In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c ) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor. Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.Keywords: parasite; PKA; inhibition; H89.The emergence and dissemination of drug-resistant malaria parasites represents one of the most important public health problems in many parts of the world today. New antimalarials are urgently required, whose rational design and development requires the identification of potential therapeutic targets. This in turns rests on a better understanding of the molecular mechanisms controlling the progression of the complex life cycle of malaria parasites, especially Plasmodium falciparum, the species responsible for the lethal form of the disease. All Plasmodium species are intracellular parasites during infection of their vertebrate hosts. Sporozoites inoculated into a host during a bite by an infected Anopheles mosquito soon invade hepatocytes, within which intense asexual division takes place (exoerythrocytic schizogony), yielding up to 40 000 merozoites in the case of P. falciparum (http:// www.malaria.org). Upon schizont rupture, these merozoites invade red blood cells, where additional rounds of asexual replication occur (erythrocytic schizogony, the phase responsible for the pathogenesis of the disease). Some merozoites, instead of undergoing a further asexual cycle, arrest their cell cycle and differentiate into male or female gametocytes. Unlike asexual forms, these sexual forms are infective to the Anopheles vector.cAMP is involved in the regulation of development of several microorganisms, and cAMP-dependent pathways exist in most eukaryotic cells. For example, in the slime mold Dictyostelium discoideum, cAMP acts as a signal for the aggregation and differentiation of cells into a multicellular organism (reviewed in [1]). In most instances, cAMP exerts its action by binding to the regulatory subunit complexed to the catalytic subunit in an inactive holoenzyme of the protein kinase A (PKA or cAMPdependent protein kinase), thereby releasing the active catalytic subunit (PKA-C), whose substrates can include other protein kinases and transcription factors. P...

Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the antiTn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.z 2000 Federation of European Biochemical Societies.

The C subunit of Dictyostelium CAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI.Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequencecomparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe-motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases.

To investigate the biological role of thioredoxin in the facultative photosynthetic bacterium Rhodobacfer sphaeroides, attempts were made to construct a thioredoxin-def icient mutant by site-specif ic mutagenesis, using the Tn903 kanamycin resistance gene for selection. In situ and Southern hybridization analyses have demonstrated that the TmA-mutation is lethal for R. sphaeroides growth under anaerobic conditions with DMSO as terminal electron acceptor and under aerobic conditions. In addition, the DNA region upstream of the trxA initiation codon is essential for aerobic growth of R.sphaeroides. An ORF of unknown function was identified in this region and is suggested to encode a product essential for aerobic metabolism of R. sphaeroides. The mechanism of thioredoxin action was also analysed by using the procedure for gene replacement to introduce a Cys33 to Ser mutation into the trxA chromosomal copy. The strain carrying this mutation produced a thioredoxin impaired in its protein-disulfide reductase activity and was also not viable. These data suggest that the physiological function of R. sphaeroides thioredoxin is redox-dependent. Thioredoxin purified from R. sphaeroides was shown to have a glutathione-disulf ide oxidoreductase activity typical of glutaredoxins. This unexpected finding suggests that R. sphaeroides thioredoxin, in contrast to Escherichia coli thioredoxin, has the potential to act in GSH-dependent processes. Thus, the fundamental role of R. sphaeroides thioredoxin in cell growth probably originates from the multiple functions it can serve in wivo.