A 17-kDa Nicotiana tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform of acid invertase

Planta. 1994;193(3):438-45. doi: 10.1007/BF00201824.

Abstract

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)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cations, Divalent / metabolism
  • Cell Wall / enzymology
  • Cells, Cultured
  • Chromatography, Ion Exchange
  • Enzyme Activation
  • Glycoside Hydrolases / antagonists & inhibitors*
  • Hydrogen-Ion Concentration
  • Isoenzymes / antagonists & inhibitors*
  • Molecular Sequence Data
  • Peptides / metabolism
  • Plant Proteins / isolation & purification
  • Plant Proteins / metabolism*
  • Plants, Toxic*
  • Protein Binding
  • Sucrose / metabolism
  • Time
  • Tobacco / enzymology*
  • beta-Fructofuranosidase

Substances

  • Cations, Divalent
  • Isoenzymes
  • Peptides
  • Plant Proteins
  • Sucrose
  • Glycoside Hydrolases
  • beta-Fructofuranosidase