Search PubMed⌕ Search

Biomedical subjects

T H Ji

Publications and source records attributed to T H Ji.

At least 91 records · Page 5Linked to original sources
PubMed1981

Photoaffinity labeling of the insulin receptor in H4 hepatoma cells: lack of cellular receptor processing.

Photoaffinity labeling techniques were used to identify insulin-binding components of the plasma membrane in insulin-responsive, monolayer-cultured hepatoma cells. The activated, photosensitive reagent, an n-hydroxysuccinimide ester of 4-azidobenzoic acid, was coupled with highly purified insulin, and the hormone derivative was subsequently iodinated, bound to cell surface receptors of intact H4 cells, and photoactivated. Ater dissolution of the cells, labeled proteins were analyzed by SDS/polyacrylamide gel electrophoresis under reducing conditions. The main labeled band exhibited an apparent molecular weight of 130,000. Two minor components of apparent mol wt 95,000 and 40,000 were also identified. Specific labeling of all 3 bands was inhibited by simultaneous incubation of the cells with native insulin, but not by the heterologous hormone, glucagon, prior to photoactivation. Binding of azidobenzoyl insulin to H4 cells was time-dependent, as was the correlated labeling of receptor components. Band-labeling by the photosensitive insulin derivative was totally light-dependent; spontaneous covalent linking of insulin and receptor was not observed. The labeled receptor-related proteins were not degraded by the cells under our experimental conditions.

Affinity Labels↗
PubMed1981

Both alpha and beta subunits of human choriogonadotropin photoaffinity label the hormone receptor.

It has been shown that a photoactivable derivative of human choriogonadotropin (hCG) labels the lutropin receptor on porcine granulosa cells [Ji, I. & Ji, T. H. (1980) Proc. Natl. Acad. Sci. USA 77, 7167-7170]. In an attempt to identify which of the hCG subunits labeled the receptor, three sets of different hCG derivatives were prepared. In the first set, hCG was coupled to the N-hydroxysuccinimide ester of 4-azidobenzoylglycine and radioiodinated. In the second set, only one of the subunits was radioiodinated, but both subunits were allowed to react with the reagent. In the third set, both the reagent and [125I]iodine were coupled to only one of the subunits. The binding activity of each hormone derivative was comparable to that of 125I-labeled hCG. After binding of these hormone derivatives to the granulosa cell surface, they were photolyzed. After solubilization, autoradiographs of sodium dodecyl sulfate/polyacrylamide gels of each sample revealed a number of labeled bands; the hCG derivatives containing 125I-labeled alpha subunit produced four bands (molecular weights 120,000 +/- 6,000, 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000) and those containing 125I-labeled beta subunit produced three bands (molecular weights 106,000 +/- 6,000, 88,000 +/- 5,000, and 83,000 +/- 4,000). Results were the same when the hormone-receptor complexes were solubilized in 0.5% Triton X-100 and then photolyzed or when the hormone was derivatized with a family of reagents having arms of various lengths. We conclude that both the alpha subunit and the beta subunit of hCG photoaffinity labeled certain membrane polypeptides and that these polypeptides are related to the hormone receptor.

Affinity Labels↗
PubMed1980

Does random collisional cross-linking occur?

In fluid membranes, mobile molecules are thought to collide at high frequencies. Concern has been expressed as to whether these colliding molecules are cross-linking during the chemical cross-linking of membrane molecules, thereby creating problems in interpreting such experiments. Hemoglobin was used as a model to test this possibility. Oligomers larger than the tetramer could be cross-linked depending on factors such as hemoglobin concentration, duration of the cross-linking reaction and the type of reagent. Under certain conditions, however, such as a hemoglobin concentration less than 150 microM or a duration of cross-linking shorter than 15 min, larger oligomers were not detectable. Analysis of these data suggests that the probability of random collisional cross-links under normal conditions is insignificant.

Chemical Phenomena↗
PubMed1980

Presence of spectrin tetramer on the erythrocyte membrane.

