Purification and some properties of alanine racemase from a bivalve mollusc Corbicula japonica.
A brackish-water mollusc, Corbicula japonica, uses large quantities of D- and L-alanine as intracellular osmotically active solutes, osmolytes, for regulation of intracellular osmolarity. We purified alanine racemase from the mantle of C. japonica to characterize its enzymological properties. The molecular masses of the enzyme were estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 140 kDa by gel filtration on high-performance liquid chromatography, suggesting the trimeric or tetrameric nature of the enzyme. Neither dialysis nor chromatographic procedures in the absence of pyridoxal 5'-phosphate led to loss of enzyme activity, although carbonyl reagents, hydroxylamine and phenylhydrazine, inhibited the activity. These results suggest that alanine racemase of the animal may bind pyridoxal 5'-phosphate tightly as a cofactor. Kinetic experiments using the partially purified enzyme revealed that alanine was the sole substrate among 17 kinds of L-amino acids tested. The Lineweaver-Burk plot for L-alanine as substrate resulted in Km value of 22.6 mM, and the value for D-alanine was 9.2 mM. Together with the previous evidence that D- and L-alanine levels of this animal change with the external salinity maintaining the D-/L-alanine ratio at unity, the present results seem to indicate that the physiological role of alanine racemase in this animal is to supply D-alanine as a main intracellular osmolyte. J. Exp. Zool. 289:1-9, 2001.
Nomura
T
T
Department of Biological Science, Faculty of Science, Hiroshima University, Kagamiyama, Higashi Hiroshima 739-8526, Japan.
Yamamoto
I
I
Morishita
F
F
Furukawa
Y
Y
Matsushima
O
O
D000409:Alanine / Q000378:metabolism; D000411:Alanine Racemase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D049872:Bivalvia / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D010659:Phenylhydrazines / Q000494:pharmacology; D013237:Stereoisomerism; D013379:Substrate Specificity
D010659:Phenylhydrazines; C030299:phenylhydrazine; D019811:Hydroxylamine; D000411:Alanine Racemase; D000409:Alanine