties of novel gramicidin S analogs possessing a tri-, tetra- or pentamethylene bridge between ornithine side chains.

Gramicidin S (GS) analogs in which the Ndelta atoms of the two Orn side chains are linked by an oligomethylene bridge [-(CH2)n-; n=3-5] were prepared via the bis(p-nitrobenzenesulfonyl) derivative [Orn(NBS)2,2']GS. For comparison the nonbridged secondary amino group-containing analog [Orn(Me)2,2']GS was also prepared. 1H NMR and CD spectral analysis indicated that these analogs adopt the same beta-sheet conformation as GS. The antimicrobial activities of these analogs were very similar, but were slightly dependent on the bridge chain length, the trimethylene-bridged analog being the most potent.

Anti-Bacterial Agents↗

PubMed1999

Choline acetyltransferase: the structure, distribution and pathologic changes in the central nervous system.

Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine, is presently the most specific indicator for monitoring the functional state of cholinergic neurones in the central and peripheral nervous systems. ChAT is a single-strand globular protein. The enzyme is synthesized in the perikaryon of cholinergic neurones and transported to the nerve terminals probably by both slow and rapid axoplasmic flows. ChAT exists in at least two forms in cholinergic nerve terminals: (i) soluble; and (ii) non-ionically membrane-bound forms. Multiple mRNA species of ChAT (R-, N-and M-types) are transcribed from different promoter regions and produced by different splicing in the mouse, rat, and human. All transcripts encode the same ChAT protein in rodents, while in human M-type mRNA has the capability to generate both large and small forms of ChAT proteins and R-and N-types ChAT mRNA generate a small form, which corresponds to the rodent ChAT. The genomic structure of ChAT is unique compared with other enzymes for neurotransmitters. The first intron of the ChAT gene encompasses the open reading frame encoding another protein, vesicular acetylcholine transporter (VAChT), which is responsible for the transportation of acetylcholine from the cytoplasm into the synaptic vesicles. The expressions of ChAT and VAChT appear to be coordinately regulated by multiple regulatory elements in cholinergic neurones. Immunohistochemical and in situ hybridization studies have revealed the localization of cholinergic neurones in the central nervous system: the medial septal nucleus, the nucleus of the diagonal band of Broca, the basal nucleus of Meynert, the caudate nucleus, the putamen, the nucleus accumbens, the pedunculopontine tegmental nucleus, the laterodorsal tegmental nucleus, the medial habenular nucleus, the parabigeminal nucleus, some cranial nerve nuclei, and the anterior horn of the spinal cord. Focally distributed cholinergic neurones project fibers to many areas in the central nervous system and construct a complicated cholinergic network, playing an important role in neuropsychic activities, such as learning, memory, arousal, sleep and movement. Central cholinergic neurones are involved in several neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis, in which disturbance of the central cholinergic system does not appear to be closely related to the etiology, but rather to the development of clinical symptoms. In addition, abnormalities of ChAT in the brain have been recently demonstrated in schizophrenia and sudden infant death syndrome.

Amino Acid Sequence↗

PubMed1999

Effects of a calcium receptor activator on the cellular response to calcium in human keratinocytes.

Changes in the concentration of extracellular calcium affect the balance between proliferation and differentiation in epidermal keratinocytes. Undifferentiated keratinocytes respond to the acute increase in the concentration of extracellular calcium with an increase of intracellular calcium concentration and inositol trisphosphate production, and, subsequently, the expression of differentiation related genes. Our previous studies demonstrated the presence of a calcium-sensing receptor in human keratinocytes, which is identical to the parathyroid calcium-sensing receptor. In this study we showed that the calcimimetic compound NPS R-467, a selective calcium-sensing receptor activator, augmented the calcium-elicited inositol trisphosphate response of cloned human keratinocyte calcium-sensing receptor expressed in human embryonic kidney cells 293. In order to define the role of the calcium-sensing receptor in calcium induced epidermal differentiation, we investigated the ability of NPS R-467 to raise intracellular Ca2+ and stimulate differentiation in normal human foreskin keratinocytes. In the presence of 0.03 mM Ca2+, NPS R-467 increased the intracellular calcium concentration response in a concentration-dependent fashion. Undifferentiated normal human foreskin keratinocyte cells responded to increased extracellular calcium concentration with increased intracellular calcium concentration. NPS R-467 potentiated this response by increasing the maximal response. Its stereoisomer, NPS S-467, was not active in raising intracellular calcium concentration. Increasing extracellular calcium concentration from 0.03 to 1.2 mM stimulated the promoter activity of the differentiation marker gene, involucrin. NPS R-467 potentiated the calcium-stimulated increase in involucrin promoter activity unlike NPS S-467 or vehicle. Northern analysis of the normal human foreskin keratinocyte cells treated with NPS R-467 demonstrated potentiation of the calcium-stimulated increases in involucrin and transglutaminase mRNA levels. These results support the hypothesis that the calcium-sensing receptor expressed in keratinocytes mediates at least part of the intracellular calcium response to extracellular calcium and calcium-induced differentiation.

