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The high specificity of malic enzyme (ME; EC 1.1.1.40) from grape berries (Vitis vinifera L.) for the naturally occurring L-enantiomer of malic acid, its very selective C4-decarboxylation, and certain allosteric properties, reported previously, favour the conjecture of a regulatory function of ME in fruit malic acid degradation. On the other hand, high ME activity was detected even during the acid-accumulating phase of berry development. Also, the in vitro reversibility of the reaction supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate, notably high CO2/HCO 3 (-) and NADPH/NADP ratios. However, a very limited incorporation of (14)C into malate and the uniform labeling pattern of the dicarboxylic acid after administration of [U-(14)C] alanine to grape berries before and after the onset of ripening, indicate that the 'reverse" reaction does not contribute essentially to grape malate synthesis. A regulatory mechanism mediating malic acid remetabolization on the basis of cosubstrate availability, comparable to the control of the hexose monophosphate shunt, is discussed.

Multiple isoforms of beta-fructofuranosidase (invertase, EC 3.2.1.26) were identified in mature green leaves of the cruciferous plant Arabidopsis thaliana (L.) Heynh. There were four major and one minor isoforms of soluble acid invertase and an additional activity which could be released from the cell wall by buffers of high ionic strength. This study reports the separation and characterisation of three soluble isoforms following ammonium sulphate and polyethylene glycol 6000 precipitations, Concanavalin A, MonoQ ion exchange, Superose 12 size-exclusion chromatography and chromatofocusing. These isoforms, designated INV1, INV2 and INV3, had isoelectric points of 4.75, 4.70 and 4.65 and a Km for sucrose of 5, 12 and 5 mM, respectively. Each had a pH optimum of 5.5, exhibited optimal activity at 45 degrees C and used sucrose as the preferred substrate. All fractions containing these isoforms contained a 52-kDa polypeptide which was specifically detected by immunoblotting with an antibody raised against deglycosylated wheat invertase. The N-terminal amino-acid sequence of this polypeptide was homologous to acid invertases isolated from other plant species. The possible origin of isoforms of soluble acid invertase is discussed.

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