Structural and binding properties of laminarin revealed by analytical ultracentrifugation and calorimetric analyses.
One of the β-1,3-glucans, laminarin, has been widely used as a substrate for enzymes including endo-1,3-β-glucanase. To obtain quantitative information about the molecular interaction between laminarin and endo-1,3-β-glucanase, the structural properties of laminarin should be determined. The results from pioneering work using analytical ultracentrifugation for carbohydrate analysis showed that laminarin from Laminaria digitata predominantly exists as a single-chain species with approximately 5% of triple-helical species. Differential scanning calorimetry experiments did not show a peak assignable to the transition from triple-helix to single-chain, supporting the notion that a large proportion of laminarin is the single-chain species. The interaction of laminarin with an inactive variant of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, E119A, was quantitatively analyzed using isothermal titration calorimetry. The binding was enthalpically driven and the binding affinity was approximately 10(6) M(-1). The results from binding stoichiometric analysis indicated that on average, E119A binds to laminarin in a 2:1 ratio. This seems to be reasonable, because laminarin mainly exists as a monomer, the apparent molecular mass of laminarin is 3.6 kDa, and E119A would have substrate-binding subsites corresponding to 6 glucose units. The analytical ultracentrifugation experiments could detect different complex species of laminarin and endo-1,3-β-glucanase.
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5, Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan. Electronic address: oda@kpu.ac.jp.
Masayuki
M
Oda
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5, Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
Yoichi
Y
Tanabe
U-Medico Inc., 1-1, Yamadaoka, Suita, Osaka 565-0871, Japan.
Masanori
M
Noda
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5, Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
Satomi
S
Inaba
U-Medico Inc., 1-1, Yamadaoka, Suita, Osaka 565-0871, Japan.
Elena
E
Krayukhina
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
Harumi
H
Fukada
U-Medico Inc., 1-1, Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Engineering, Osaka University, 1-1, Yamadaoka, Suita, Osaka 565-0871, Japan.
Susumu
S
Uchiyama
D039903:Actinobacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000378:metabolism*; D005936:Glucans / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D005936:Glucans; C008247:laminaran; D004693:Glucan Endo-1,3-beta-D-Glucosidase
Analytical ultracentrifugation; Carbohydrate-binding; Differential scanning calorimetry; Isothermal titration calorimetry; β-1,3-glucan