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Biomedical subjects

T H Ji

Publications and source records attributed to T H Ji.

At least 73 records · Page 4Linked to original sources
PubMed1986

Disulfides of the lutropin receptor.

Affinity cross-linking of the lutropin receptor with 125I-human choriogonadotropin (hCG) on porcine granulosa cells produced four distinct homone-receptor complexes under reducing conditions. They contain 18-, 24-, 28-, and 34-kDa components (Ji, I., Bock, J. H., and Ji, T. H. (1985) J. Biol. Chem. 260, 12815-12821). Photoaffinity labeling and cross-linking produced 136-, 102-, and 74-kDa hCG-receptor complexes under reducing conditions and the 136-kDa complex under nonreducing conditions. In addition, the unreduced 102-kDa complex was seen in photoaffinity labeling but not in cross-linking. When the unreduced 136-kDa complex was reduced, the 102- and 74-kDa complexes were generated, indicating release of the 34- and the 28-kDa components in two steps. When the unreduced 102-kDa complex was reduced, the 74-kDa complex was produced, indicating the release of a 28-kDa component. The 74-kDa complex could not be reduced but was cleaved by alkaline treatment to produce the hCG alpha beta dimer. The results indicate that the 24-kDa component is released from the 74-kDa complex, since the apparent mass of the hCG alpha beta dimer on gels is 50 kDa. The 24-kDa component appears to be the initial site for photoaffinity labeling or cross-linking and to be disulfide linked to the 28-kDa component which is in turn disulfide linked to the 34-kDa component. These intercomponent disulfides exist in some receptors but not all. Formation of the disulfide-linked 136-kDa band required the presence of a sulfhydryl-blocking agent, N-ethylmaleimide. In particular, the 34-kDa component was vulnerable to reduction. There was no significant evidence of disulfides between the hormone and any of the receptor components.

Affinity Labels↗
PubMed1986

Structural homologies in the lutropin/human choriogonadotropin receptor and the follitropin receptor on porcine granulosa cells.

In order to examine the structure of the human choriogonadotropin (hCG) receptor and the follitropin (FSH) receptor on porcine granulosa cells, the hormone receptors were photoaffinity-labeled or affinity-cross-linked. The resulting hormone-receptor complexes were analyzed by alkaline cleavage of cross-links, reduction of disulfides, and peptide maps. The results revealed striking similarities in the structure of the hormone receptors. Both appear to be oligomeric; the hCG receptor has at least four components of 18, 24, 28, and 34 kDa, whereas the FSH receptor shows three distinct components of 18, 22, and 34 kDa. The 24- and the 22-kDa components are the sites for the primary photoaffinity labeling or affinity cross-linking by hCG and FSH, respectively. These components were linked by intercomponent disulfides. Reduction of cross-linked complexes revealed that in the hCG receptor the 24-, the 28-, and the 34-kDa components were disulfide-linked sequentially in a linear form as were the 22-, the 18-, and the 34-kDa components in the FSH receptor. The peptide maps of cross-linked hCG-receptor and FSH-receptor complexes, however, were distinct, indicating that the hCG receptor and the FSH receptor were not identical.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of the gonadotropin receptor with native, asialo, and deglycosylated choriogonadotropin.

