IN-VITRO RATES OF RUMEN PROTEOLYSIS OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE (RUBISCO) FROM LUCERNE LEAVES, AND OF OVALBUMIN, VICILIN AND SUNFLOWER ALBUMIN-8 STORAGE PROTEINS

Laboratory systems were developed, based upon in-vitro incubations wit h rumen fluid, to examine the rate of proteolysis (ie degradation) of sunflower albumin 8 (SFA 8; 24% sulphur amino acids; SAA) from sunflow er seeds, ovalbumin (6% SAA) from chicken egg white, ribulose-1,5-bisp hosphate carboxylase (Rubisco; 3% SAA) from lucerne leaves, and vicili n (0% SAA) from pea seeds. After fractionation by SDS-PAGE, proteins w ere analysed by either Western blotting, using specific antibodies (SF A 8, vicilin and ovalbumin) or by Coomassie blue staining (Rubisco). P roteolysis of the large subunit of Rubisco occurred very quickly and a s two components, with half-lives of 11.6 and 1.5 h. The small subunit of Rubisco was more resistant to degradation, with a half-life of 17. 3 h. Vicilin was degraded extremely rapidly (half-life 0.16 h). SFA 8 and ovalbumin both showed resistance to degradation, but by two differ ent mechanisms. Ovalbumin was not degraded at all during the initial 1 6 h of incubation, but then degraded with a half-life of 8.7 h. SFA 8 (mol wt 12100) disappeared very rapidly, with a half-life of 3.0 h. Th e degradation of SFA 8 was associated with the appearance of a polypep tide (mol wt 8000), which was extremely resistant to degradation, with a half-life of 69.3 h. It was concluded that both the number of disul phide linkages and tertiary structure were important in determining re sistance of proteins to rumen degradation, and that incorporation of S FA 8 and ovalbumin proteins into forages using genetic engineering tec hniques would be likely to increase the quantity of SAA bypassing rume n fermentation.