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).(ABSTRACT TRUNCATED AT 250 WORDS)
Weil
M
M
Botanisches Institut, Johann Wolfgang Goethe-Universität, Frankfurt, Germany.
Krausgrill
S
S
Schuster
A
A
Rausch
T
T
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000378:metabolism; D002473:Cell Wall / Q000201:enzymology; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010947:Plants, Toxic; D011485:Protein Binding; D013395:Sucrose / Q000378:metabolism; D013995:Time; D014026:Nicotiana / Q000201:enzymology*; D043324:beta-Fructofuranosidase
D002413:Cations, Divalent; D007527:Isoenzymes; D010455:Peptides; D010940:Plant Proteins; D013395:Sucrose; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase