Studies on tissue transglutaminases: interaction of erythrocyte type-2 transglutaminase with GTP

Ca2+ and GTP are the main modulators of type-2 transglutaminases. To study the interaction of the enzyme with GTP, we have employed periodate-oxidized GTP as an affinity-label probe. Dialdehyde GTP bound irreversibly to type-2 transglutaminase in a time-dependent way with 1:1 stoichiometry at complete modification. The reaction took place in the absence, but was more rapid in the presence, of cyanoborohydride. Native GTP prevented incorporation of dialdehyde GTP, and Ca2+ significantly slowed down the reaction rate. The modified enzyme displayed decreased sensitivity to Ca2+, with a sigmoid saturation curve. We conclude that type-2 transglutaminase has a single GTP-binding site, the modification of which by dialdehyde GTP mimics nucleotide binding to the enzyme.

Synthesis of spin-labelled 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl)-phosphatidylcholine

Chemical synthesis of spin-labelled lecithin 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)-stearoyl)-phosphatidylcholine was achieved by reaction of equimolar amounts of purified egg yolk lyso-lecithin with commercially available 16-(N-oxyl-4′,4′-dimethyloxazolidine)stearic acid activated with N,N-carbonyldiimidazole. After purification by silicic acid column chromatography, spin-labelled lecithin was obtained in 52.2% yield and had an ester:phosphorus:spin molar ratio of 2.0:1.1:1.0. When dimyristoyl-phosphatidylcholine and dimyristoyl-phosphatidylcholine:cholesterol (10:1) liposomes, containing 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl)-phosphatidylcholine as a probe, were examined for thermotropic changes monitored by electron spin resonance spectroscopy, transition temperatures of 24.0 and 24.2 °C, respectively, were obtained in a very good agreement with previously reported values obtained with different probes and by different techniques, and with our own differential scanning calorimetry measurements. The potential usefulness of synthetic 2-(16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl)-phosphatidylcholine as a spin-label probe in studies of lipid–protein interactions in biological membranes was discussed.