In order to examine the subunit structure of membrane-associated spectrin, human erythrocyte membranes were cross-linked with a variety of photosensitive heterobifunctional reagents by flash photolysis millisecond cross-linking (Kiehm, D.J., and Ji, T.H. (1977) J. Biol. Chem. 252, 8524-8531). Cross-linking of ghosts produced a series of new bands with apparent molecular weights of approximately 420,000, 710,000, and 910,000, as well as a band at the top of the gels. Evidence is presented that these three bands represent spectrin dimer, trimer, and tetramer, respectively. We propose that spectrin tetramer exists on the membrane as the predominant structural unit and that dimer is not a repeating structural unit.

Cross-Linking Reagents↗
PubMed1980

Macromolecular photoaffinity labeling of the lutropin receptor on granulosa cells.

Lutropin, a pituitary hormone, and human choriogonadotropin bind to the same receptors in the ovary and elicit identical responses. A photoactivable derivative of human choriogonadotropin was used to identify the lutropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycylglycine, and iodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself did but with a slightly lower Ka, 2.98 X 10(9) M-1 compared with 5.1 X 10(9) M-1 for 125I-hormone. The binding could be blocked with untreated hormone or lutropin but not with follitropin, prolactin, insulin, or bovine serum albumin. Its alpha and beta subunits could be crosslinked to produce alpha beta dimer by photolysis, the extent of crosslinking being dependent upon the reagent concentration used for the derivatization: 22.8% at 50 microM, 37.3% at 100 microM, and 67.2% at 150 microM. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels under reducing conditions, three new bands of lower electrophoretic mobility appeared in addition to alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis and the presence of both cells bearing the receptor and the 125I-hormone derivative. It could be blocked by excess untreated hormone. The three bands correspond to molecular weights, 96,000 +/- 6,700, 66,000 +/- 4,600, and 63,000 +/- 4,400. Because the hormone has a high carbohydrate content and such glycoproteins are known to exhibit anomalous electrophoretic mobilities, these estimates must be tentative.

Affinity Labels↗
PubMed1980

A photochemical crosslinking study of the subunit structure of membrane-associated spectrin.

The subunit structure of membrane-associated spectrin was investigated by crosslinking human erythrocyte membranes with a variety of cleavable photosensitive heterobifunctional reagents by flash photolysis millisecond crosslinking. Crosslinked complexes were analyzed on agarose-polyacrylamide gels whose high exclusion limit the resolution of molecular with Mr larger than 10(6). Crosslinking of membrane ghosts produced a series of new bands with apparent molecular weights of approximately 420 000, 170 000 and 910 000, as well as a band at the top of the gels. No significant bands were detectable between the 910 000-Mr band and the material at the gel top. The molecular weights of the three bands correspond to spectrin dimer, trimer, and tatramer, suggesting a limiting tetrameric stoichiometry for membrane-associated spectrin. The rate of formation of spectrin oligomers was examined by increasing crosslinking time. Dimers accumulate before trimer and tetramer, and as tetramer increased, dimer diminished. A simple kinetic model is employed which is also consistent with spectrin tetramer existing on the cytoplasmic surface of the erythrocyte membrane as the basic structural unit.

Azides↗
PubMed1979

Cross-linking of fibronectin to sulfated proteoglycans at the cell surface.

Fibronectin is a major surface protein of normal animal cells but is absent from many transformed cells. Addition of fibronectin to transformed cells causes increased cell substrate adhesion and changes in the morphology and cytoskeleton of the cells. We have coupled fibronectin to photoactivable chemical cross-linkers and have added it to cells to identify those molecules to which it binds. In this way, fibronectin can be cross-linked to sulfated proteoglycans at the cell surface. The cross-linking is specific for fibronectin. The fibronectin-proteoglycan complex is sensitive to chondroitinase ABC and AC and to trypsin. Addition of fibronectin also affects binding of hyaluronic acid to the cells. These results suggest that fibronectin interacts with proteoglycans at the cell surface. The existence of such interactions may have implications for the role of fibronectin and proteoglycans in cell adhesion.

