Studies on the Polymorphism of Thiaminase I in Seawater Fish

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Thiaminase I from the seawater fish Fisturalia petimba was characterized, and pI polymorphism of the enzyme was first described, together with the active subfragment of thiaminase I. The liver of F. petimba contained thiaminase I of at least three different pIs and the major fraction exhibited pI 5.7. The most evident difference among pI isozymes was the size of the active subfragments into which they were dissociated. pI 5.7 enzyme dissociated into subfragments of 25kDa, while pI 7-9 enzymes dissociated into approx. 22kDa. The reaction rate measured by pyridine as the second substrate was three times higher in pI 7-9 enzymes compared with pI 5.7 enzyme. The degree of cadmium inhibition, when aniline was the co-substrate, also showed obvious differences between pI isozymes. When the major pI fraction was further purified by the difference in hydrophobicity, a smaller active fragment of approx. 22kDa appeared, indicating the possibility that the difference in the size of active subfragment between isozymes is a result of partial fragmentation. The pI 5.7 enzyme was purified 250times and the size of the most purified preparation was found to be 106kDa by gel filtration analysis. The purified preparation gave an active 25kDa subfragment by SDS-PAGE, together with a 15kDa non-active subfragment. The enzyme was, thus, inferred to contain active subfragments together with the 15kDa non-active fragments. Amino acid sequencing of the 25 kDa active subfragment revealed, together with the fully processed N-terminal sequence, two N-terminal peptides with extra Pro-Ser and Gly-Pro-Ser attached to it, and the NCBI non-redundant database did not show significant similarity to other known proteins. On the other hand, the molecular mass of the holoenzyme from the viscera of the seawater fish Engraulis japonica was estimated to be approx. 100kDa by gel filtration chromatography. An SDS-PAGE analysis revealed that it contained an active subfragment of 22kDa.

Studies on the Polymorphism of Thiaminase I in Seawater Fish

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