G-PROTEINS AND CA2-MODULATED PROTEIN-KINASES OF A PLASMA MEMBRANE-ENRICHED FRACTION AND ISOLATED EYESPOT APPARATUSES OF SPERMATOZOPSIS-SIMILIS (CHLOROPHYCEAE)()

Knowledge about possible elements involved in signal transduction and adaptational phenomena during phototaxis of flagellate green algae is limited. In order to identify such putative elements we present here: (i) the isolation and characterisation of an enriched plasma membrane fraction of Spermatozopsis similis, and (ii) a comparison of the distr ibution of Ca2+-dependent putative kinases and G proteins, as recognis ed by antibodies directed against mammalian G(alpha)-subunits, in the plasma membrane preparation and isolated eyespot apparatuses of this g reen alga. In western blot analyses a 54 kDa protein reacted with an a ntiserum against a conserved sequence of G protein alpha-subunits (ant i-alpha common). A 43 kDa protein was recognised by a serum against th e G(alpha)-subunit of transducin. These proteins were enriched in the eyespot fraction and to a lesser extent in the plasma membrane fractio n. Two polyclonal antisera against C-terminal peptides of animal G(alp ha)-subunits also labelled a 43 kDa and 54 kDa protein in the eyespot preparations. Additionally four putative Ca2+-dependent kinases or the ir catalytic subunits (47, 48, 77 and 82 kDa) were identified in the p lasma membrane fraction by a gel kinase assay following electrophoreti c separation. The 82 kDa protein was specifically enriched in the plas ma membrane fraction, while the other putative kinases were also enric hed in the eyespot fraction but not in other subcellular fractions. Mo noclonal antibodies directed against a soybean Ca2+-dependent protein kinase cross-reacted with a 61 kDa protein in the plasma membrane and a 60 kDa protein in the soluble fraction. Only a very faint response w as observed in the fraction enriched in eyespot apparatuses. Our resul ts indicate (i) a low cross-contamination of the eyespot apparatus pre paration by normal plasma membrane constituents and (ii) the regulator y potential for the identified kinases and G proteins in blue/green-li ght-dependent photoresponses of Spermatozopsis similis.