An ELISA-based assay for detergent-solubilized cellular beta 1,4-galactosyltransferase activity. Use of a polyacrylamide derivative with GlcNAc-beta side chains as a solid phase acceptor substrate.
In our previous paper [Oubihi et al. (1998) Anal. Biochem., 257, 169-175], we have shown that a polyacrylamide-derived synthetic glycopolymer with GlcNAcbeta side chains, termed PAP(GlcNAcbeta), is useful as a solid phase acceptor substrate for the ELISA-based analyses of soluble beta1,4(-)galactosyltransferase (GalT) activity in milk. This method is now used to assay detergent-solubilized cellular GalT. The glycopolymer coated on polystyrene plates was shown to be highly stable against the non-ionic and ionic detergents tested (0 approximately 5% solutions of Triton X-100 and SDS). Such stability made it possible to incubate the ELISA plate with detergent-solubilized GalT and to wash the ELISA plate with SDS solution after the GalT reaction, leading to high accuracy and sensitivity of this assay. The GalT activity was assayed using this method for 1% Triton X-100 extracts of various tissue samples of mice and several cultured cell lines. The results showed that the specific GalT activity of tissue extracts was low in brain and intestine, and high in ovary, muscle, and kidney. As for the cultured cell lines, COS7, COMMA-1D and C2C12 cells showed high specific activity, while CHO and MDCK cells showed low activity. The myoblast C2C12 had a slight increase in GalT activity during starvation-induced cell differentiation. On the other hand, GaIT-I transcript estimated by RT-PCR rather decreased during C2C12 cell differentiation, suggesting a differentiation-dependent switch in GalT isozymes. Taken all together, the ELISA-based assay using PAP(GlcNAcbeta) as a solid phase acceptor substrate was demonstrated to be a useful method for the assay of membrane-bound galactosyltransferases.
Oubihi
M
M
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
Oshima
K
K
Aoki
N
N
Kobayashi
K
K
Kitajima
K
K
Matsuda
T
T
D000180:Acrylic Resins; D000818:Animals; D019556:COS Cells; D002454:Cell Differentiation; D002460:Cell Line; D003902:Detergents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D051379:Mice; D009237:N-Acetyllactosamine Synthase / Q000032:analysis* / Q000235:genetics; D017830:Octoxynol; D011108:Polymers; D011137:Polystyrenes; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D013379:Substrate Specificity; D014020:Tissue Extracts; D000099166:Polyacrylamides
D000180:Acrylic Resins; D003902:Detergents; D009237:N-Acetyllactosamine Synthase; D017830:Octoxynol; D011108:Polymers; D011137:Polystyrenes; D012967:Sodium Dodecyl Sulfate; D014020:Tissue Extracts; D000099166:Polyacrylamides