PubMed · 4911965
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1970. Further progress on bladder cancer.. https://pubmed.ncbi.nlm.nih.gov/4911965/
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Transglutaminase is a calcium-dependent enzyme which catalyzes amine incorporation and cross-linking of proteins. To isolate the amine acceptor protein substrates of transglutaminase in mammalian livers, a biotin-labeled primary amine substrate of transglutaminase, 5-(biotinamido) pentylamine, was used for biotin labeling of proteins in the liver extracts by endogenous transglutaminase activity. The biotin-labeled proteins were isolated and recovered by biotin-avidin-affinity chromatography. The obtained proteins were separated by SDS-PAGE. Proteins with molecular masses of 15, 24, 35, 40, 44, 93, and 134 kDa were the main components of labeled proteins in mouse liver extract. In rat and guinea pig liver extracts, 32-, 38-, 40-, 44-, and 134-kDa proteins and28-, 40-, 44-, 55-, 60-, 91-, and 134-kDa proteins were the main components of labeled proteins, respectively.Using amino-terminal amino acid sequence analyses and sequence homology searches, the 38-kDa protein from rat liver was identified as a subunit of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and the 28-kDa protein from guinea pig liver was identified as a subunit of glutathione S-transferase (class theta) (EC 2.5.1.18). Both the glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle and glutathione S-transferase (class pi) from human placenta also could be amine acceptors in the amine incorporation catalyzed by guinea pig liver transglutaminase. These results suggest that these enzymes can be modified posttranslationally by cellular transglutaminase.
Oligodeoxynucleotide N3'-->P5' phosphoramidates are promising candidates for antisense therapeutics, as well as for diagnostic applications. We recently reported a new method for the synthesis of these oligonucleotide analogs which makes use of a phosphoramidite amine-exchange reaction in the key coupling step. We report herein an improved set of monomers that utilize a more reactive, hindered phosphoramidite to produce optimal yields in a single coupling step followed by oxidation, thereby eliminating the need for the previously reported couple-oxidize-couple-oxidize approach. On the 10 micromol scale, the synthesis is performed using only 3.6 equivalents (equiv.) of monomer. An improved oxidation reagent consisting of hydrogen peroxide, water, pyridine and THF is also introduced. Reported here for the first time is the use of a reverse-phase purification methodology employing a ribonucleotide purification handle that is removed under non-acidic conditions, in contrast to the conventional dimethoxytrityl group. The synthesis and purification of uniformly modified N3'-->P5' phosphoramidate oligodeoxy-nucleotides, as well as their chimera containing phosphodiester and/or phosphorothioate linkages at predefined positions, using these new methodologies are included herein. The results of31P NMR studies that led to this improved amine-exchange methodology are also described.
A previous study [Ferson, Wray and Fisher (1996) Mol. Microbiol. 22, 693-701] has shown that transposon-mediated disruption of a protein 47% identical to the Escherichia coli GABA (4-aminobutyrate) transporter abolishes the ability of nitrogen-limited culture conditions to induce expression of a GABA transport activity in Bacillus subtilis. Here it is demonstrated directly that the B. subtilis GABA permease (gabP) gene can complement the transport defect in the gabP-negative E. coli strain. Unexpectedly, the ligand-recognition profile of the B. subtilis GabP was found to differ substantially from that of the highly homologous E. coli GabP. Unlike the E. coli GabP, the B. subtilis GabP: (i) exhibits approx. equal preference for the 3-carbon (beta-alanine, Km=9.6 microM) and the 4-carbon (GABA, Km=37 microM) amino acids, and (ii) resists inhibition by bulky, conformationally constrained compounds (e.g. nipecotic acid, guvacine), which are active against GABA transporters from brain. The present study shows additionally that the B. subtilis GabP can translocate several open-chain GABA analogues (3-aminobutyrate, 3-aminopropanoate, cis-4-aminobutenoate) across the membrane via counterflow against [3H]GABA. Thus, consistent with the idea that the ligand-recognition domain of the B. subtilis GabP is less spacious than that of the close homologue from E. coli, the former exhibits more stringent requirements than the latter for substrate recognition and translocation. These distinct functional characteristics of the E. coli and B. subtilis GABA transporters provide a basis by which to identify ligand-recognition domains within the amine-polyamine-choline transporter superfamily.