The structure of Canavalia, a common bean (Canavalia ensiformis) protein homologous to phaseolin, the main seed storage protein of Phaseolus vulgaris, has been investigated by X-ray crystallography and found to be a hexamer composed of three identical pairs of similar but not identical subunits. related by a perfect triple-axis and pseudo dyad axes (strict C3 and pseudo D3). One member of each subunit pair is derived from the amino-terminal half of a 49,000 molecular weight precursor polypeptide and the other from its carboxy-terminal half.
Thus, crystallographic evidence indicates that the parent polypeptide is a tandem duplicate and is structurally redundant (McPherson A. 1982 J Biol Chem 255: 10472). Various physical and chemical properties of the protein were investigated in both the uncleaved and cleaved forms. These included native molecular weights, amino acid analysis, number of exposed sulfhydryl groups, carbohydrate content, metal ion analysis, crystallization behaviour, and protein fate in developing seeds. The purified precursor protein was also found to possess a substantial level of α-d-mannosidase activity and appears to share a number of other physical and chemical properties with that enzyme.
Materials and Methods
- Crowth of plants
Bean (Canavalia ensiformis) seeds were purchased at Sigma Chemical Co. (Poole, Dorset, UK). The seeds were individually planted at the University of California, Riverside soil mix I1 (Chandler, 1957). After germination, the plants were transferred to large pots containing the same type of soil and grown to maturity in an air-filtered greenhouse under controlled control temperature from 25 to 3 ° C. The seeds were harvested 40 DAF (after about 5 months of growth) and is removed from their pods and linings were stripped. The beans were washed with sterile water and immediately frozen in liquid nitrogen and stored at -7OOC.
- Synthesis of single-stranded cDNA from total RNA
The frozen beans were ground to a fine powder in the presence of liquid nitrogen in a Waring blender. The total RNA was isolated by methods described by Kirby (1968) and Chirgwin et al. (1979). The ssDNA was generated from total RNA by transcription (Verma, 1981) and the ssDNA was quantified measuring the incorporation of [cY- ~ * P] ~ CTP (Krug et al., 1987).
- Preparation of oligonucleotide primers for PCR and Direct sequencing
Oligonucleotide primers were synthesized using phosphoramidite chemistry on an Applied Biosystems DNA synthesizer (model 380), or prepared in Biotechnology Instrumentation facility at the University of California, Riverside, using an Applied Biosystems DNA synthesizer (model 394). The oligonucleotide products were further purified by Applied Biosystems Oligonucleotide Purification Cartridges as described by the manufacturer.