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Association of tubulin carboxypeptidase with microtubules in living cells.

ABSTRACT: Tubulin carboxypeptidase is the enzyme that releases the C-terminal tyrosine from alpha-tubulin, converting tyrosine-terminated (Tyr) to detyrosinated (Glu) tubulin. The present study demonstrates that this enzyme is associated with microtubules in living cells. We extracted cultured cells (COS-7) with Triton X-100 under microtubule-stabilizing conditions and found tubulin carboxypeptidase activity in the cytoskeleton fraction. We ruled out, by using several control experiments, the possibility that this result was due to contamination of the isolated cytoskeletons by non-associated proteins contained in the detergent fraction or to an artifact in vitro during the extraction procedure. The associated carboxypeptidase activity showed characteristics similar to those of brain tubulin carboxypeptidase and different from those of pancreatic carboxypeptidase A. In comparison with cultures at confluence, those at low cell density contained small (if any) amounts of carboxypeptidase activity associated with microtubules. In addition, the enzyme was shown to be associated only with cold-labile microtubules. The tubulin carboxypeptidase/microtubule association was also demonstrated in Chinese hamster ovary, NIH 3T3 and PC12 cells. Interestingly, this association was not observed in cultured embryonic brain cells. Our results demonstrate that tubulin carboxypeptidase is indeed associated with microtubules in living cells. Furthermore, the findings that this association occurs with a subset of microtubules and that its magnitude depends on the degree of confluence of the cell culture indicate that it could be part of the mechanism that regulates the tyrosination state of microtubules.

SUBMITTER: Contin MA

PROVIDER: S-EPMC1220178 | biostudies-other | 1999 Apr

REPOSITORIES: biostudies-other

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Project description:We report the results of experimental investigations involving photobiomodulation (PBM) of living cells, tubulin, and microtubules in buffer solutions exposed to near-infrared (NIR) light emitted from an 810 nm LED with a power density of 25 mW/cm2 pulsed at a frequency of 10 Hz. In the first group of experiments, we measured changes in the alternating current (AC) ionic conductivity in the 50-100 kHz range of HeLa and U251 cancer cell lines as living cells exposed to PBM for 60 min, and an increased resistance compared to the control cells was observed. In the second group of experiments, we investigated the stability and polymerization of microtubules under exposure to PBM. The protein buffer solution used was a mixture of Britton-Robinson buffer (BRB aka PEM) and microtubule cushion buffer. Exposure of Taxol-stabilized microtubules (~2 μM tubulin) to the LED for 120 min resulted in gradual disassembly of microtubules observed in fluorescence microscopy images. These results were compared to controls where microtubules remained stable. In the third group of experiments, we performed turbidity measurements throughout the tubulin polymerization process to quantify the rate and amount of polymerization for PBM-exposed tubulin vs. unexposed tubulin samples, using tubulin resuspended to final concentrations of ~ 22.7 μM and ~ 45.5 μM in the same buffer solution as before. Compared to the unexposed control samples, absorbance measurement results demonstrated a slower rate and reduced overall amount of polymerization in the less concentrated tubulin samples exposed to PBM for 30 min with the parameters mentioned above. Paradoxically, the opposite effect was observed in the 45.5 μM tubulin samples, demonstrating a remarkable increase in the polymerization rates and total polymer mass achieved after exposure to PBM. These results on the effects of PBM on living cells, tubulin, and microtubules are novel, further validating the modulating effects of PBM and contributing to designing more effective PBM parameters. Finally, potential consequences for the use of PBM in the context of neurodegenerative diseases are discussed.

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Association of tubulin carboxypeptidase with microtubules in living cells.

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