Purification and calcium dependence of transglutaminases from sheep hair follicles

Abstract

GTP cyclohydrolase I (EC 3.5.4.16) has been purified for the first time from a higher plant, spinach leaves. The purified preparation appeared to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight of this enzyme was estimated at 135,000 by gel filtration and the subunit molecular weight was estimated at 35,000 by SDS-PAGE. The latter method also suggested that this enzyme was composed of four identical subunits. The enzyme was stable to heat treatment at 50°C for 10min, and the activity was maintained for at least six months when stored at −30°C. The enzyme had an optimum pH of around 8.0 in Tris buffer. The Hill coefficient of the enzyme was calculated to be 2.2. The pI of the enzyme was measured as 5.1 by chromatofocusing. 1H- and 13C-NMR spectroscopic analyses of cell wall microfibril β-l,3-xylan from Caulerpa brachypus were performed in detail. The total assignment of all 1H- and 13C-NMR signals in the β-l,3-xylan was achieved by evaluating the 2D C-H COSY, DQF-COSY, and NOESY spectra. The results, including information on through-space interaction between the xylopyranose residues, were confirmed. The determination of the glycosidic linkages by NMR spectroscopic analyses agrees well with the results from a chemical analysis.he use of tissue fractions and cellulose-related compounds. The most active enzyme induced by the crude fiber fraction and Avicel was β-glucosidase, among the cell wall degrading enzymes tested. The β-glucosidase was very inducible in the strains with strong pathogenicity, and intensively degraded the fiber fraction made from apple fruit tissues. The same degradation of the cell wall fraction was demonstrated with the purified enzyme.To study the calcium sensitivity of sheep hair follicle transglutaminase, which was reportedly calcium-independent [H. W. Harding and G. E. Rogers, Biochemistry, 11, 2858–2863 (1972)], the enzyme was purified from a homogenate of merino sheep hair follicles and its calcium dependence was examined. As a result of purification, two types of transglutaminases (DEAE-unabsorbed and absorbed transglutaminase, DU-TG and DA-TG, respectively) were obtained. The molecular mass of DU-TG was 77 and 82kDa by SDS-PAGE and gel filtration, respectively, while that of DA-TG was 40 and 80 kDa. Each enzyme was obviously calcium dependent and contained (a) cysteine residue(s) in the active site, like other known mammalian transglutaminases. Maximum activation of DU-TG and DA-TG was observed at 1 and 0.1 mm CaCl2, respectively. © 1997, Taylor & Francis Group, LLC. All rights reserved.