Isolation, expression and characterization of the gene for an ADP-ribosylation factor from the human malaria parasite, Plasmodium falciparum.
We have isolated an ADP-ribosylation factor (ARF) gene from the human malarial parasite, Plasmodium falciparum. The gene (P. falciparum arf1) has four introns and the exons encode a protein of 181 amino acids with high similarity to the mammalian class I ARF proteins 1-3 (> or = 74% amino acid identity). Southern hybridization suggests there is at least one additional arf in the P. falciparum genome. Northern analysis identified a single P. falciparum arf1 mRNA of 1.8 kb in the asexual blood stage form of the parasite. The P. falciparum arf1 mRNA levels are developmentally regulated, reaching a maximum during nuclear division towards the end of the intraerythrocytic cycle. P. falciparum arf1 cDNA was isolated by reverse-transcriptase polymerase chain reaction and used to express a recombinant protein in Escherichia coli. Recombinant P. falciparum ARF1 protein was purified with stoichiometric amounts of bound GDP, although intrinsic guanose triphosphatase activity of the protein could not be detected. The protein stimulated cholera-toxin-catalyzed ADP-ribosyltransferase activity in a reaction that was dependent upon the addition of both dimyristoylglycerophosphocholine and cholate. The protein bound GTP with first-order kinetics with an apparent rate constant, k', of 0.0145 (+/- 0.0019) min-1. These results suggest that P. falciparum ARF1 is a member of the class 1 ARF family and provide additional evidence for the existence of a classical secretory pathway in P. falciparum.
Stafford
W H
WH
Division of Parasitology, National Institute for Medical Research, Mill Hill, London, UK.
Stockley
R W
RW
Ludbrook
S B
SB
Holder
A A
AA
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002772:Cholera Toxin / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011943:Receptors, Adrenergic, beta / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D002352:Carrier Proteins; D018076:DNA, Complementary; D011943:Receptors, Adrenergic, beta; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000262:Adenylyl Cyclases