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-disrup ted Agrobacterium tumefaciens-transformed Nicotiana tabacum L. cells a nd purified to homogeneity. More than 90% of total cellular invertase activity (measured at pH 4.8) was recovered in the NaCl-eluted fractio n whereas for the cytoplasmic marker glucose-6-phosphate dehydrogenase 96% of total activity could be extracted from the tissue after salt-e lution, indicating absence of appreciable stress-induced cell disrupti on. Likewise, appreciable contamination of CWI with vacuolar acid inve rtase could be excluded. Tobacco CWI cross-reacted with an antiserum d irected against deglycosylated carrot CWI; however, during some purifi cation steps CWI enzyme activity did not correlate with CWI immunosign al. In fractions of low CWI activity and strong immunosignal, a putati ve inhibitor peptide with an apparent M(r) of 17 kDa was detected afte r 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 (pl 9.5) glycoprotein. Gel-permeation chromatography i ndicated that enzymatically active CWI is a monomer. Deglycosylation o f the denatured CWI by treatment with endo-beta-N-acetylglucosaminidas e, peptide-N-glycosidase F and trifluoromethanesulfonic acid indicated the presence of two high-mannose and two complex glycans. In partiall y purified CWI fractions the carrot CWI anti-serum cross-reacted with one other tobacco cell-wall peptide (M(r) 28 kDa). To address the poss ibility of a second invertase isoenzyme cross-reacting with the carrot antiserum, intact CWI and the 28-kDa peptide were digested in vitro w ith Staphylococcus aureus V8 protease and cyanogen bromide. A comparis on of the resulting peptide patterns identified the .28-kDa polypeptid e 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 be ing a proteolytic degradation product.