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Acid invertase in Nicotiana tabacum crown-gall cells: Molecular properties of the cell-wall isoform

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Abstract

Cell-wall invertase (CWI; EC 3.2.1.26) was salt-eluted from non-disrupted Agrobacterium tumefaciens-transformed Nicotiana tabacum L. cells and purified to homogeneity. More than 90% of total cellular invertase activity (measured at pH 4.8) was recovered in the NaCl-eluted fraction whereas for the cytoplasmic marker glucose-6-phosphate dehydrogenase 96% of total activity could be extracted from the tissue after salt-elution, indicating absence of appreciable stress-induced cell disruption. Likewise, appreciable contamination of CWI with vacuolar acid invertase could be excluded. Tobacco CWI cross-reacted with an antiserum directed against deglycosylated carrot CWI; however, during some purification steps CWI enzyme activity did not correlate with CWI immunosignal. In fractions of low CWI activity and strong immunosignal, a putative inhibitor peptide with an apparent Mr of 17 kDa was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining (Weil et al. 1994, Planta 193, 438–445). The CWI of transformed tobacco cells has an apparent Mr of 69 kDa (SDS-PAGE) and is a basic (pI 9.5) glycoprotein. Gel-permeation chromatography indicated that enzymatically active CWI is a monomer. Deglycosylation of the denatured CWI by treatment with endo-β-N-acetylglucosaminidase, peptide-N-glycosidase F and trifluoromethanesulfonic acid indicated the presence of two high-mannose and two complex glycans. In partially purified CWI fractions the carrot CWI antiserum cross-reacted with one other tobacco cell-wall peptide (Mr 28 kDa). To address the possibility of a second invertase isoenzyme cross-reacting with the carrot antiserum, intact CWI and the 28-kDa peptide were digested in vitro with Staphylococcus aureus V8 protease and cyanogen bromide. A comparison of the resulting peptide patterns identified the 28-kDa polypeptide as a cleavage product of CWI. Running electroeluted CWI (69 kDa) on a second SDS-polyacrylamide gel led to substantial formation of the 28-kDa peptide. This suggests that the intrinsic 28-kDa cleavage product is the result of an intrinsic lability of tobacco CWI, rather than being a proteolytic degradation product.

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Abbreviations

Con A:

concanavalin A

CWI:

cell-wall invertase

Endo H:

endo-β-N-acetylglucosaminidase

Glc-6-P-DH:

glucose-6-phosphate dehydrogenase

1-OMG:

methyl α-d-glucopyranoside

p17:

17 kDa peptide

pI:

isoelectric point

PNGase F:

peptide-N-glycosidase F

TFMS:

trifluoromethanesulfonic acid

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Authors and Affiliations

  1. Botanisches Institut, J.W.Goethe-Universität, Siesmayerstrasse 70, D-60323, Frankfurt, Germany

    Marion Weil & Thomas Rausch

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  1. Marion Weil
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  2. Thomas Rausch
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Additional information

This work was supported by grants from the Deutsche Forschungsgemeinschaft. The gift of an antiserum directed against carrot cellwall invertase from Dr. Arnd Sturm (Friedrich-Miescher-Institut, Basel, Switzerland) is kindly acknowledged. Furthermore, the authors thank Sigrid Ranostaj for excellent technical assistence.

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Weil, M., Rausch, T. Acid invertase in Nicotiana tabacum crown-gall cells: Molecular properties of the cell-wall isoform. Planta 193, 430–437 (1994). https://doi.org/10.1007/BF00201823

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