ABSTRACT
We describe an improved, rapid and cost effective assay for measuring O6-alkylguanine-DNA alkyltransferase (AGT) levels in large numbers of small biological samples. The assay is based on the use of a synthetic O6-methylguanine oligonucleotide bound to magnetic beads via a biotin-streptavidin linkage and bound to its complement. A 35S label is incorporated into the free end of the duplex. The basis of the assay lies in the observation that the restriction enzyme PvuII will not cleave the bead-bound duplex oligonucleotide having an O6-methylguanine within the restriction sequence. However, on removal of the methyl group by AGT present in cell extracts prior to incubation with PvuII, the 35S-labelled oligonucleotide is cleaved by the restriction enzyme, releasing a 35S-labelled 8 bp fragment. Due to the stoichiometric nature of the AGT reaction, quantitation of AGT is easily achieved by counting an aliquot of the supernatant after pelleting the unrestricted bead complexes with a magnet. AGT levels measured in certain cell lines and human lymphocytes by the reported assay are comparable to other methods. The assay can be performed in basic laboratories and allows for the rapid processing of many samples simultaneously, which could prove useful in clinical and epidemiological studies.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/antagonists & inhibitors , Methyltransferases/analysis , Base Sequence , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/metabolism , Escherichia coli/enzymology , Feasibility Studies , Humans , Hydrolysis , Immunomagnetic Separation , Lymphocytes/enzymology , Methyltransferases/blood , Molecular Sequence Data , O(6)-Methylguanine-DNA Methyltransferase , Oligonucleotides/metabolism , Phenotype , Sensitivity and SpecificityABSTRACT
1. Nerve growth factor from Bitis arietans venom was isolated in high yield and purified to homogeneity using a rapid two-step procedure involving gel exclusion chromatography and reversed-phase HPLC. 2. On polyacrylamide gel electrophoresis in SDS, the NGF migrates as a 25 kDa homodimer and is thus atypical of other Viperid NGFs. 3. Evidence suggests that, unlike mammalian beta NGFs, the subunits of the Bitis arietans homodimer are covalently linked by a disulphide bond(s). 4. Partial sequence analysis shows that only 6 out of the first 21 amino acids are identical with those of cobra NGF including cys-14 and val-21 which are known to be important for NGF activity.