Human choriogonadotropin (hCG) is a heterodimeric hormone composed of an alpha and a beta subunit. hCG and its asialo (ashCG) and deglycosylated (dghCG) forms vary in their ability to stimulate hormone responsive adenylate cyclase. ashCG is a partial agonist, and dghCG is an antagonist. Photoactivatable moieties were coupled to hCG, ashCG, and dghCG, and the derivatives were radioiodinated. Competitive binding studies indicate that all of the derivatives had a similar affinity for the gonadotropin receptor on porcine granulosa cell membranes. Radiolabeled derivatives were used to photoaffinity label the gonadotropin receptor. Radiolabeled complexes were separated by NaDodSO4/PAGE. All of the derivatives produced similar autoradiographic patterns, except that dghCG produced an additional 48-kDa complex. To investigate the structure of the complexes further, peptide mapping of proteolytic digests was used. All, except for the 48-kDa complex, generated similar peptide maps indicating a relationship between those complexes in which the smaller components are part of the larger. The 48-kDa complex contained both subunits of 40-kDa dghCG. Therefore, this complex is expected to contain an additional component of 8 kDa. The complex was generated whether the hormone-receptor complex was photoaffinity labeled on cells, on isolated membranes, or after solubilizing in detergent. Formation was blocked by excess hCG and did not occur in the absence of UV irradiation. We conclude that the hCG derivatives are able to photoaffinity label the hCG receptor but that the dghCG derivative can photoaffinity label an additional component that was not observed when derivatives of hCG or ashCG were used to label the receptor.

Adenylyl Cyclases↗
PubMed1985

Photoaffinity labeling of the follitropin receptor.

A photoactivatable derivative of human follitropin was used to identify the follitropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycine, and radioiodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself, but with a slightly lower Ka, 1.12 X 10(10) M-1 compared with 1.4 X 10(10) M-1 for the 125I-hormone. The binding could be blocked with untreated hormone. Its alpha and beta subunits could be cross-linked to produce alpha beta dimer by photolysis. 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, two new bands (106 and 61 kDa) of lower electrophoretic mobility appeared in addition to the alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis, and the 125I-hormone derivative specifically bound to cells bearing the receptor. Binding could be blocked by excess untreated follitropin but not with human choriogonadotropin and thyrotropin. Under nonreducing conditions, one major band (104 kDa) of cross-linked complexes appeared. Upon reduction with dithiothreitol and second-dimensional electrophoresis, the 104-kDa band produced two smaller complexes of 75 and 61 kDa, indicating the loss of two components and the existence of intercomponent disulfides. Successful production of the 104-kDa complex requires blocking of free sulfhydryl groups with N-ethylmaleimide. It is, however, independent of various protease inhibitors or the temperature and the time period of hormone incubation with cells or the plasma membrane fraction. The mass estimates and the interaction with the hormone of the photoaffinity-labeled components are discussed.

Affinity Labels↗
PubMed1985

Composition and peptide maps of cross-linked human choriogonadotropin-receptor complexes on porcine granulosa cells.

Radioiodinated human choriogonadotropin was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to the hormone receptor on porcine granulosa cells and the solubilized sample was electrophoresed. Cross-linked samples revealed four additional bands of slower electrophoretic mobility in addition to the hormone alpha, beta, and alpha beta dimer bands. The four bands corresponded to masses of 68, 74, 102, and 136 kDa whereas the alpha beta dimer band corresponded to 50 kDa. Formation of the four bands requires the 125I-hormone to bind specifically to the receptor with subsequent cross-linking. Binding can be prevented by excess of native hormone but not by follitropin. A monofunctional analog of the cross-linking reagent failed to produce the four bands. They were also produced by cross-linking Triton X-100-solubilized hormone-receptor complexes. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of the cross-linked sample were treated with reagents that cleave covalent cross-links and then electrophoresed in a second dimension gel, 18-, 24-, 28-, and 34-kDa components were released, in addition to the alpha and beta subunits of the native hormone. Simultaneous peptide mapping of the cross-linked complexes in the gel matrix with Staphylococcus V8 protease or papain revealed progressive proteolysis to generate terminal fragments of 30 or 27 kDa, respectively. These fragments were unique to and commonly present in the 74-, 102-, and 136-kDa hormone-receptor complexes but were not produced by proteolysis of the cross-linked human choriogonadotropin (hCG) alpha beta dimer or the hCG alpha subunit. Apparently, the radioactively labeled segment(s) of the alpha subunit of 125I-hCG was cross-linked to the 24-kDa component. The results demonstrate the protein nature of the receptor and suggest that 125I-hCG was initially cross-linked to the 24-kDa component to generate the 74-kDa complex, then the 28- and 34-kDa components were sequentially cross-linked to the 24-kDa component in the 74-kDa complex to generate the 102- and 134-kDa complexes.

