Gamma-glutamyl transpeptidase (γ-GTP), an enzyme purported to regulate amino acid transport, is expressed at high levels in blood-brain-barrier (BBB) capillary endothelium, but is a property that is variably expressed in cell culture. The ability of endothelial cells to form capillary-like tubes under different culture conditions has been reported by several investigators. In this study, a rat cerebral resistance vessel endothelial cell strain (RV-150 ECT) , that spontaneously forms cellular aggregates and connecting tubes, has been shown to contain a subpopulation of γ-GTP positive cells, was evaluated for the spatial relationship between γ-GTP and its mRNA, and the tube-forming structures in culture.Cells were grown on coverslips and fixed in acetone for enzymecytochemistry or in 3% paraformaldehyde and 0.5% glutaraldehyde in PBS pH 7.4 for immunocytochemistry and in situ transcription (IST), respectively. The enzyme substrate/acceptor were γ-glutamyl-4-methoxy-2-naphthyl-amine and glycylglycine. The primary antibody was affinity purified goat anti-γ-GTP IgG. The primers for the IST were 25-nt oligonucleotides complementary to γ-GTP mRNA. After the primers were hybridized to the mRNA the mRNA was transcribed in situ in a mixture of dNTPs containing DIG-labeled dUTP and reverse transcriptase.
Statistical optimization of culture conditions of mesophillic gamma-glutamyl transpeptidase from Bacillus altitudinis IHB B1644
