Linda Tanigoshi

Purification of Plasmodium lophurae cathepsin D and its effects on erythrocyte membrane proteins

The cathepsin D of Plasmodium lophurae was purified using a combination of CM-Sephadex, pepstatin-agarose and Sephadex G-100 chromatography. The plasmodial enzyme was distinct from that of the host red cell and bovine spleen in its low isoelectric point (pI 4.3). The cathepsin D of P. lophurae, as well as plasmodial extracts demonstrating such proteinase activity, were able to digest the membrane proteins of duckling and human red cells at pH 7.4; proteolysis was not inhibited in phosphate-buffered saline by 100 microM pepstatin. Membrane proteins most susceptible to proteolysis were those of the cytoskeleton, notably bands 1 and 2 (spectrin), bands 2.1-2.6 (spectrin-binding proteins) and band 3. Membrane protein degradation by crude plasmodial extracts was partially inhibited by a combination of 10 mM FeCl3, and 10 mM phenylmethylsulfonyl fluoride in phosphate-buffered saline. The changes induced in erythrocyte membrane proteins by exposure to plasmodial cathepsin D parallel the alterations observed in red cell membranes obtained from malaria infected cells. Since the action of the plasmodial protease was confined to the inner surface of the red cell membrane, it is possible that protease-induced modifications in the red cell cytoskeleton could lead to merozoite release.

Incorporation of 14C-amino-acids by malaria (Plasmodium lophurae) IV. In vivo utilization of host cell haemoglobin☆

Abstract 1. 1. The avian malarjal parasite. Plasmodium lophurae , was grown in vivo through one asexual cycle m duck erythrocytes containing radioactive haemoglobin. 2. 2. Radioactivity was recovered in the parasite proteins (uncontaminated by host cell haemoglobin or malaria pigment), thus demonstrating the in vivo utilization of host cell haemoglobin by the malaria parasite for synthesis of plasmodial proteins.

Incorporation of 14C-amino acids by malarial plasmodia (Plasmodium lophurae): VI. Changes in the kinetic constants of amino acid transport during infection☆

Abstract The majority (10 of 17) of amino acids tested entered the mature duck erythrocyte by a saturable, non-uphill transport system, whereas for the erythrocyte-free malarial parasite, Plasmodium lophurae, the converse was true: most amino acids entered the parasite by simple diffusion. Only five amino acids (glutamic and aspartic acids, cysteine, lysine, arginine) showed mediated entry into P. lophurae. The pattern of mediated amino acid transport into the duck erythrocyte was altered upon infection, e.g., either entry was by diffusion or there was a reduced affinity for the amino acid. Transport characteristics similar to those found in the malaria-infected erythrocyte were produced by treating normal duck red cells with a cell-free extract of malaria-infected erythrocytes and quinine (a depressor of red cell ATP). It is suggested that depletion of host cell ATP as well as elaboration of as yet unidentified substances by the parasite promote the changes in permeability seen in the malaria-infected cell.

The glutamate dehydrogenase of Plasmodium lophurae (Avion malaria)

Abstract The agent of avian malaria, Plasmodium lophurae , contains an NADP-glutamate dehydrogenase (EC 1.4.1.2) which is capable of oxidizing glutamate to α-ketoglutarate. This glutamate dehydrogenase exists as a single electrophoretic form in both the free parasite and in the malaria-infected cell; the normal erythrocyte contains two other electrophoretic forms of low activity. It is suggested that the oxidation of glutamate, and its further metabolism via the Krebs tricarboxylic acid cycle may provide an additional energy source for the parasite.

Plasmodium lophurae: Immunization of Pekin ducklings with different antigen preparations

Abstract Pekin ducklings were vaccinated with Freunds complete adjuvant plus free Plasmodium lophurae parasites, erythrocytes infected with P. lophurae schizonts, or parasite membrane vesicles. Approximately 50% of the vaccinated ducklings were resistant to challenge with this malarial parasite. However, little protection was afforded by immunization of ducklings with a parasite-specific histidine-rich protein.

Incorporaion of 1c-amino-acids by malaria (plasmodium lophurae): II. Migration and incorporation of amino-acids

Abstract 1. 1. The avian malaria. Plasmodium lophurae, malaria-infected erythrocytes, and normal duck erythrocytes suspended in a protein-free medium resembling the in vivo environmental milieu showed 4 patterns of accumulation of radioactive amino-acids. 2. 2. In vitro, malaria parasites were capable of utilizing exogenously supplied amino-acids for the synthesis of parasite proteins. The protein synthetic activity found for the malariainfected cell could, in general, be represented as the sum of the activities of the uninfected cell and that of the free parasite. 3. 3. A hypothesis is presented which proposes that the rate of amino-acid incorporation by the parasite depends on three factors: (a) the free amino-acid levels, (e) the rate of amino-acid entry, and (c) the abundance of the amino-acid in the protein being synthesized.

Plasmodium lophurae: Glucose-1-14C and glucose-6-14C catabolism by free plasmodia and duckling host erythrocytes☆

Abstract 1. 1. The in vitro catabolism of glucose-1- 14 C and glucose-6- 14 C by the avian malaria Plasmodium lophurae was studied. 2. 2. Free parasites and malaria-infected erythrocytes principally formed lactate from glucose; smaller quantities of glutamate, aspartate and alanin were also recovered. The C-6/C-1 ratio for these catabolites was 1. 3. 3. Continuous monitoring of the release of 14 CO 2 showed that greater amounts of 14 CO 2 was recovered from the C-1 than from the C-6 of labeled glucose. 4. 4. Erythrocyte-free P. lophurae also metabolized pyruvate- 14 C and produced considerable quantities of 14 CO 2 ; in addition, radioactivity was recovered in citrate, malate, succinate and fumarate. There was no evidence for acetate formation from pyruvate by free parasites.

Alterations in sodium and potassium in red blood cells and plasma during the malaria infection (Plasmodium lophurae)

Abstract 1. 1. During the course of infection of Plasmodium lophurae (avian malaria) in white Pekin ducklings the red blood cell sodium concentration was significantly elevated in parasitized animals (14·0±1·32 mM; mean ± S.E.M.) as compared to normal animals (8·8 ± 0·72 mM); the plasma potassium level was also significantly increased in malarious ducklings (5·4 ± 0·29 mM) when compared to normal ducklings (2·9 ± 0·18 mM). 2. 2. Cation concentrations of P. lophurae more closely resembled those of the duck erythrocyte than of duck plasma.

Incorporaion of 1c-amino-acids by malaria (plasmodium lophurae): III. Metabolic fate of selected amino-acids

Abstract 1. 1. Erythrocyte-free Plasmodium lophurae (avian malaria) directly incorporate carbon 14 from arginine, lysine, and methionine into the parasite proteins; malaria-infected cells incorporate 14 C-proline mainly into the proteins of the parasitized cell. 2. 2. 14 C-Glutamic acid is principally metabolized to 14 COa by the free parasite and is not involved to a large degree in the biosynthesis of proteins.

Failure to protect ducklings against malaria by vaccination with histidine-rich protein

Abstract Vaccination of white Pekin ducklings, bred either on the east (New York) or the west (California) coast of the USA, with a histidine-rich protein provided no significant protection against subsequent challenge with 2 × 10 7 Plasmodium lophurae per kg body-weight.