The structure and properties of the isozymes of cassava linamarase

Abstract

Purified linamarase (EC.3.2.1.21) from cassava petiole, stem and root cortex were shown to have native M(,r) of 600,000 - 2,000,000 on Sepharose 4B chromatiography. Structure was analysed by electrophoresis under both denaturing and non-denaturing conditions. Linamarase from all three sources showed subunit M(,r) of about 63,000 after denaturation by 1% SDS and heat. Analysis of the enzme in acid-urea gels and triton-acid-urea gels showed that linamarase gave the same single band for enzyme from three sources, suggesting linamarase consisted of a single peptide chain. On isoelectric focusing in agarose gels without denaturing agent, linamarase from petiole and stem gave one major (pI 4.6) band, while linamarase from root cortex gave a double band (pI 4.5-4.6). Under non-denaturing conditions, the purified linamarase appeared as a ladder pattern with more than 6 active bands in alkalind PAGE but failed to show a ladder pattern, appearing as a single broad band in cellulose acetate electro phoresis. Since the latter has no sieving effect, these results suggested that linamarase is present as aggregates with various MW sizes. The presence of a ladder pattern in fresh extract was confirmed and suggested the possibility that such aggregates existed in vivo, with the prominents forms consisting of even subunit numbers, namely 4, 6, 8, 10, and larger multiples. On 2 dimensional non-denaturing PAGE, stem linamarase showed little interconcersion of aggregated forms. Data obtained from the partial separation of high MW forms and low MW forms on Sepharose 4B chromatography and demonstration of effect of urea on linamarase aggregates suggest that existence of linamarase aggregates of appropriate size are needed for improved specificity towards linamarin. Re-evaluation of the different forms eluted at different pH (pH 4.3. 3.3 and 2.9 on chromatofocusing showed no diffisoelectric focusing, suggesting that all three forms may have the same pI. In addition, the kpattern obtained on non-denaturing PAGE suggested that the different chromatofocusing forms may be due to the differences in aggregate size. Amino acid analysis showed no difference between linamarase from the three tissues. The composition showed that the content of acidic amino, acids (Glx + Asx) was double the content of basic amino acids (Lys + Arg + His), consistent with the enzymes low pI. Furthermors, the content of hydrophobic amino scids (Ala, Val, Leu, Ile, Pro, Phe, Tyr) was high and approachen 50% of the total amino acids.