Aniline Compounds↗

PubMed1999

Epithelial sodium channels are upregulated during epidermal differentiation.

Terminal differentiation of epidermal keratinocytes is linked to transmembrane ion flux. Previously, we have shown that amiloride, an inhibitor of epithelial sodium channels, blocks synthesis of differentiation-specific proteins in normal human keratinocytes. Here, we have identified the specific subunits of amiloride-sensitive human epithelial sodium channels in relation to differentiation of cultured human keratinocytes, as well as to epidermal development. As assessed by northern hybridization, RNase protection assay, and reverse transcription-polymerase chain reaction, transcripts encoding functional alpha and regulatory beta subunits of human epithelial sodium channels were expressed both in cultured keratinocytes and in epidermis at levels comparable with the kidney. The mRNA expression of both human epithelial sodium channel-alpha and -beta increased during calcium-induced keratinocyte differentiation. Whereas the beta subunit of human epithelial sodium channel was induced by elevated concentrations of calcium, the alpha subunit increased with duration of culture. The regulatory gamma subunit was less abundant but also expressed in epidermis. Both human epithelial sodium channel-alpha and -beta were localized throughout the nucleated layers of human adult epidermis, but these channels were not detected in early stages of fetal epidermal development. This co-ordinated expression of subunits suggests that epithelial sodium channels may play an important part in both epidermal differentiation and skin development, presumably by modulating ion transport required for epidermal terminal differentiation.

Blotting, Western↗

PubMed1999

Propofol decreases the clearance of midazolam by inhibiting CYP3A4: an in vivo and in vitro study.

OBJECTIVE: To examine the effect of propofol on the pharmacokinetics of midazolam in vivo and to elucidate the mechanism of the pharmacokinetic changes of midazolam by propofol with the use of human liver microsomes and recombinant CYP3A4. METHODS: In an in vivo, double-blind randomized study, 24 patients received 0.2 mg/kg midazolam and either 2 mg/kg propofol (propofol group) or placebo (placebo group) for induction of anesthesia. In the propofol group, continuous infusion of propofol at 9 mg/kg/h was started immediately after the bolus infusion of propofol and was maintained for an hour. In the placebo group the same dose of soybean emulsion as a placebo was given and infused intravenously for an hour instead of propofol. In an in vitro study the effect of propofol on the metabolism of midazolam was studied with human liver microsomes and recombinant CYP3A4. RESULTS: In the propofol group the mean clearance of midazolam was decreased by 37% (P = .005) and the mean elimination half-life was prolonged by 61% (P = .04) compared with the placebo group. The mean plasma concentrations of 1'-hydroxymidazolam were lower in the propofol group than in the placebo group at 5, 10, 15, 20, and 30 minutes after midazolam was administered (P < .05). The mean (+/-SD) Michaelis-Menten constant for midazolam 1'-hydroxylation by human liver microsomes was 5.6 +/- 3.3 micromol/L. The formation of 1'-hydroxymidazolam was competitively inhibited by propofol, and the mean inhibition constant was 56.7 +/- 16.6 micromol/L. The mean Michaelis-Menten constant and mean inhibition constant values for midazolam 1'-hydroxylation by recombinant CYP3A4 were 4.0 micromol/L and 61.0 micromol/L, respectively, consistent with the mean values obtained from human liver microsomes. CONCLUSION: Propofol decreases the clearance of midazolam, and the possible mechanism is the competitive inhibition of hepatic CYP3A4.

Adult↗

PubMed1999

Fentanyl inhibits metabolism of midazolam: competitive inhibition of CYP3A4 in vitro.

