43. Relative staining strength of esterase bands changing according to staining agents

Dongxu LIU, Zixian LI, Xuexing GUO and Xide CAO

Chengdu Institute of Biology, Academia Sinica, Chengdu 610015, China

The relative staining strength of isozyme bands has been considered to represent the relative activity and content of corresponding isozymes. We found, however, that the relative staining strength differed according to staining agents.

A male-sterile line 765-5A of O. sativa, an O. grandiglumis strain, and their hybrids obtained by the embryo-rescue technique, 230-1, 230-2 and 230-3, were used. The vertical polyacrylamide gel thin-layer electrophoresis and staining procedures for esterase isozymes (Beijing Agric. University 1988) were employed, and flag-leaf blades fully expanded in the field were examined by 1) alpha-naphthyl acetate (a-NA) and fast blue RR salt (RR), 2) alpha-NA and fast blue B salt (B), 3) beta-naphthyl acetate (beta-NA) and RR, 4) beta-NA and B, 5) alpha-NA, beta-NA and RR, and 6) alpha-NA, beta-NA and B. The staining solution was prepared as follows: 100mg alpha-NA, beta-NA, RR or B was dissolved in 3ml aceton, and 100ml phosphate buffer at pH 6.4 was added, and then was filtered into a staining basin. After electrophoresis, the gel plates were incubated in newly prepared staining solutions at 37 deg C for 30 min. when stained with RR as the coupling agent, and for 20 min. when stained with B.

A total of 47 anodic bands were observed, which were numbered 1A to 47A according to their decreasing mobilities. The bands with changing staining strength in different agents are shown in Table 1. 

Table 1. Relative staining strength of some esterase bands differing
according to staining agents
==============================================================================
                        RR                      B
Band =============================== =============================== Carriera  
    alpha-NA beta-NA alpha-NA+betaNA alpha-NA betaNA alpha-NA+betaNA
===============================================================================
2A       +      +            +         ++       ++          ++           E
3A       +      +            +         ++       ++          ++        B, C, E
4A       +      +            +         ++       ++          ++           D
7A       +      -            +/-       ++       -           +/-          D
8A       +      +            ++        ++       ++          ++++         D
14A      +++    -            +++       +++      -           +++          B
17A      +++    -            +++       +++      -           +++          B
18A      +      +            ++        ++       ++          ++++     C, D, E
==============================================================================
+++++ Maximum staining strength,   -  Null.
a: B-230-1, C-230-2, D-230-3, hybrids from 765-5A x O. grandiglumis.
  E-O. grandiglumis.
Most of the bands were stained by both alpha-NA and beta-NA, but 7A, 14A and 17A were stained only when alpha-NA was used as the substrate. This suggests that the substrate specificity of alpha-NA is lower than that of beta-NA. Irrespective of coupling agents, the relative staininc, strength of 7A decreased and that of 8A and of 18A increased when alpha-NA and beta-NA were mixed. Further, the relative staining strength of bands 2A, 3A, 4A and 18A increased when stained with B, as compared with those stained with RR, although the staining time was 20 min. with B and 30 min. with RR. This suggests that B is more sensitive than RR.

Generally, the higher the enzyme content and activity, the higher the staining strength and the wider the band in the zymogram. However, isozymes represent a group of enzymes which catalyse the same reaction. They may have different structures and optimum conditions of reaction such as pH and temperature.