Azides↗
PubMed1977

A novel approach to the identification of surface receptors. The use of photosensitive hetero-bifunctional cross-linking reagent.

Methyl 4-azidobenzoimidate, a photosensitive hetero-bifunctional cross-linking reagent, was synthesized and characterized. This reagent has an imidoester at one end, which reacts spontaneously with primary amines, and an arylazide at the other end, which reacts with a variety of chemical groups upon photolysis by ultraviolet radiation. The reagent molecules were attached to concanavalin A by reactions between imidoester groups of the reagents and free amino groups of the lectin. These activated lectins were purified on a Sephadex G-25 column and showed the binding affinity to an affinity column, glucosylated Sepharose, and to the human erythrocyte ghost membrane. The activated lectins were incubated with the membranes and then unbound lectins were removed by washing. The lectins bound to receptors in the membranes were irradiated with a shortwave ultraviolet lamp to photolyze arylazides attached to the lectins, thus cross-linking the lectins and receptors together. Then the membranes were solubilized and electrophoresed. On gels, the intensity of the lectin receptor band diminished slightly and concomitantly a new band of a higher molecular weight appeared. When 125I-labeled concanavalin A was used, the new band contained the radioactivity. The extent of the appearance of the new band and the decrease of the receptor band were reduced significantly when the ultraviolet irradiation was omitted or the activated lectins were incubated with the membranes in the presence of the lectin inhibitor, alpha-methylmannoside. The irradiation of nonactivated, receptor-bound concanavalin A did not cause those changes. When the activated lectins alone were irradiated with ultraviolet, the band of the lectin dimer appeared whereas nonirradiated lectins appeared mostly as monomers. It is concluded that a small fraction of the activated lectins were cross-linked to receptors in the membrane upon photolysis. In this study, only 8 reagent molecules were attached to a tetramer of the lectin, compared with the presence of approximately 40 available free amino groups. The efficiency of such cross-links of ligands to receptors may be increased by employing longer versions of the hetero-bifunctional cross-linking reagents and also by attaching more of the reagent molecule to ligands.

Azides↗
PubMed1976

Crosslinking of lectins and receptors in membranes with hetero-bifunctional crosslinking reagents.

125I-Con A was reacted with the imidoester group of methyl 4-azidobenzoimidate and isolated from excess reagents on a Sephadex G-25 column. The hetero-bifunctional crosslinking reagent has two dissimilar functional groups, imidoester and arylazide, instead of two identical functional groups found in conventional homo-bifunctional crosslinking reagents. The two functional groups react under different conditions and it is possible to react one first and to activate the other later. Here the reagents were attached to the lectins by the imidoester reactions, and the arylazides now attached to the lectins were intended for the later use to crosslink the lectins and receptors. Human erythrocyte ghosts were incubated with the activated lectins, unbound lectins removed, and irradiated with UV to photolyze arylazides, thus crosslinking the lectins to the receptors in membranes. Upon the solubilization and subsequent electrophoresis of the sample, a new band appeared on the gel. 125I-Con A was detected in band A, the new band, and band 3, one of the membrane glycoproteins, diminished in parallel to the appearance of band A. The concurrent appearance of band A accompanied by 125I-Con A and decrease of band 3 were significantly reduced, when ghosts were incubated with the activated lectins but were not irradiated, ghosts were incubated with nonactivated 125I-Con A and irradiated, or ghosts were incubated with the activated concanavalin A in the presence of alpha-methylmannoside, the lectin inhibitor, and irradiated. The inhibitor removed a significant amount of the activated lectins bound to membranes, but failed to do so the activated lectins crosslinked into band A.

Azides↗
PubMed1974

Crosslinking of the glycoproteins in human erythrocyte membranes.

The glycoproteins of human erythrocyte membranes were crosslinked with dimethyl adipimidate dihydrochloride. On sodium dodecyl sulfate-polyacrylamide gel electrophoregrams of the crosslinked solubilized membranes, at least three new glycoprotein complexes appeared in addition to the normal glycoprotein species. One of the new glycoprotein complexes was shown to contain two of the three species of membrane glycoproteins.

Adipates↗