Using fluorogenic substrates and polyacrylamide gels we detected in cell-free extracts of Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei only a single aminopeptidase. A comparative study of the aminopeptidase activity in each extract revealed that the enzymes have similar specificities and kinetics, a near-neutral pH optima of 7.2 and are moderately thermophilic. Each has an apparent molecular weight of 80,000 +/- 10,000, determined by high performance liquid chromatography on a calibrated SW500 column. Whilst the P. c. chabaudi and P. berghei activity co-migrate in native polyacrylamide gels, that of P. falciparum migrates more slowly. The three enzymes can be selectively inhibited by ortho-phenanthroline and are thus metallo-aminopeptidases; however, in contrast to other aminopeptidases the metal co-factor does not appear to be Zn2+.

Erythrocyte invasion assays are described for two species of rodent malaria, namely Plasmodium berghei and P.c. chabaudi. These invasion assays are simple, are carried out using a candle jar and allow a number of assays to be performed simultaneously. Our results demonstrate that both rodent malaria species show an in vitro preference for reticulocytes although the preference of P. c. chabaudi for these cells is not as marked as that of P. berghei. The details of our invasion assays and our results obtained are discussed.

RPMI-1640 is routinely used as the basal medium for the in vitro maintenance of malaria parasites. In this study we tested several commercially available nutritional media in a Plasmodium chabaudi chabaudi erythrocyte invasion assay and showed that three media, BME Basal Medium--modified, Dulbecco's Modified Eagle Medium, and William's Medium E, improved the level of merozoite invasion when compared with RPMI-1640. These media improve the rate of maturation of newly invaded rings to young trophozoites. Radioisotope incorporation by trophozoites maintained in these three media was also improved when compared to trophozoites maintained in RPMI-1640. BME Basal Medium--modified, or a combination of three parts BME Basal Medium--modified with one part William's Medium E, supported higher levels of erythrocyte invasion by merozoites. We suggest that either of these media replace the currently used RPMI-1640 for in vitro studies on P. c. chabaudi.

100 200 Time (min) Fig. 1. Effect of the peptide sialin and its analogues I (Gly-Gly-Lys-Lys) and 2 (Ah-Gly-Lys-A@ at 5.0 mM on the pH of salivary sediment incubated with 2.8 mM-glucose at 37°C' A , Glucose; 0, glucose plus siahn; *, glucose plus analogue 1;., glucose plus analogue 2.final concentrations of 2.8 mM (glucose) and 5.0 mM (peptide) in a total volume of 1.8 ml. The pH of the peptide solutions was adjusted to 7.0 before addition to the sediment suspension. The suspensions were incubated at 37°C with ResultsThe pH changes recorded are shown in Fig. 1. The response curves found for sediment plus glucose and for sediment plus glucose plus sialin are as expected. With analogue 2, pH recovery occurred earlier than with sialin, indicating that hydrophobicity may be important in peptide uptake. In the assay with analogue 1, pH recovery also occurred earlier and a higher final pH was recorded. This result was unexpected and appears to show that sialin does not function simply by release of arginine in the bacterium.1.

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