Determination of specific activities and kinetic constants of biotinidase and lipoamidase in LEW rat and Lactobacillus casei (Shirota)

Enzyme kinetic parameters, such as Km, Vmax (or V), kcat/Km, and Ki (by biotin or lipoic acid) for biotinidase and lipoamidase were determined in Lewis (LEW) rat and Lactobacillus casei (Shirota) using fluorimetric high-performance liquid chromatography (HPLC). It was found that the final protein concentration below 0.1 mg/ml is sufficient to obtain linear hydrolytic reaction and to determine the Michaelis–Menten type kinetic parameters (Km, V, Ki). We applied this HPLC enzyme assay method onto the rat and some bacteria. The highest specific activities (Vs) for biotinidase were found in Lactobacillus casei (Shirota) and rat kidney. It was also found that the largest Ki by product for biotinidase and lipoamidase were present in the Lactobacillus casei (Shirota). There has been found specie (between rat and mouse) differences and tissue (organ) differences, together with tissue region differences and sex differences in some tissues. Summary of the distributions of both enzymes in LEW rat was also presented. Therefore, this HPLC determination method for the enzyme kinetic parameters in tissues is expected to be an indispensable tool for the investigation of the various diseases in humans.