Fentanyl decreases clearance of midazolam administered i.v., but the mechanism remains unclear. To elucidate this mechanism, we have investigated the effect of fentanyl on metabolism of midazolam using human hepatic microsomes and recombinant cytochrome P450 isoforms (n = 6). Midazolam was metabolized to l'-hydroxymidazolam (l'-OH MDZ) by human hepatic microsomes, with a Michaelis-Menten constant (K(m)) of 5.0 (SD 2.7) mumol litre-1. Fentanyl competitively inhibited metabolism of midazolam in human hepatic microsomes, with an inhibition constant (Ki) of 26.8 (12.4) mumol litre-1. Of the seven representative human hepatic P450 isoforms, CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4, only CYP3A4 catalysed hydroxylation of midazolam, with a K(m) of 3.6 (0.8) mumol liter-1. Fentanyl competitively inhibited metabolism of midazolam to l'-OH MDZ by CYP3A4, with a Ki of 24.2 (6.8) mumol litre-1, comparable with the Ki obtained in human hepatic microsomes. These findings indicate that fentanyl competitively inhibits metabolism of midazolam by CYP3A4.

Anesthetics, Intravenous↗

PubMed1999

Role of human N-acetyltransferases, NAT1 or NAT2, in genotoxicity of nitroarenes and aromatic amines in Salmonella typhimurium NM6001 and NM6002.

Human NAT1 and NAT2 genes were subcloned into pACYC184 vector and the plasmids thus obtained were introduced into Salmonella typhimurium O-acetyltransferase-deficient strain NM6000 (TA1538/1, 8-DNP/pSK1002), establishing new strains NM6001 and NM6002, respectively. We compared the sensitivities of these two strains with those of NM6000 towards carcinogenic nitroarenes and aromatic amines in the SOS/umu response. The induction of umuC gene expression by these chemicals in the presence and absence of the S9 fraction was assayed by measuring the cellular beta-galactosidase activity expressed by the umuC"lacZ fusion gene in the tester strains. 2-Nitrofluorene and 2-aminofluorene induced umuC gene expression more strongly in the NM6001 strain than in the NM6002 strain. In contrast, induction of umuC gene expression by 1, 8-dinitropyrene, 6-aminochrysene and 2-amino-3,5-dimethylimidazo[4, 5-f]quinoline was weaker in the NM6001 strain than in the NM6002 strain. 1-Nitropyrene, 2-amino-6-methyl-dipyrido[1,2-a:3', 2'-d]imidazole, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3-methyl-9H-pyrido[2,3-b]indole were found to induce umuC gene expression at similar extents in both strains. These results suggest that the newly developed strains can be employed for the studies on mechanisms of genotoxicity of a variety of nitroarenes and aromatic amines, along with the assessment of cancer risk to humans.

Arylamine N-Acetyltransferase↗

PubMed1999

A rare case of sarcoidosis with bilateral leg lymphedema as an initial symptom.

A 55-year-old man was admitted to our hospital because of bilateral leg lymphedema. He also showed subcutaneous nodules and CT scan disclosed multiple enlarged lymph nodes at thoracic, abdominal, and inguinal areas. Biopsy of the inguinal lymph node and the subcutaneous nodule revealed noncaseating epithelioid cell granuloma, a finding consistent with sarcoidosis. Lymphedema was attributed to the blockade of lymph flow by the systemic lymph node involvement of the disease. Within 1 week after the initiation of steroid therapy, his leg edema disappeared. Lymphedema could be the initial symptom of systemic sarcoidosis.

Diagnosis, Differential↗

PubMed1999

Structures of the deoxy and CO forms of haemoglobin from Dasyatis akajei, a cartilaginous fish.

The three-dimensional structures of the deoxy- and carbonmonoxyhaemoglobin (Hb) from Dasyatis akajei, a stingray, have been determined at 1.6 and 1.9 A resolution, respectively. This is one of the most distantly related vertebrate Hbs to human HbA. Both structures resemble the respective forms of HbA, indicating that the alpha2beta2-type tetramer and the mode of the quaternary structure change are common to Hbs of jawed vertebrates. Larger deviations between D. akajei Hb and human HbA are observed in various parts of the molecule, even in the E and F helices. Significant mutations and/or conformational changes are also observed around the haems, in the C-terminal region of the beta subunit, in the alpha1beta2 interface and in the organic phosphate-binding site of HbA. Despite these structural differences, the oxygen affinity, haem-haem interaction, Bohr effect and organic phosphate effect of D. akajei Hb are all only moderately reduced. Compared with human HbA, the overall r.m.s. deviation of main-chain atoms in the helical regions of bony fish Hbs is smaller than that of D. akajei Hb.

