Auxin-Induced Changes in the Molecular Weight Distribution of Cell Wall Xyloglucans in Avena Coleoptiles

The effect of auxin on the molecular weight (Mw) distributionof cell wall xyloglucans was investigated by gel permeationchromatography using coleoptile segments of Avena sativa L.cv. Victory, and the following results were obtained.

1. The water-insoluble hemicellulose (HC-A) mainly consisted ofxyloglucans. Iodine staining method revealed that relativelylarge amounts of xyloglucans were present in the water-solublehemicellulose (HC-B) and water-soluble polysaccharide (WS) fractions.
2. IAA did not cause remarkable changes in xyloglucan contentsin the hemicellulose, but significantly increased the xyloglucancontent in the WS fraction.
3. IAA substantially decreased theweight-average Mw of HC-A. Thiseffect became apparent within30 min of the incubation period,and was not affected by the0.15 M mannitol or 2% sucrose applied.Hydrogen ions also causeda decrease in the weight-average Mwof HC-A; its effect beingreversible.
4. Neither IAA nor hydrogen ions caused any remarkablechangesin the weightaverage Mw of water-soluble xyloglucansin theHC-B.

These results suggest that cell wall xyloglucans have an importantrole in auxininduced cell wall loosening in oat coleoptile cells. (Received May 10, 1984; Accepted August 20, 1984)     

Growth Regulation of Dark-grown Dwarf Barley Coleoptile by the Endogenous IAA Content

Growth curves of dark-grown coleoptiles of 11 isogenic coleoptilardwarf strains of barley (Hordeum vulagare L. cv. Akashinriki:uzu, 5, 77, 97, 105, 125, 131, 133, 136, 145 and 148) were simulatedwith a logistic equation and the endogenous IAA contents ofthe barley strains were determined. Growth analysis of the dwarfbarley coleoptiles revealed that the final coleoptile lengthwas correlated with the growth rate on the 2nd day after germination(r=0.897), when the growth rate was about maximum. The endogenousIAA Content of the barley strains, measured fluorometrically,indicated that on the 2nd day, the dwarf strains contained lessendogenous IAA than the normal Strain. The IAA content on the2nd day was correlated to the growth rate on the 2nd day (r=0.907,except for Strain 145) and the final coleoptile length (r=0.933,except for strains 77 and 145). The correlation, however, wasnot significant on the 3rd day. These results suggested thatthe dwarfism of the dark-grown coleoptiles of the barley Strainsexamined is primarily controlled by the endogenous IAA content. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University, Osaka 558, Japan. (Received February 1, 1982; Accepted April 13, 1982)     

Reaction of S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine with sulfite: Synthesis of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid and application to the determination of sulfite

A procedure for the simultaneous preparation of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid is described. The method is based on the quantitative reaction between sulfite and S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine. The yield was 95% for S-sulfo-l-cysteine and 91% for l-alanine 3-sulfinic acid. The reaction was also applied to the quantitative determination of sulfite in biological materials. In this procedure, sulfite reacts with S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine. Separation of the reaction product, S-sulfo-l-cysteine, is done by ion-exchange fractionation, and it is determined with acid ninhydrin reagent 2 (M. K. Gaitonde, 1967, Biochem. J.104, 627–663). The recovery was 96.8 ± 0.3%.     

Rat hepatic uroporphyrinogen III cosynthase: Purification,properties, and inhibition by metal ions

Rat hepatic uroporphyrinogen III cosynthase has been isolated and purified 50-fold with a 36% yield by ammonium sulfate fractionation and sequential chromatography on DEAE-Sephacel and Sephadex G-100SF. Inhibition of uroporphyrinogen III formation with increasing porphobilinogen concentration was observed. Cosynthase was shown to be thermolabile, and a time-dependent loss of enzyme activity during reaction with uroporphyrinogen I synthase and porphobilinogen was observed. The pH optimum for the complete system (synthase and cosynthase) was pH 7.8 in 50 mm Tris-HCl or 50 mm sodium phosphate buffer. Various metals (KCl, NaCl, MgCl₂, CaCl₂) increased formation of Uroporphyrinogen III. Heavy metals including ZnCl₂, CdCl₂, and CuCl₂ were shown to selectively inhibit cosynthase activity, whereas other metals (HgCl₂, PbCl₂) were less selective and inhibited both synthase and cosynthase at similar concentrations.     

43Ca NMR studies of Ca2+-Tetrahymena calmodulin complexes

⁴³Ca NMR spectra of Ca²⁺-Tetrahymena calmodulin(Tet. CaM.) complexes have been observed under various conditions. Off-rate of Ca²⁺ from Tet. CaM. is estimated to be approx. 2.7 × 10³ s^?1 under a certain assumption. Relaxation rates of ⁴³Ca NMR of Ca²⁺-Tet. CaM. are remarkably increased(by one order in magnitude) by adding trifluoperazine(TFP), a potent calmodulin antagonist. Relaxation parameters estimated suggest that Ca²⁺ mobility is reduced by the TFP binding. A stoichiometry of TFP is two moles per Tet. CaM. molecule. The relaxation rates of ⁴³Ca NMR signals are increased by adding excessive Mg²⁺ to the Ca²⁺-Tet. CaM. solutions. The addition of Mg²⁺ to the Ca²⁺-Tet. CaM. complex decreases apparent pKa value of the complex as well.     

Purification and characterisation of β-N-acetylhexosaminidase from the butter clam, saxidomus purpuratus

A β-N-acetylhexosaminidase [EC 3.2.1.30] has been purified ～98-fold from an extract of the digestive organs of Saxidomus purpuratus by using ammonium sulfate fractionation, and chromatography on Toyopearl HW-50, CM-cellulose, and Sepharose 4B. The purified enzyme, the molecular weight of which was estimated to be ～66,000 by gel filtration, was composed of two sub-units of molecular weight 30,000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified enzyme had a pH optimum of 3.8 and an optimum temperature of 55°, and its activity was enhanced ～2-fold in the presence of 0.1m sodium chloride. The Michaelis constants toward p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucoside and -galactoside were 1.2 × 10^?4 and 1.3 × 10^?4m, respectively.     

Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants

We have measured the specific uptake of ¹²⁵I-labelled diphtheria toxin in the presence of methylamine by a number of cell lines with different sensitivities to diphtheria toxin. The results show a strong correlation between the toxin sensitivities of the cell lines and the amount of specific uptake. The specific association of labelled toxin with cells was clearly demonstrated even with CHO cells, a cell line with relatively low sensitivity. Thus, CHO cell mutants that are resistant to diphtheria toxin could be classified as toxin-binding or non-binding cells by this method.