Homoserine dehydrogenase (HSDH) is the third enzyme in the pathway leading to threonine, methionine and isoleucine in the aspartate family of amino acids. In maize, HSDH is feedback-controlled by threonine.
HSDH was extracted from shoot and internodes of floury-a and normal BP etiolated seedlings 3 days old. The extraction procedure of Bryan and Lochner (Plant Physiol. 68:1400-1405, 1981) was used but DTE was replaced by 5.0 MM of 2-mercaptoethanol. Extraction and all subsequent operations were carried out at 5 C. Weighed tissues were ground using a mortar and pestle. Homogenates were filtered through two layers of cheesecloth and centrifuged at 18,000 xg for 30 min. Crude supernatants were analyzed by discontinuous gel electrophoresis (Davies), with the HSDH activity being located on the gels by the nitroblue tetrazolium dye precipitation procedure of Matthews et al. (Plant Physiol. 55:991-998, 1975).
Electrophoretic analysis of HSDH from extracts of 72-hour-old maize shoot and internodes indicated the presence of 3 to 4 enzyme forms (Fig. 1). Tissue-specific and genotype-specific differences in the proportion of the enzymes have been observed. Bands 3 and 4 were the major enzyme forms independent of the tissue and genotype. However, the ratio of Bands 3 and 4 has been changed from about 2:1 in the floury-a crude extracts (A-B) to 1:1 in the BP crude extracts (C-D). Band 1, the HSDH threonine resistant form, was difficult to localize in the BP crude extracts. However, in the floury-a extracts, Band 1 appeared as a faint band. Band 2 was clearly localized in all floury-a electrophoretic patterns independent of the tissue (root tissue, shoot tissue, whole kernel, internodes) and the age of seedlings. On the other hand, Band 2 was very difficult to localize and differentiate from Band 1 in the BP crude extracts.
These preliminary results suggest that changes in the HSDH are not only both tissue and species specific, as has been previously proposed, but also could be genotype-specific. These results could also explain in part some of the unusual responses of the floury-a material to lysine-threonine feedback regulation.
Figure 1. Gel electrophoretic analysis of HSDH. A: floury-a shoots; B: floury-a internodes; C: BP shoots; D: BP internodes. Bands have been numbered 1 to 4 in order of decreasing mobility. These bands were not detected when duplicate gels were incubated in the absence of homoserine after electrophoresis. A to D 5% acrylamide gels. Equal amounts of protein were applied to each gel.
Miguel Angel Rapela
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