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S Krausgrill

Publications and source records attributed to S Krausgrill.

4 recordsLinked to original sources
PubMed1998

Cloning of a tobacco apoplasmic invertase inhibitor. Proof of function of the recombinant protein and expression analysis during plant development.

Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.

Amino Acid Sequence↗
PubMed1996

Sucrose protects cell wall invertase but not vacuolar invertase against proteinaceous inhibitors.

Vacuolar (VI) and cell wall invertases (CWI) of higher plants can be inactivated in vitro and, possibly, in vivo by proteinaceous inhibitors. The respective mechanisms have not yet been compared. Therefore, partially purified CWI from transformed tobacco cells and VI from tomato fruit were preincubated with invertase-inhibitor fractions isolated from the same tissues. Both inhibitors were able to inhibit both invertases. However, VI was fully inhibited within less than 1 min by both inhibitors, whereas inactivation of CWI was much slower. Furthermore, CWI, but not VI, was strongly protected against inhibition by sucrose. A polyclonal antiserum directed against the tobacco inhibitor (I(NT)) cross-reacted with a 19 kDa polypeptide in the partially purified tomato inhibitor (I(LE)) fraction. The results indicate that I(NT) and I(LE)have similar structural properties, whereas the mechanism of inactivation is clearly different for CWI and VI.

Agrobacterium tumefaciens↗
PubMed1994

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)

Amino Acid Sequence↗