Polyclonal anybodies against the 18- and 20-kDa subunits of nucleoside diphosphate (NDP) kina.se of the ciliate protozoan Paramecium tetraurelia were used to determine the subcellular distribution of the enzyme in Paramecium. NDP kinase was present in soluble and microsomal fractions of cell bodies, in the Triton extract of cilia, and in the deciliation supernatant. The antibodies also recognized a 44-kDa protein present only in the paniculate fraction of cell bodies. In thin sections of Paramecium, the antibody specifically labeled the ridges of cortical units (either the inner alveolar membrane or part of the epiplasm), and the heterochromatin in the macronucleus. Purified pellicle (containing the plasma membrane and associated cytoskeletal elements) contained only the 44-kDa cross-reactive band which we believe to be a novel NDP kinase; the 18- and 20-kDa NDP kinase subunits were not detectable in this fraction. Purified pellicles also contained an NDP kinase with a specific activity about that expected from the amount of antigen present. When purified pellicle is incubated with [γ-32p] ATP, a protein of 44 kDa is labeled, and the label can be chased with excess ADP or IDP. This is the labeling pattern expected for an NDP kinase with the typical Ping-Pong mechanism. The label is lost after mild treatment with heat or acid, but is stable in basic solution, indicating that the enzyme employs a phosphohistidine intermediate. Cross-reactive proteins of 40-50 kDa were also detected in Xenopus and two cultured human cell lines.
|Original language||English (US)|
|State||Published - Dec 1 1996|