Regulation of cytoplasmic tubulin carboxypeptidase activity in vitro by cations and sulfhydryl-modifying compounds

Abstract

alpha-tubulin subunits within microtubules (MTs) can be post-translationally detyrosinated by a tubulin-specific carboxypeptidase (TCP) activity to form biochemically distinct MTs. Attempts to characterize and purify TCP have suffered from the inability to detect low levels of activity and to distinguish TCP from other, competing enzyme activities. We recently developed an assay for TCP [Webster et al. (1992) Biochemistry 31:5849] that uses taxol-stabilized MTs as the substrate. In this study, we exploited the increased sensitivity and specificity of this new assay to explore the effects of various agents that might act to either stimulate or inhibit this enzyme in vitro. We tested a variety of both monovalent and divalent cations for their ability to affect TCP, and tested whether the cations were affecting the enzyme, the substrate, or both. We found that TCP displayed salt-sensitive binding to MTs, characteristic of other, more well characterized MT-associated proteins. While both calcium and magnesium stimulated TCP activity over a narrow concentration range (2-10 mM), they inhibited activity at higher concentrations. Other divalent cations tested, including zinc, copper, and cobalt, inhibited TCP at virtually all concentrations tested, but to different levels (zinc > copper > cobalt). Most of the zinc-induced TCP inhibition was attributed to the interference with the normal binding of TCP to MTs. In addition, we examined the involvement of free sulfhydryl groups (which are important for the activities of many types of enzymes) in TCP activity by the addition of sulfhydryl-modifying compounds during the assay, and found that their addition reduced TCP activity mainly (but not solely) by their action on the extract that contained the TCP. Finally, we tested the ability of DL-benzylsuccinic acid, a potent inhibitor of carboxypeptidase A, to inhibit TCP. While carboxypeptidase A has been found, in other studies, to be inhibited by micromolar concentrations, TCP was affected only at concentrations above 20 mM, adding another proof that carboxypeptidase A and TCP are distinct enzyme activities.

Alteration of the C-terminal Amino Acid of Tubulin Specifically Inhibits Myogenic Differentiation

“…In Vitro Assay of Tubulin Carboxypeptidase Activity-Preparation of brain TCP, [ 14 C]tyrosine-labeled MT substrate, and the TCP assay, were described previously (25,26). Briefly, [ 14 C]tyrosine-labeled tubulin dimers were self-assembled in MHM (25 mM MOPS, 25 mM HEPES, pH 7.5, 5 mM MgCl 2 ) for 20 min, stabilized by Taxol (20 M, 5 min), and centrifuged, all at 37°C (436,000 ϫ g, 4 min, Optima TL centrifuge; Beckman Instruments) to yield a MT substrate pellet.…”

“…For the first incubation step, i.e. binding of TCP to MT substrate, the pellet from the previous step was incubated in MHM (10 min, 37°C) with 5 volumes of crude brain TCP and 20 M Taxol (26), and the mixture was centrifuged as before, except at 25°C. For the second incubation step, measuring TCP activity, the pellet from the previous step was resuspended in MHM (30 min, 37°C), proteins were precipitated with 10% trichloroacetic acid (w/v) at 4°C, centrifuged (245,000 ϫ g, Optima TL), and radioactivity of trichloroacetic acid-soluble (i.e.…”

Tubulin Post‐Translational Modifications

“…The carboxypeptidase described by Webster et al [38], which undergoes a 60-80% decrease in activity upon reduction in pH to 5.4, appears to modify polymerized, as opposed to soluble tubulin, although substrates were not compared under identical conditions. Rather than mixing soluble tubulin with cell-free extracts, it would have been preferable to strip the carboxypeptidase from microtubules, this appears to be possible [39], and test the enzyme so obtained with soluble tubulin under conditions the same as those employed in assays where microtubules were used. A tubulin-reactive carboxypeptidase has been partially purified from Artemia and shown to be more active with soluble tubulin than taxol-stabilized microtubules, but its role in tubulin modification in vivo is uncertain A tubulin-reactive carboxypeptidase from rat brain has affinity for microtubules [37,40,421 and centrifugation of samples to remove microtubules depletes carboxypeptidase activity in the resulting supernatants [37].…”

Environmental Degradation at a Public Park in Southern Brazil as Revealed Through a Genotoxicity Test (MN) on Peripheral Blood Cells from Poecilia vivipara (Teleostei)

“…Tubulins (components of the mitotic spindle) are possibly responsible for genotoxic effects (MN) due to the negative interference of heavy metals on motor proteins . Zinc, copper, and cobalt reduce the numbers of sulfhydryl groups in the tubulins, which may be responsible for their effects on tubulin polymerization (Webster and Oxford 1996). Taking into consideration all of these possibilities of genotoxic intervention, the high frequency of micronuclei demonstrated in the present study suggests that copper could be acting as an aneugenic and/or clastogenic agent.…”