Sucrose protects cell wall invertase but not vacuolar invertase against proteinaceous inhibitors

Abstract

Vacuolar (VI) and cell wall invertases (CWI) of higher plants can be inactivated in vitro and, possibly, in vivo by proteinaceous inhibitors. The respective mechanisms have not yet been compared. Therefore, partially purified CWI from transformed tobacco cells and VI from tomato fruit were preincubated with invertase-inhibitor fractions isolated from the same tissues. Both inhibitors were able to inhibit both invertases. However, VI was fully inhibited within less than 1 rain by both inhibitors, whereas inactivation of CWI was much slower. Furthermore, CWI, but not VI, was strongly protected against inhibition by sucrose. A polyclonal antiserum directed against the tobacco inhibitor (INT) cross-reacted with a 19 kDa polypeptide in the partially purified tomato inhibitor (ILE) fraction. The results indicate that INT and ILE have similar structural properties, whereas the mechanism of inactivation is clearly different for CWI and VI.Key words: Cell wall invertase; Vacuolar invertase; Invertase inhibitor; Substrate protection; Solanaceae millimolar concentrations and exhibits a pronounced pH-dependence between 4 and 6, (iii) CWI may be protected against inhibition by sucrose (half maximum protection at 1.2 mM), and (iv) CWI and lit form tight complexes in sucrose-starved cells. INT has been purified to homogeneity and a polyclonal antiserum has been raised [5,18]. In view of (i) the inconsistencies of previous studies on the localization of invertases and inhibitors, and (ii) the significant structural differences between CWI and VI, we have addressed the following question: do invertase inhibitors inactivate CWI and VI by the same mechanism? We have partially purified inhibitors from tomato fruit [20], ILE, and transformed tobacco cells [18], INT, and characterized the inactivation of two plant invertases, i.e. VI from tomato fruit (in this tissue 3 highly similar VI isoforms are co-expressed [3]) with a Km of 7.4-7.9 mM and a pI of about 5, and CWI from tobacco cells with a Km of 0.6 mM and a pI of 9.5 [7]. The results indicate conspicuous differences in the mechanisms of inactivation.

Cloning of a Tobacco Apoplasmic Invertase Inhibitor1

“…However, the effective inhibitor concentration required for full inhibition could de facto be significantly lower since the degree of on-column renaturation of the recombinant Nt-inh1-protein could not yet be determined. A significant substrate protection was observed when Suc was present during the preincubation, in agreement with the inhibition characteristics determined earlier for the partially purified INH protein isolated from transformed tobacco cells (Weil et al, 1994;Sander et al, 1996). To further confirm the specificity of invertase inhibition, we analyzed the effect of the recombinant inhibitor .…”

“…The inhibition of tobacco CWI and tomato fruit VI by tobacco INH isolated from tobacco cells (Weil et al, 1994;Sander et al, 1996) could be faithfully reproduced by the recombinant Nt-inh1 protein expressed in E. coli (Figs. 5 and 6; Table I).…”

“…Tobacco CWI and tomato fruit VI were prepared as previously described (Weil and Rausch, 1994;Sander et al, 1996). Inhibition assays followed the protocol of Weil et al (1994).…”

Atividade amidolítica e de invertase ácida solúvel em tubérculos de batata armazenados sob duas condições de temperatura

“…Esses pesquisadores observaram que o nível do inibidor da invertase diminui quando exposto a baixas temperaturas aumentando novamente com a exposição a altas temperaturas. Sabe-se que a localização da invertase e o sítio de aumento na produção de hexose podem causar profundos efeitos na fisiologia do tubérculo (Sonnewald et al, 1997).…”

“…The activity of specific invertase was higher in tubers of the autumn crop due to the exposure to low temperatures before harvesting. lular conduz a uma drástica redução no conteúdo de sacarose do tubérculo e a um correspondente aumento no conteú-do de glicose (Sonnewald et al, 1997). Em muitas espécies de plantas, a atividade da invertase ácida aumentou em resposta a fitohormônios (Morris e Arthur, 1984;Ehness e Roitsch, 1997).…”