Kemptide (sequence: LRRASLG) is a synthetic peptide holding the consensus recognition site for the catalytic subunit of the cAMP-dependent protein kinase (PKA). cAMP-independent protein kinases that phosphorylate kemptide were stimulated in Trypanosoma equiperdum following glucose deprivation. An enriched kemptide kinase-containing fraction was isolated from glucose-starved parasites using sedimentation throughout a sucrose gradient, followed by sequential chromatography on diethylaminoethyl-Sepharose and Sephacryl S-300. The trypanosome protein possesses a molecular mass of 39.07–51.73 kDa, a Stokes radius of 27.4 Ǻ, a sedimentation coefficient of 4.06 S and a globular shape with a frictional ratio f/fo = 1.22–1.25. Optimal enzymatic activity was achieved at 37 °C and pH 8.0, and kinetic studies showed Km values for ATP and kemptide of 11.8 ± 4.1 and 24.7 ± 3.8 µm, respectively. The parasite enzyme uses ATP and Mg2+ and was inhibited by other nucleotides and/or analogues of ATP, such as cAMP, AMP, ADP, GMP, GDP, GTP, CTP, β,γ-imidoadenosine 5′-triphosphate and 5′-[p-(fluorosulfonyl)benzoyl] adenosine, and by other divalent cations, such as Zn2+, Mn2+, Co2+, Cu2+, Ca2+ and Fe2+. Additionally, the trypanosome kinase was inhibited by the PKA-specific heat-stable peptide inhibitor PKI-α. This study is the first biochemical and enzymatic characterization of a protein kinase from T. equiperdum.

Virulence factors from the ROP2-family have been extensively studied in Toxoplasma gondii, but in the closely related Neospora caninum only NcROP2Fam-1 has been partially characterized to date. NcROP40 is a member of this family and was found to be more abundantly expressed in virulent isolates. Both NcROP2Fam-1 and NcROP40 were evaluated as vaccine candidates and exerted a synergistic effect in terms of protection against vertical transmission in mouse models, which suggests that they may be relevant for parasite pathogenicity. NcROP40 is localized in the rhoptry bulbs of tachyzoites and bradyzoites, but in contrast to NcROP2Fam-1, the protein does not associate with the parasitophorous vacuole membrane due to the lack of arginine-rich amphipathic helix in its sequence. Similarly to NcROP2Fam-1, NcROP40 mRNA levels are highly increased during tachyzoite egress and invasion. However, NcROP40 up-regulation does not appear to be linked to the mechanisms triggering egress. In contrast to NcROP2Fam-1, phosphorylation of NcROP40 was not observed during egress. Besides, NcROP40 secretion into the host cell was not successfully detected by immunofluorescence techniques. These findings indicate that NcROP40 and NcROP2Fam-1 carry out different functions, and highlight the need to elucidate the role of NcROP40 within the lytic cycle and to explain its relative abundance in tachyzoites.

Starch is globally important as a source of food and, in addition, has a wide range of industrial applications. Much of this agriculturally produced starch is synthesized in developing seeds, where its biological function is to provide energy for seedling establishment. Storage starch in developing seeds is synthesized in heterotrophic plastids called amyloplasts and is distinct from the transient synthesis of starch in chloroplasts. This article reviews our current understanding of storage starch biosynthesis occurring in these organelles and discusses recent advances in research in this field. The review discusses starch structure and granule initiation, emerging ideas on the evolution of the pathway, the enzymes of starch synthesis, and the post-translational modification and regulation of key enzymes of amylopectin biosynthesis.

Calcium (Ca2+) is thought to play a major role in the photoresponse of both vertebrates and invertebrates, but the mechanisms through which Ca2+ exerts its effects are unclear. In many systems, some effects of Ca2+ on cellular processes are thought to be mediated via activation of calcium/calmodulin protein kinase (CaCAM-PK) and the phosphorylation of specific proteins. Thus, protein substrates for CaCAM-PK in photoreceptor cells may be important in mediating the effects of Ca2+ on the photoresponse.

In this study, we identify eight substrates for CaCAM-PK found in both the ventral and lateral eyes of Limulus. We focus on a characterization of one of these, a 46-kD substrate. We show that its subcellular distribution in ventral photoreceptors and its isoelectric forms are identical to the 46-kD light-stimulated phosphoprotein (46A) described by Edwards et al. (1989). Furthermore, we present evidence that 46A is unique to photoreceptor cells, and that it is present throughout the cell. Based on the results of this study, and the previous study by Edwards et al. (1989), we propose that 46A is involved in mediating the effects of Ca2+ on Limulus photoreceptor cell function, and that it may be involved in dark adaptation.

The protein intermediates of the photoresponse and the modulation of this response in invertebrate photoreceptors are largely unknown. As a first step toward identifying these proteins, we have examined light-stimulated changes in protein phosphorylation in preparations of Limulus photoreceptors. Here we show that light modulates the level of phosphorylation of three proteins associated with Limulus ventral photoreceptors: the upper band of a 46-kD protein doublet (46A) and a 122-kD protein, which become more heavily phosphorylated in response to light, and the lower component of the 46-kD doublet (46B), which is phosphorylated in dark-adapted cells, but not in cells maintained in the light. In dark-adapted preparations, 46A is phosphorylated within 30 s after a flash of light and dephosphorylates over a period of many minutes. It is also a major substrate for calcium/calmodulin-dependent protein kinase (Wiebe et al., 1989); therefore, we speculate that 46A is involved in some aspect of dark adaptation. Interestingly, the level of phosphorylation of 46A is the same when measured from preparations maintained in complete darkness or ambient light for at least 1.5 h. The 122-kD phosphoprotein is the same protein which becomes phosphorylated in response to efferent innervation to Limulus eyes (Edwards et al., 1988) and the efferent neurotransmitter, octopamine (Edwards and Battelle, 1987). It may be involved in the increase in retinal sensitivity and the enhanced response of photoreceptors to light that is initiated by efferent innervation. Its role in light-stimulated processes is not clear. The level of phosphorylation of 46B may be most relevant to the long-term state of adaptation of the photoreceptor cell to light and dark.

