Loss of liposome binding of NADH dehydrogenase from alkalophilic Bacillus on subtilisin digestion

Abstract

Alkalophile NADH dehydrogenase consisting of two 65-kDa subunits was changed by subtilisin into an enzyme species consisting of two 38-kDa subunits. The amino acid composition and enzyme activity per molecule of the subtilisin-treated enzyme were almost the same as those of the native enzyme, respectively. On mixing with phospholipid liposome, the conformation of the native enzyme was changed, as suggested by the changes in the type of Arrhenius plot and of CD spectrum and enzyme activity. These conformational properties of the subtilisin-treated enzyme, on the other hand, were not affected by liposome. Gel filtration of the subtilisin-treated enzyme mixed with the liposome showed no binding of the protein to liposome.

Tryptic Digestion of NADH Dehydrogenase from Alkalophilic Bacillus1

“…On the other hand, the CD spectrum and enzyme activity of SE were not affected by the phospholipids (15). These results may confirm the liposome-binding property of NE.…”

“…The enzyme species (TE3) with almost the same mobility as SE had the molecular size of 40 kDa per monomer (Fig. lb) and the specific activity of 130 units/mg protein, which was similar to that of SE (130 units/mg protein) (15). The enzyme species (TE2) with mobility between those of TE1 and TE3 was found to consist of two kinds of subunits (40 and 65 kDa) (Fig.…”

“…As shown in Fig. 8, it was difficult to identify the region(s) or hydrophobic amino acid cluster(s) because NE and SE (and therefore TE3 also) contain many lysyl residues (32 and 22 mol Lys/subunit) (15).…”

“…The enzyme from Bacillus stearothermophilus is monomeric (6) and that from alkalophilic Bacillus dimeric (12). Each of the two subunits (65 kDa) of the alkalophile enzyme contains the catalytic and liposome-binding regions (15). On subtilisin digestion, the polypeptide exhibiting the liposome-binding property is digested into small peptide fragments and an enzyme 1 A part of this work was presented at 4th Federation of Asian and Oceanian Biochemists Congress held on November 30-December 5, 1986, Singapore, and was published elsewhere (1).…”

“…Present addresses: 'Mitsubishi Kasei Industries Ltd., Midori-ku, Yokohama, Kanagawa 227. 4 species of two subunits (38 kDa) is formed (15). Recently it was found that three protein species were formed on tryptic digestion.…”