Occurrence of immunoreactive 80 kDa and non-immunoreactive diacylglycerol kinases in different pig tissues

We surveyed diacylglycerol kinase in different pig tissues by using rabbit antibody immunospecific to the brain 80 kDa enzyme [Kanoh, Iwata, Ono & Suzuki (1986) J. Biol. Chem. 261, 5597-5602]. Among the other tissues examined, the immunoreactive 80 kDa enzyme was found only in the thymus and, to a much lesser extent, in the spleen, although this enzyme species was widely distributed in a variety of brain regions. Other tissues such as platelets, kidney, heart and liver contained little, if any, immunoreactive enzymes. Gel filtration of cytosolic enzymes from several tissues revealed the presence of three major activity peaks, apparently corresponding to 280, 120 and 80 kDa. Thymus and spleen contained the immunoreactive 80 kDa species together with non-immunoreactive 280 kDa enzyme. In the case of platelets, the kinase consisted almost exclusively of non-immunoreactive 120 kDa species with some 280 kDa enzyme. In an attempt to characterize the different kinase forms, the thymus enzyme was chosen for further studies because of its high activity. No immunoreactive proteins were detected in Western-blot analysis when the 280 kDa enzyme was solvent-extracted, proteinase-treated or preincubated in the presence of Ca2+. In comparison with the 80 kDa species, the 280 kDa enzyme was much more heat-stable and less dependent on deoxycholate in the assay mixture. Although the purification of different forms of the kinase is required to confirm the presence of isoenzymes, the results show that there exist several immunologically distinct diacylglycerol kinase species.