Animals↗
PubMed1985

Composition of cross-linked 125I-follitropin-receptor complexes.

Both of the alpha and beta subunits of intact human follitropin (FSH) were radioiodinated with 125I-sodium iodide and chloramine-T and could be resolved on sodium dodecyl sulfate-polyacrylamide gels. Radioiodinated FSH was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to its membrane receptor on the porcine granulosa cell surface as well as to a Triton X-100-solubilized form of the receptor. Cross-linked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65, 83, and 117 kDa, in addition to the hormone bands. The hormone alpha beta dimer band corresponded to 43 kDa. Formation of the three bands requires the 125I-hormone to bind specifically to the receptor with subsequent cross-linking. Binding was prevented by an excess of the native hormone but not by other hormones. A monofunctional analog of the cross-linking reagent failed to produce the three bands. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of cross-linked complexes were treated to cleave covalent cross-links and then electrophoresed in a second dimension, 18-, 22-, and 34-kDa components were released, in addition to the alpha and beta subunits of the hormone.

Animals↗
PubMed1985

Intersubunit disulfides of the follitropin receptor.

The electrophoretic mobility of radioiodinated follitropin (FSH) alpha and beta subunits as well as the alpha beta dimer changed markedly depending on the concentration of reducing agents such as dithiothreitol. The changes were more dramatic in the beta subunit than in the alpha subunit. 125I-FSH, complexed to the receptor on porcine granulosa cells or in Triton X-100 extracts, was cross-linked with a cleavable (nondisulfide) homobifunctional reagent, solubilized in sodium dodecyl sulfate without reducing agents, and electrophoresed. The cross-linked sample revealed three bands of high molecular mass, in addition to the hormone subunit and dimer bands. The band of lightest mass, 110 kDa, was the major band and the other two of 76 and 62 kDa were barely noticeable. Upon reduction with dithiothreitol, the 110-kDa band decreased while the 76- and 62-kDa bands increased, indicating the existence of disulfides between components of the 110-kDa complex. Formation of the disulfide-linked complexes requires 125I-FSH, specifically bound to the hormone receptor and cross-linking, and can be prevented with an excess of native FSH but not human choriogonadotropin. Complex formation was independent of blocking free sulfhydryl groups with N-ethylmaleimide. When the cross-linked complexes were reduced in the gel matrix and analyzed on fresh gels, the 76- and 62-kDa complexes were generated from the 110-kDa band, indicating the loss of two components. The lost components were estimated to be at 14 and 34 kDa. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22-, 14-, and 34-kDa components to the FSH alpha beta dimer. The results of reduction of the cross-linked complexes demonstrate the existence of disulfide linkage between the three components.

Animals↗
PubMed1985

Aggregation state of the gonadotropin receptor.

The aggregation state of the gonadotropin receptor has been examined by coupling fluorescence energy transfer donor and acceptor fluorophores to hCG and LH. Energy transfer is observed at low (4 degrees C) but not at high (37 degrees C) temperature. Energy transfer could also be detected with receptor solubilized in the presence of hormone at the lower temperature only. Solubilization of receptor in the absence of hormone and subsequent addition of hormone conjugates revealed no energy transfer. These results are consistent with stabilization of receptor complexes at low temperatures, but presumptive hormone induced receptor dissociation under physiological conditions.

Chemical Phenomena↗
PubMed1985

Radioiodination of a photoactivatable heterobifunctional reagent.