Amino Acid Sequence↗

PubMed1999

High-molecular-weight fibronectin synthesized by adenoid cystic carcinoma cells of salivary gland origin.

To understand the morphogenesis of characteristic cribriform structures and the frequent invasion of salivary adenoid cystic carcinomas (ACC) along such basement membrane-rich structures as peripheral nerves, we have isolated fibronectin (FN) from the culture media of ACC3 cells established from a parotid ACC and characterized its glycosylation and alternative splicing status. FN isolated from ACC3 cells (ACC-FN) showed a molecular mass of 315 kDa in SDS-PAGE and was less heterogeneous and larger than plasma FN (pFN) or FNs from other cell sources. Differential enzymatic treatments of immunoprecipitated ACC-FN with neuraminidase, peptide-N-glycosidase F and endo-alpha-N-acetylgalactosaminidase revealed that ACC-FN was composed of a polypeptide chain of 270 kDa, with 10 kDa each of N-linked and O-linked oligosaccharide chains. Reverse transcription polymerase chain reaction (RT-PCR), in-situ hybridization, and immunofluorescence studies showed that most ACC-FNs contained ED-A, ED-B and IIICS regions in the molecules. This alternative splicing status of ACC-FN seemed to contribute to its less heterogeneous and larger molecular form. Cell attachment assay demonstrated that ACC-FN was more potent than pFN in adhesion of ACC3 cells. The results indicated that ACC-FN may function as a substrate for attachment of ACC3 cells, or that ACC3 cells trap and retain ACC-FN in their pericellular space. This isoform of FN may play an important role in the mode of invasion of ACC and the formation of stromal pseudocysts in the characteristic cribriform structure of ACC.

Alternative Splicing↗

PubMed1999

Extremely acute phenytoin-induced peripheral neuropathy.

PURPOSE: Peripheral neuropathy is a rare adverse effect associated with phenytoin (PHT), and it usually occurs after the prolonged use of PHT. Acute PHT-induced peripheral neuropathy is extremely rare. METHODS: An 18-year-old girl was admitted for the control of epilepsy. Just a few hours after the administration of PHT, she complained of distal lower-extremity paresthesia in a stocking distribution and motor weakness: the Achilles tendon reflex was absent. RESULTS: Electrophysiological studies revealed slightly reduced sensory-conduction velocity and mild prolongation of distal latency in the lower extremities. After the discontinuation of PHT, these symptoms disappeared gradually, and sensory-conduction velocity and distal latency became normal. CONCLUSIONS: Although it has been reported that peripheral neuropathy occurred after treatment with PHT for a week, there has been no report of a patient such as ours, who developed peripheral neuropathy just a few hours after the initial administration of PHT. The underlying mechanism remains unknown; however, we should pay attention to such extremely acute peripheral neuropathy when using PHT.

Acute Disease↗

PubMed1999

Clinical diversity in acute necrotizing encephalopathy.

Acute necrotizing encephalopathy is a novel disease entity, proposed by Mizuguchi et al in 1995, that shows a characteristic selective and symmetric involvement of the thalamus, brain stem, and cerebellum. It usually leaves sequelae. The etiology of acute necrotizing encephalopathy is unknown. We describe here six patients aged 6 months to 5 years (four boys and two girls). Four cases were typical, and the patients' cranial computed tomographic scans and magnetic resonance imaging showed irreversible symmetric involvement of the thalamus, brain stem, and cerebellum. Three of the patients died, and one was left with severe sequelae. In the other two patients, who had selective reversible thalamic involvement, the disease was mild; one also showed transient unilateral thalamic involvement. These patients recovered completely. We consider the illness in these two patients to fit the criteria of the mild form of acute necrotizing encephalopathy. We believe that acute necrotizing encephalopathy has some clinical diversity, as is seen in other neurologic disorders, and that a mild form could exist.

Atrophy↗

PubMed1999

Pyridoxine-dependent seizures in an older child.

