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

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 M_r 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 M_r 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--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 (M_r 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.Abbreviations Con A concanavalin A - CWI cell-wall invertase - Endo H endo--N-acetylglucosaminidase - Glc-6-P-DH glucose-6-phosphate dehydrogenase - 1-OMG methyl -d-glucopyranoside - p17 17 kDa peptide - pI isoelectric point - PNGase F peptide-N-glycosidase F - TFMS trifluoromethanesulfonic acidThis 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.