Cytosolic and cell-wall-bound acid invertases from leaves of Urtica dioica L.: a comparison

Abstract

Two different forms of acid invertase (EC 3.2.1.26) were extracted from expanding leaves of the stinging nettle (Urtica dioica L.). One form was soluble and could be localized within the cytosol, whereas the other was ionically bound to the cell-wall and could not be detected in protoplasts. Both forms were purified, the latter to homogeneity. Western blotting with antibodies against the pure enzyme from cell walls was positive with the cell-wall enzyme but negative with the soluble form of acid invertase. Both forms are glycoproteins with identical molecular weights of 58 kDa. The Km values for sucrose (raffinose) are 5 mM (4.8 mM) for the soluble and 1.2 mM (3.6 mM) for the cell-wall-bound enzyme. The pH optimum of the latter is slightly more acidic (4.5) than that of the soluble invertase (5.5). Both forms could easily be distinguished by their isoelectric points which were determined at pH 4.6 for the soluble and pH 9.3 for the wall-bound enzyme. When extraction and purification were carried out in the absence of protease inhibitors, both acid invertases showed microheterogeneity ('multiple forms'). However, with benzamidine and phenylmethylsulfonylfluoride as protease inhibitors each invertase produced only one protein band upon isoelectric focusing and gel electrophoresis, respectively.

Induction of Apoplastic Invertase of Chenopodium rubrum by D-Glucose and a Glucose Analog and Tissue-Specific Expression Suggest a Role in Sink-Source Regulation

“…The activities were considerably lower than the activity of the extracellular invertase. The observed differential regulation of the activity of the two acidic invertases supports protein data (Fahrendorf and Beck, 1990) that the intracellular form is not the precursor of the cell-wall-bound extracellular isoenzyme.…”

Cloning of cDNA for a cell wall-bound acid invertase from tomato (Lycopersicon esculentum) and expression of soluble and cell wall-bound invertases in plants and wounded leaves of L. esculentum and L. peruvianum.

“…Soluble acid invertases are believed to control levels of hexoses in sink (Schaffer et al, 1987;Yelle et al, 1991;Klann et al, 1993Klann et al, , 1996Ohyama et al, 1995;Davies and Robinson, 1996;Zrenner et al, 1996) and source (Scholes et al, 1996) tissues. Cell wall-bound (extracellular or apoplastic) acid invertases are ionically bound to cell walls (Hisajima and Arai, 1978;Krishnan et al, 1985;Karuppiah et al, 1989;Fahrendorf and Beck, 1990); they have acidic pH optima and basic pIs. It has been proposed that cell wall-bound acid invertases participate in phloem unloading (Miron and Schaffer, 1991;Dali et al, 1992;Ruan and Patrick, 1995;Ho, 1996;Brown et al, 1997), generation of sink strength (Weber et al, 1995(Weber et al, , 1996Cheng et al, 1996;Vizzotto et al, 1996), and gravitropism (Wu et al, 1993).…”

Tissue level localization of acid invertase in leaves: an hypothesis for the regulation of carbon export

“…Acid invertase occurs in both a soluble form localized in the vacuole, and a cell-wall-bound form (Lyne & ap Rees, 1971;Leigh et al, 1979;Vattuone, Prado & Sampietro, 1981;Faye, Moutassim & Ghorbel, 1986;Fahrendorf& Beck, 1990;RufTner et al, 1990;Ranwala, Suematsu & Masuda, 1992;Salzer & Hager, 1993;Walker & Pollock, 1993). It has been suggested that acid invertase could act as an effector of partitioning by influencing sucrose metabolism via futile cycling (Huber, 1989), or by modulation of the transport path where phloem loading involves an apoplastic stage (Foyer, 1987).…”

“…A range of molecular weights for proteins crossreacting with the antibodies were detected by the more sensitive ECL detection protocol. There are other indications of multiple forms of acid invertase resulting from post-translationai modification in radish (Faye et al, 1986) or from proteolysis in Urtica dioica (Fahrendorf & Beck, 1990). The use of different measurement techniques has also resulted in a range of estimates of native molecular weight (Pollock & Lloyd, 1977;Krishnan, Blanchette & Okita, 1985).…”