The N-hydroxysuccinimide ester of 4-azidosalicylic acid, a photoactivable heterobifunctional reagent, can be radioiodinated. The low efficiency (3%) of the radioiodination by a previously published method (I. Ji and T. H. Ji, 1982, Anal. Biochem. 121, 286-289) has been increased to 63% by substituting the solvent, acetone, with others such as aqueous acetonitrile, dimethylformamide, or dimethyl sulfoxide. The resulting 125I reagent was used for derivatizing human choriogonadotropin. The radioactive hormone derivative was crosslinked to the alpha beta dimer upon photolysis.

Azides↗
PubMed1983

Chemical crosslinking of cell membranes.

The complexity of cell membranes makes the resolution of their macromolecular topology one of the more challenging problems in modern molecular and cellular biochemistry. Despite the difficulties inherent in any such analysis, a surprisingly simple yet powerful approach exists that has consistently yielded valuable results. This method is chemical crosslinking, in which cell membranes are treated with crosslinking reagents (usually bifunctional) which produce covalent linkages between membrane components. The resultant complexes are usually then separated and identified by electrophoresis. This review is intended to provide a guide to the investigator who is unfamiliar with this approach. The overall strategy of crosslinking is discussed including selection of reagents, conditions to optimize crosslinking and the cleavage of crosslinked complexes to regenerate the original target for identification purposes. The crosslinking of biological membranes is then reviewed with special emphasis on recent advances including macromolecular photoaffinity labeling, kinetic analysis to probe symmetry properties and potential artifacts that may complicate interpretation of results. Examples of specific applications of crosslinking to membranes are presented in tabular form. The final portion of the review discusses the synthesis and properties of the most widely employed crosslinking reagents. Available reagents are summarized in a series of comprehensive tables. It is hoped that our discussion will provide the uninitiated investigator with sufficient information to ascertain the applicability of chemical crosslinking to particular areas of interest.

Affinity Labels↗
PubMed1981

Synthesis and application of cleavable photoactivable heterobifunctional reagents.

Three different cleavable photoactivable heterobifunctional reagents have been synthesized and characterized by proton NMR, 13C NMR, mass spectroscopy, infrared spectroscopy, and thin-layer chromatography. They are the N-hydroxysuccinimide ester of 3-[(4-azidophenyl)dithio]-propionic acid, methyl 3-[(4-azidophenyl)dithio]propionimidate, and N-[(4-azidophenyl)thio]phthalimide. Concanavalin A was coupled with one of the reagents, the N-hydroxysuccinimide ester of 3-[(4-azidophenyl)dithio]propionic acid, and radioiodinated. The [125I]lectin derivative and [125I]lectin showed similar specific binding to the receptors on the human erythrocyte membrane. Upon photolysis, subunits of the [125I]lectin derivative were cross-linked to produce dimer, trimer, and tetramer. These cross-linked complexes were readily cleaved by reducing the disulfide bond of the reagent.

Azides↗
PubMed1981

Structure of the lutropin receptor on granulosa cells. Photoaffinity labeling with the alpha subunit in human choriogonadotropin.

Ovarian granulosa cells have specific receptors for lutropin and human choriogonadotropin (hCG). To investigate the nature of the receptor, several different photo-cross-linkable derivatives of radioiodinated hCG were incubated with porcine granulosa cells and hormone derivative-receptor complexes extracted with 0.5% Triton X-100. The extracted complexes appeared in a single peak on gel permeations and in rat zonal sedimentation studies. The estimated molecular weight was found to be 250,000-370,000 with an associated s value of 7.7 +/- 0.3. When Triton X-100 extracts were examined by gel electrophoresis, the autoradiograph revealed four bands. Three were conspicuous, corresponding for molecular weights of 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000, but a fourth band, corresponding to a molecular weight of 120,000 +/- 6,000, was less well defined. Reducing agents, 20 mM dithiothreitol and 2% beta-mercaptoethanol, acid, and 3 M urea did not affect the production of the bands. Furthermore, the bands once formed were resistant to these agents, indicating 1) covalent nondisulfide bond formation in photoaffinity labeling and 2) noncovalent association of photoaffinity-labeled membrane components with the natural hormone receptor.

Animals↗