MICROTUBULAR PROTEIN IN ITS POLYMERIZED OR NONPOLYMERIZED STATES DIFFERENTIALLY MODULATES IN-VITRO AND INTRACELLULAR FLUXES CATALYZED BY ENZYMES OF CARBON METABOLISM

The fluxes through HK/G6PDH and PK/LDH coupled-enzymatic reactions wer e quantified in the presence of physiological concentrations (1-15 mu M) of polymerized or non-polymerized microtubular protein (MTP) from r at brain and in a permeabilized yeast cell system. In vitro enzymatic fluxes were increased by either polymerized or nonpolymerized brain MT P mainly in the lower range of MTP concentration. At fixed MTP concent rations in the flux stimulatory range of HK/G6PDH (1 mg/ml MTP) or PK/ LDH (0.4 mg/ml MTP), a hyperbolic and sigmoidal response to NADP and P EP, respectively, was detected. That dependence varied according to th e polymeric status of MTP. The specificity of the phenomenon observed in vitro, was tested for the PK/LDH and HK/G6PDH enzymatic couples in the presence of neutral polymers such as glycogen (less than or equal to 10 mg/ml), poly(ethylene glycol) (up to 10% w/w) or G-actin (less t han or equal to 1 mg/ml). In permeabilized Saccharomyces cerevisiae ce lls, the PK-catalyzed flux was sensitive to microtubule disruption by nocodazole (15 mu g/ml). The HK/G6PDH system was not affected by nocod azole showing values of kinetic parameters close to those obtained in vitro in the presence of polymerized brain MTP. Indirect immunofluores cence with specific antibodies against tubulin allowed to confirm the microtubules disruption in the presence of nocodazole in permeabilized yeast cells under the same conditions in which enzymes were assayed i ntracellularly. The experimental evidence is in agreement with the obs erved phenomenon of increase in fluxes in the enzymatic reactions assa yed to be specifically induced by MTP either in vitro or in situ. The results presented are discussed in terms of the assembly of large supr amolecular structures as a supraregulatory mechanism of synchronizatio n of systemic cellular processes such as metabolic fluxes. (C) 1994 Wi ley-Liss, Inc.