Type XIII collagen is a type II transmembrane protein predicted to consist of a short cytosolic domain, a single transmembrane domain, and three collagenous domains flanked by noncollagenous sequences. Previous studies on mRNAs indicate that the structures of the collagenous domain closest to the cell membrane, COL1, the adjacent noncollagenous domain, NC2, and the C-terminal domains COL3 and NC4 are subject to alternative splicing. In order to extend studies of type XIII collagen from cDNAs to the protein level we have produced it in insect cells by means of baculoviruses. Type XIII collagen ␣ chains were found to associate into disulfidebonded trimers, and hydroxylation of proline residues dramatically enhanced this association. This protein contains altogether eight cysteine residues, and interchain disulfide bonds could be located in the NC1 domain and possibly at the junction of COL1 and NC2, while the two cysteine residues in NC4 are likely to form intrachain bonds. Pepsin and trypsin/chymotrypsin digestions indicated that the type XIII collagen ␣ chains form homotrimers whose three collagenous domains are in triple helical conformation. The thermal stabilities (T m ) of the COL1, COL2, and COL3 domains were 38, 49 and 40°C, respectively. The T m of the central collagenous domain is unusually high, which in the light of this domain being invariant in terms of alternative splicing suggests that the central portion of the molecule may have an important role in the stability of the molecule. All in all, most of the type XIII collagen ectodomain appears to be present in triple helical conformation, which is in clear contrast to the short or highly interrupted triple helical domains of the other known collagenous transmembrane proteins.The collagens are classically divided into two major groups, fibrillar and nonfibrillar, depending on their structural and functional characteristics, and the heterogeneous group of nonfibrillar collagens can be further divided into several subgroups (1, 2). Type XIII collagen belongs to the group of membraneassociated collagens, together with the hemidesmosomal component type XVII collagen. These two nonfibrillar collagen types are not structurally homologous except that both have been predicted to have a transmembrane domain near their N terminus. Immunoprecipitation of biotinylated type XIII collagen from surface-labeled HT-1080 cells, subcellular fractionation, and immunofluorescence staining have been used to demonstrate that type XIII collagen molecules are indeed located in the plasma membranes of these cells (3). Type XIII collagen molecules are presumed to reside in the plasma membrane in a "type II" orientation with a cDNA-derived predicted structure consisting of a short N-terminal cytosolic domain, a single transmembrane domain, and a large, mainly collagenous ectodomain. The N-terminal noncollagenous domain, NC1, 1 of the human ␣1(XIII) chain encompasses a 38-residue cytosolic domain, a 23-residue transmembrane domain, and the first 60 residues of the noncollagenous ...

No abstract

No abstract

The cDNA coding for rat Sadenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50) has been cloned into a plasmid expression vector, pKK-223-3, and expressed in E. co&. The authenticity of the expressed protein has been demonstrated by reactivity with antibodies specific for rat AdoMetDC, by size analysis on SDS gels visualized with i~rnuno~~~~~io~~ and, finaEy, by catalytic activity. The expression of the enzyme rest&s in a decrease in the activity of the bacteria] enzyme suggesting the repiacement of the bacterial enzyme by the rat AdoMetDC. Similarly, the addition of exogenous spermidine to the growth medium reduces bacterial enzyme activity affecting only marginally the expression of the recombinant protein.

No abstract