A 17-KDA NICOTIANA-TABACUM CELL-WALL PEPTIDE ACTS AS AN IN-VITRO INHIBITOR OF THE CELL-WALL ISOFORM OF ACID INVERTASE

When cell-wall invertase (CWI) from Nicotiana tabacum L. cell-suspension cultures, either non-transformed or transformed with Agrobacterium tumefaciens, was salt-eluted from intact cells and purified on Sulfopropyl-Sephadex (SPS) by pH-gradient elution, the enzyme lost about 50% of its activity during a 1-h incubation at pH 4.8. However, Western-blot analysis indicated no appreciable enzyme degradation. Re-chromatography of CWI peak fractions on SPS using NaCl-gradient elution showed the presence of a 17-kDa peptide (p17) in fractions with low CWI activity but strong CWI immunosignal (Weil and Rausch 1994, Planta 193, 430-437). When separating CWI from p17 by Concanavalin A (Con A)-Sepharose chromatography, inhibition could be restored by incubating the inhibitor-containing fraction with inhibitor-free CWI. More than 90% of CWI could be inhibited, suggesting that all CWI was susceptible to p17 binding. The presence of divalent metal ions (Ca2+, Mg2+, Zn2+) during pre-incubation of CWI with p17 reduced CWI inhibition substantially. Also, sucrose protected CWI against inhibition by p17 (half-maximum protection at 1.3 mM). Binding of p17 to CWI during a 1-h pre-incubation was pH-dependent, pH 4.5 causing maximum inhibition, whereas at pH 6.5 no inhibition was observed. Gel-permeation chromatography revealed that the native inhibitor acts as a monomer. Immunoprecipitation of CWI co-precipitated p17, confirming direct binding of p17 to CWI. When fractions containing CWI and p17 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting a diffuse immunosignal of 86-90 kDa was observed (in addition to the prominent CWI signal at 69 kDa). Equilibration of this zone with urea-containing sample buffer prior to a second SDS-PAGE run resulted in a strong immunosignal at 87 (+/- 2) kDa, suggesting that during one step in the formation of the p17-CWI complex the two polypeptides became firmly aggregated. The distribution of CWI and glucose-6-phosphate dehydrogenase activities between the cell-wall protein fraction and salt-eluted cells shows that cells retained their structural integrity, thus indicating co-localization of p17 and CWI in situ (Weil and Rausch 1994). We have purified p17 to homogeneity and its N-terminus has been sequenced, revealing no similarity to other known protein sequences. Possible physiological roles of p17 are discussed.