Tae H. Ji

Macromolecular photoaffinity labeling with radioactive photoactivable heterobifunctional reagents

Abstract Three new photoactivable heterobifunetional reagents which can be radioidinated have been synthesized. One of the radioactive reagents, the N -hydroxysuccinimide ester of 4-azidosalicylic acid was coupled to concanavalin A and the lectin derivative was used for the photoaffinity labeling of the lectin receptor on the erythrocyte ghost membrane.

Interference by detergents, chelating agents, and buffers with the Lowry protein determination.

Abstract EDTA distorts the Lowry method even at the concentration of 0.5 m m . The effect of EDTA, glycine, glycylglycine, Tricine, and Tris on the Lowry method can be calibrated only at a fixed concentration of the chemicals. The effect of succinic acid, sodium citrate, and Bicine on the Lowry method changes depending on the concentration of the chemicals, but the changes were not as significant as in the case of the chemicals mentioned above. Sodium phosphate at pH 8.9, sodium dodecyl sulfate, sucrose, and urea do not affect the Lowry method when a reference containing only the chemical is used.

Analyses of ovine corpora lutea for tumor necrosis factor mRNA and bioactivity during prostaglandin-induced luteolysis

It has been suggested that tumor necrosis factor (TNF) participates in the mechanism of regression of the corpus luteum. We measured luteal expression of TNF alpha mRNA and biological activity during prostaglandin-induced luteolysis in sheep. Initiation of functional luteolysis was marked by a sharp decline in concentrations of progesterone in luteal tissue beginning 4 h after administration of luteolysin. Structural regression of corpora lutea was manifested by a reduction in glandular weight at 16 h. A luteal cytotoxic factor with TNF alpha-like bioactivity was isolated after the decrease in tissue progesterone had occurred, but before evidence of luteal resorption. We were unable to detect temporal alterations in TNF alpha mRNA in luteal samples by classical Northern blot or in situ hybridization analyses. These results imply that luteal TNF alpha is derived primarily as a preformed entity from an extraovarian source, such as infiltrating leukocytes. These results raise the possibility that this cytokine might not be involved in the early stages of luteal regression in the ewe, yet could play a secondary role, perhaps in the subsequent opsonization and removal of degenerating cells.

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.

Gene, interaction, signal generation, signal divergence and signal transduction of the LH/CG receptor

Trophoblastic neoplasms and choriocarcinoma cells express high levels of the hCG receptor. The hCG receptor is encoded by a single gene in chromosome 2p21‐p16, spanning over −70 kb with 11 exons and 10 introns. Multiple mRNA species are produced from the gene utilizing two proximal promoters and several Sp‐1 elements as well as proximal and distal suppressors. In fact, regulatory proteins which bind to one of these suppressors are expressed less in choriocarcinoma cell lines than in placenta.

Molecular mechanism of LH/CG receptor activation

It is known that the N-terminal half of the LH/CG receptor is responsible for high hCG binding whereas the C-terminal half is capable of receptor activation. Our results suggest that initial hCG binding at the high affinity site in the N-half receptor induces conformational adjustments. This leads to low affinity secondary contacts of the complex of hCG/the N-half receptor with the C-half receptor. This low affinity secondary contact is responsible for activating the receptor. This is based on the following observations. The C-terminal tail of hCG alpha is known to be involved in activation of the LH/CG receptor. In addition to hCG, we examined the C-terminal three residues (His90-Lys91-Ser92) of the common alpha subunit of FSH and TSH. The results show their differential roles in the three hormones. Ser92 is important for binding and cAMP induction of TSH but not for hCG and FSH. Lys91 is important for binding and cAMP induction of hCG, and cAMP induction but not binding of FSH. It is not important for binding or cAMP induction of TSH. His90 is important for all three hormones. When all three residues were truncated, FSH and TSH lose their affinity for binding and cAMP induction, whereas hCG is still capable of binding but not cAMP induction. Therefore, the three amino acids contribute differently in receptor binding and cAMP induction of hCG, FSH and TSH. Our data also indicate that the evolution of the alpha subunit has been constrained in order not to impair any of the hormones. This suggests that each hormone can be independently engineered to improve the potency. To chemically identify the contact site of the alpha C-tail of hCG in the LH/CG receptor, a decamer peptide corresponding to the alpha subunit sequence from His83 to Ser92 (peptide alpha 81-92) was derivatized with UV sensitive reagent, ABG and radio-iodinated. The resulting ABG-125I-peptide alpha 83-92 was capable of binding and activating the LH/CG receptor. Furthermore, it specifically photoaffinity-labeled the LH/CG receptor. In addition, the amino group of alpha Lys91 of peptide alpha 83-92 is crosslinked to a carboxyl group of the receptor, an indication of close association. Reciprocal mutagenesis of alpha Lys91 and Asp397 in exoloop 1 of the LH/CG receptor suggests the complementary of this pair in receptor activation but not the high affinity interaction of hCG and the receptor. In addition, Lys583 of exoloop 3 is also crucial for receptor activation. To test the conformational adjustment, ABG was attached to hCG alpha and reassociated with untreated beta to produce ABG-125I-alpha/beta. The extent of inter-subunit crosslinking of ABG-125I-alpha/beta bound to the receptor was two to three fold less than unbound ABG-125I-alpha/beta. This result indicates structural change at the subunit interface in response to hCG binding to the receptor.

Electrofractionation : a technique for detecting and recovering biomolecules

Electrofractionation (EF) is a technique that allows electrophoretic materials to be detected and recovered following electrophoresis. The EF apparatus utilizes the resolving power of electrophoresis and the mobile phase of liquid chromatography to create a continuous elution system. Our data indicate that EF is able to detect and recover oligonucleotides, as DNA fragments, proteins, and presumably other material that can be analyzed by electrophoresis. EF shares functional similarities with high-performance electrophoresis chromatography (HPEC) but operates by a different strategy and at a fraction of the cost. Moreover, EF can be constructed largely from standard laboratory equipment. The simplicity, rapid analysis times, low cost, and high recovery yields of EF make this system a practical alternative to the conventional detection and purification methods used for biomolecules.