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. 2002 Sep;130(1):374-9.
doi: 10.1104/pp.005587.

Successive glycosyltransfer activity and enzymatic characterization of pectic polygalacturonate 4-alpha-galacturonosyltransferase solubilized from pollen tubes of Petunia axillaris using pyridylaminated oligogalacturonates as substrates

Kazumasa Akita  1 , Takeshi Ishimizu, Tatsuya Tsukamoto, Toshio Ando, Sumihiro Hase
Affiliations

Successive glycosyltransfer activity and enzymatic characterization of pectic polygalacturonate 4-alpha-galacturonosyltransferase solubilized from pollen tubes of Petunia axillaris using pyridylaminated oligogalacturonates as substrates

Kazumasa Akita et al. Plant Physiol. 2002 Sep.

Abstract

Polygalacturonate 4-alpha-galacturonosyltransferase (pectin synthase) was solubilized from pollen tubes of Petunia axillaris and characterized. To accomplish this, an assay method using fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as acceptor substrates was developed. When the pollen tube enzyme was solubilized with 0.5% (v/v) Triton X-100 and was incubated with PA-OGA and UDP-galacturonic acid (UDP-GalUA), successive transfer activity of more than 10 GalUAs from UDP-GalUA to the nonreducing end of PA-OGA was observed by diethylaminoethyl high-performance liquid chromatography. This activity was time- and enzyme concentration-dependent. The optimum enzyme activity was observed at pH 7.0 and 30 degrees C. Among the PA-OGAs investigated, those with a degree of polymerization of more than 10 were preferred as substrates. The crude pollen tube enzyme had an apparent K(m) value of 13 microM for the PA-OGA with a degree of polymerization 11 and 170 microM for UDP-GalUA. The characteristics of the P. axillaris pollen tube enzyme and the usefulness of fluorogenic PA-OGAs for the assay of this enzyme are discussed.

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Figures

Figure 1
Figure 1
DEAE anion-exchange HPLC of PA-OGAs. A mixture of PA-OGAs was analyzed by DEAE HPLC. The peak eluted at 35 min was identified as PA-OGA of DP 4 by matrix-assisted laser-desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Arrows and numbers indicate the PA-OGA elution position and DP of the PA-OGA eluted, respectively.
Figure 2
Figure 2
DEAE HPLC of products obtained with PGA-GalUAT using PA-OGA of DP 14. A, The enzyme reaction was conducted in the presence of 15 μm PA-OGA of DP 14 and 8.7 microunits of the crude enzyme for 0 to 60 min. Arrows and numbers indicate the PA-OGA elution position and DP of the PA-OGA eluted, respectively. B, Amounts of products plotted against incubation time. PA-OGAs of DP 15 (○), 17 (●), 19 (□), 21 (▪), 23 (▵), 25 (▴), and 27 (⋄) are shown.
Figure 3
Figure 3
Time- and enzyme concentration-dependence of the enzyme reaction. A, Time course of GalUA incorporation into PA-OGA of DP 14. B, Relationship between enzyme concentration and incorporation of GalUA into PA-OGA. The enzyme reaction was continued for 30 min.
Figure 4
Figure 4
Effects of DP of acceptors on PGA-GalUAT activity. The reaction was conducted with 2.6 microunits of the crude enzyme and various PA-OGAs of the following DP: 5 (10.8 μm), 6 (14.2 μm), 7 (13.2 μm), 8 (12.3 μm), 9 (13.4 μm), 10 (13.2 μm), 11 (11.7 μm), 12 (14.2 μm), 13 (13.4 μm), 14 (12.1 μm), and 15 (14.0 μm). The initial transfer activities are shown relative to that of the PA-OGA of DP 14.
Figure 5
Figure 5
Effects of pH on PGA-GalUAT activity. The crude enzyme (3.3 microunits) was incubated with 7.9 μm PA-OGA of DP 12. The buffers used were 100 mm MES-NaOH (–●–), HEPES-NaOH (–▪–), and Tris-HCl (–▴–). The enzyme activity at pH 7.0 was taken as 1.0.
Figure 6
Figure 6
Effect of temperature on PGA-GalUAT activity. The crude enzyme (4.3 microunits) and 6.8 μm PA-OGA of DP 15 were incubated for 30 min. The activities are shown relative to that at 30°C.
Figure 7
Figure 7
Effect of Mn2+ concentration on PGA-GalUAT activity. The crude enzyme (1.6 microunits) was incubated with 7.9 μm PA-OGA of DP 12. Each reaction mixture contained 0.13 mm EDTA derived from the extraction buffer. The activities relative to 5 mm Mn2+ are shown.
Figure 8
Figure 8
Galacturonidase digestion of products obtained with PGA-GalUAT. A, PA-OGA of DPs 12 and 13 were prepared from the reaction with 2.3 microunits of the crude enzyme, and 26.3 μm PA-OGA of DP 11. B, The enzymatic products (PA-OGAs of DP 12 and 13) shown in A were collected and treated with the soluble fraction containing galacturonidase activity from P. axillaris pollen tubes for 1 and 4 h at 28°C. Arrows and numbers indicate the PA-OGA elution position and DP of the PA-OGA eluted, respectively.
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