The case of a 12-year-old girl with pyridoxine-dependent seizures is reported. She developed status epilepticus just after birth and ordinary antiepileptic drugs were administered without effect. Her seizures ceased only on the administration of pyridoxine. Status epilepticus associated with the withdrawal of pyridoxine occurred three times, and ceased only after the renewed administration of pyridoxine. She now has mental retardation and mild spastic diplegia. The reported cases of pyridoxine-dependent seizures usually have been neonates and infants. Older patients were not fully investigated, and so we have reviewed these cases of pyridoxine-dependent seizures. As known previously, pediatricians should not forget that pyridoxine should be continued for life.

Child↗

PubMed1999

Expression and signal transduction of calcium-sensing receptors in cartilage and bone.

We previously showed that Ca2+-sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca2+]([Ca2+]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (approximately 140-190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca2+]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca2+]o reduced PTH-induced cAMP formation. Raising [Ca2+]o also increased intracellular free [Ca2+] in SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca2+]o may couple to signaling pathways important in skeletal metabolism.

Animals↗

PubMed1999

The interaction of TSH receptor autoantibodies with 125I-labelled TSH receptor.

Detergent-solubilized porcine TSH receptor (TSHR) has been labeled with 125I using a monoclonal antibody to the C-terminal domain of the receptor. The ability of sera containing TSHR autoantibody to immunoprecipitate the labeled receptor was then investigated. Sera negative for TSHR autoantibody (as judged by assays based on inhibition of labeled TSH binding to detergent-solubilized porcine TSHR) immunoprecipitated about 4% of the labeled receptor, whereas sera with high levels of receptor autoantibody immunoprecipitated more than 25% of the labeled receptor. The ability to immunoprecipitate labeled TSHR correlated well with ability of the sera to inhibit labeled TSH binding to the receptor (r = 0.92; n = 63), and this is consistent with TSHR autoantibodies in these samples being directed principally to a region of the receptor closely related to the TSH binding site. Preincubation of labeled TSHR with unlabeled TSH before reaction with test sera inhibited the immunoprecipitation reaction, providing further evidence for a close relationship between the TSHR autoantibody binding site(s) and the TSH binding site. This was the case whether the sera had TSH agonist (i.e., thyroid stimulating) or TSH antagonist (i.e., blocking) activities, thus, providing no clear evidence for different regions of the TSHR being involved in forming the binding site(s) for TSHR autoantibodies with stimulating and with blocking activities. The ability of TSHR autoantibodies to stimulate cyclic AMP production in isolated porcine thyroid cells was compared with their ability to immunoprecipitate labeled porcine TSHR. A significant correlation was observed (r = 0.58; n = 50; P < 0.001) and the correlation was improved when stimulation of cyclic AMP production was compared with inhibition of labeled TSH binding to porcine TSHR (r = 0.76). Overall, our results indicate that TSHR autoantibodies bind principally to a region on the TSHR closely related to the TSH binding site, and this seems to be the case whether the autoantibodies act as TSH agonists or antagonists.

Animals↗

PubMed1999

Analysis of carbohydrate residues on recombinant human thyrotropin receptor.

An investigation of the sugar groups on recombinant human TSH receptors (TSHR) expressed in CHO-K1 cells and solubilized with detergents is described. Western blotting studies with TSHR monoclonal antibodies showed that the receptor was present principally as two bands with approximate molecular masses of 120 and 100 kDa. Further blotting studies using lectins and/or involving treatment with different glycosidases indicated that the 100-kDa band contained about 16 kDa of high mannose-type sugars, and the 120-kDa band contained about 33 kDa of complex-type sugars. It was possible to separate the 120- and 100-kDa components of the TSHRs by lectin affinity chromatography. In particular, Galanthus nivalis lectin, which binds high mannose-type sugars, bound the 100-kDa band, but not the 120-kDa band, whereas Datura stramonium lectin, which binds complex-type sugars, bound the 120-kDa band, but not the 100-kDa band. 125I-Labeled TSH binding studies with the various lectin column fractions showed that TSH-binding activity was principally associated with the complex-type sugar containing the 120-kDa form of the receptor rather than the high mannose-containing 100-kDa form. During peptide chain glycosylation, high mannose-type sugar residues are attached first and then modified by the formation of complex type structures to form the mature glycoprotein. Our data suggest that in the case of the TSH receptor, this type of posttranslational processing has an important role in forming the TSH-binding site.

Animals↗