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Purification and characterization of a DNA-binding heterodimer of 52 and 100 kDa from HeLa cells.

ABSTRACT: In studies of protein binding to the upstream region of the human proliferation-associated antigen p120 gene, a heterodimer of 52 and 100 kDa proteins was purified from HeLa cells. A 1:1 ratio of p52 and p100 was constant throughout the purification. The heterodimer was localized to cell nuclei, as shown by immunofluorescence. The pI values of the p52 and p100 were 7.8 and 8.6 respectively. The peptide sequences obtained for p52 (QSNKTFNLEKQNHTPRKKHQ and PLRGKQLRVRFAAHSASLTVR) and for p100 (PGGPKPGGGPGLSTPGGHPKPPHRGGGEPPRGRQ and GPGPGQSGPKPPIPPPPPHQQ) were not found in the computer databanks. One p52 peptide sequence, PLRGKQLRVRFA, shows considerable sequence similarity to a conserved motif in topoisomerase II of multiple species. The p52/100 heterodimer bound to different DNA probes. The binding was competed by poly(dI-dC), sonicated salmon sperm DNA, and circular or linearized plasmid DNA. The optimal DNA binding for the heterodimer was at pH 7-9 with low salt. The DNA-binding subunit of the heterodimer was the p100 polypeptide, as shown by u.v.-cross-linking assays and Southwestern blots.

SUBMITTER: Zhang WW

PROVIDER: S-EPMC1132410 | biostudies-other | 1993 Feb

REPOSITORIES: biostudies-other

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Project description:Thiadiazoles are one of the most widely utilized agents in medicinal chemistry, having a wide range of pharmacologic activity. Microtubules (MTs) have always remained a sought-after target in rapidly proliferating cancer cells. We screened for the growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on cancer cells and identified NMK-TD-100, as the most potent agent. Cell viability experiments using human cervical carcinoma cell line (HeLa cells) indicated that the IC50 value was 1.42±0.11 µM for NMK-TD-100 for 48 h treatment. In further study, we examined the mode of interaction of NMK-TD-100 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. NMK-TD-100 induced arrest in mitotic phase of cell cycle, caused decline in mitochondrial membrane potential and induced apoptosis in HeLa cells. Immunofluorescence studies using an anti-α-tubulin antibody showed a significant depolymerization of the interphase microtubule network and spindle microtubule in HeLa cells in a concentration-dependent manner. However, the cytotoxicity of NMK-TD-100 towards human peripheral blood mononuclear cells (PBMC) was lower compared to that in cancer cells. Polymerization of tissue purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC50 value of 17.5±0.35 µM. The binding of NMK-TD-100 with tubulin was studied using NMK-TD-100 fluorescence enhancement and intrinsic tryptophan fluorescence of tubulin. The stoichiometry of NMK-TD-100 binding to tubulin is 1:1 (molar ratio) with a dissociation constant of ~1 µM. Fluorescence spectroscopic and molecular modeling data showed that NMK-TD-100 binds to tubulin at a site which is very near to the colchicine binding site. The binding of NMK-TD-100 to tubulin was estimated to be ~10 times faster than that of colchicine. The results indicated that NMK-TD-100 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided insights into its potential of being a chemotherapeutic agent.

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Purification and characterization of a DNA-binding heterodimer of 52 and 100 kDa from HeLa cells.

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