Autophosphorylation by protein kinases has been implicated as an important control mechanism in signal transduction and growth regulatory pathways in mammalian cells. We have set out to investigate whether any such autophosphorylating protein kinase activities can be found in Trypanosoma brucei. In order to do this, we have developed a system for characterizing such protein kinase activities using an in vitro assay. This assay was carried out by fractionation of trypanosome lysates using isoelectric focusing gel electrophoresis followed by incubation of the gel in γ32P-labelled nucleotide triphosphate and subsequent autoradiography. We have identified two classes of autophosphorylating protein kinase activities. In the first class all were dependent on ATP as the phosphate donor substrate and were all found to have a molecular size of 60 kDa. Differences in the activity of these protein kinases were observed between the bloodstream and procyclic life-cyle stages. Furthermore, the addition of mammalian epidermal growth factor to bloodstream stage lysates stimulated an additional activity. The second class of autophosphorylating protein kinases utilized GTP as the phosphate donor and were all found to be 90 kDa in size. Stage-specific differences were also observed in the activity of these protein kinases.

Reversible phosphorylation is catalysed by protein kinases that transfer the γ-phosphate from ATP to amino acid residues of proteins. The process can be reversed by protein phosphatases. Phosphorylation can dramatically activate or inhibit enzymes and affect protein-protein interaction. Through phosphorylation protein kinases can amplify and propagate cellular signals. In plants and now seeds, protein kinases involved in hormone, defence and environmental stress responses are being identified. Increasingly, these protein kinases are being cloned and characterized, demonstrating the major role of reversible protein phosphorylation in seeds.

Molecular interplay during snail-schistosome interactions is poorly understood and there is much to discover concerning the effect of snail host molecules on molecular processes in schistosomes. Using the Biomphalaria glabrata – Schistosoma mansoni host-parasite system, the effects of exposure to haemolymph, derived from schistosome-resistant and susceptible snail strains, on protein tyrosine phosphorylation in miracidia have been investigated. Western blotting revealed several tyrosine phosphorylated proteins in this larval stage. Exposure of miracidia to haemolymph from susceptible snails for 60 min resulted in a striking, 5-fold, increase in the tyrosine phosphorylation of a 56 kDa (p56) S. mansoni protein. In contrast, haemolymph from resistant snails had little effect on protein tyrosine phosphorylation levels in miracidia. Confocal microscopy revealed that tyrosine phosphorylation was predominantly associated with proteins present in the tegument. Finally, treatment of miracidia with the tyrosine kinase inhibitor genistein significantly impaired their development into primary sporocysts. The results open avenues for research that focus on the potential importance of phospho-p56 to the outcome of schistosome infection in snails, and the significance of protein tyrosine kinase-mediated signalling events to the transformation of S. mansoni larvae.

An early difference in net protein phosphorylation activity occurred in dormant and after-ripened wheat (Triticum aestivum L.) grains within the first hour of imbibition at 30°C. Embryos from dry, dormant and after-ripened caryopses exhibited a high degree of protein phosphorylation, particularly of four proteins (68, 54, 51 and 42 kDa). Upon hydration, protein phosphorylation activity in dormant embryos decreased within 1 h and reached minimal phosphorylation activity by 5 h. Protein phosphorylation activity in after-ripened (germinable) embryos was not suppressed and remained high during germination. If the hydrated dormant embryos were dried, protein phosphorylation activity was restored. Application of the protein phosphatase inhibitor, okadaic acid, partially overcame dormancy and slowed the decrease in phosphorylation activity. These results suggested that reduction of protein phosphorylation activity slowed the rate of germination.

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.

Summary 221

I. Introduction 221

1. The role of sucrose in plants 221

2. C4photosynthesis 222

II. Pathway of sucrose synthesis in C4plants 223

III. Control of sucrose synthesis in C4plants 225

1. The diurnal cycle in C3plants 225

2. The diurnal cycle in C4plants 227

3. Abiotic stress and sucrose synthesis 230

4. Integration of sucrose synthesis with other metabolic pathways 232

IV. Future prospects 233

Acknowledgements 234

References 234

Sucrose has a central role in the metabolism of all higher plants. Here we discuss the location of sucrose synthesis in the leaves of C4 plants, the control of flux through the pathway and its response to environmental conditions, and finally some of the prospects for the genetic manipulation of sucrose metabolism in C4 plants. Much of our knowledge about sucrose synthesis in plants comes from C3 species; it is evident, from those studies of C4 species that are available, that there are many similarities between sucrose synthesis in C3 and C4 plants. However, some of the basic regulatory mechanisms common to both C3 and C4 plants seem to have been adapted for the specialized photosynthetic metabolism of C4 plants. There are also some other important differences: for example, some C4 species possess photosynthetic cells with little or no capacity for sucrose synthesis. For these reasons it is not always appropriate to extrapolate directly from C3 to C4 plants. Even where data are available from one C4 species, usually maize, it should not be assumed that all C4 plants are necessarily the same in every respect. Although C4 plants constitute a small fraction of the plant kingdom as a whole, they include a disproportionate number of agriculturally important species. C4 plants also have some advantages over C3 plants for studies of the regulation of cell-specific gene expression and the control of metabolism. For these reasons we suggest that they are worthy of more attention by researchers.