Keiichi Kawano

Adsorption of human lysozyme onto hydroxyapatite. Identification of its adsorbing site using site-directed mutagenesis.

To elucidate hydroxyapatite‐protein interaction, mutant human lysozymes in which the surface charge was modified by site‐directed mutagenesis were used. Five mutant human lysozymes (K1A, K13A, K33A, R10A, R14A) were expressed in yeast. The chromatographic behavior of these lysozymes was studied with a HPLC hydroxyapatite column. Elution molarities of K1A and R14A mutants were greatly lowered. While Lys‐13 and Arg‐10 are located around Lys‐1 and Arg‐14, K13A and R10A mutants bound onto hydroxyapatite stronger than K1A and R14A mutants. In combination with an X‐ray crystal structure of human lysozyme, it is concluded that the adsorbing site of human lysozyme is at the back of the active site and that Arg‐14, Lys‐1, Arg‐10 and Lys‐13 play important roles in binding.

Interaction of aluminum ion with ATP. Mechanism of the aluminum inhibition of glycerol kinase and its reversal by spermine

Aluminum ion inhibited yeast glycerol kinase competitively with respect to the substrate MgATP. The K value of the enzyme for aluminum ion was about 3 μM. Spermine at physiological concentrations prevented glycerol kinase from the inhibition by aluminum ion. Nuclear magnetic resonance spectroscopy showed the specific elimination by spermine of aluminum from the metal-ATP complex, but no dissociation of MgATP complex by spermine. Inhibition by aluminum ion of glycerol kinase as well as hexokinase can reduce the utilization of energy fuel in yeast. Change in polyamine concentration may control energy production in vivo, and is responsible for the development of age-related aluminum toxicity.