A metallo-dependent cysteine proteinase of Cryptosporidium parvum associated with the surface of sporozoites.
A proteinase of 24 kD was found associated with sporozoites of Cryptosporidium parvum. Optimal hydrolysis of azocasein, casein, bovine serum albumin, and gelatin occurred at a pH of 6.5-7.0. Activity against azocasein was inhibited by ethylenediaminotetraacetic acid (EDTA), iodoacetic acid (IAA), trans-epoxysuccinyl-L-leucylamido(4-guanido) butane (E-64), and phosphoramidon, suggesting that the enzyme was a metallo-dependent cysteine proteinase. Both serine and aspartate protease inhibitors failed to inhibit enzyme activity. The enzyme was partially purified by preparative isoelectric focusing of parasite membrane proteins. Polyclonal antiserum to parasite membrane proteins was generated in rats. The enzyme-containing fraction was subjected to SDS-PAGE and probed with antiserum, and the antibodies against the protease were eluted directly from nitrocellulose blots. An indirect immunofluorescence assay using these monospecific antibodies revealed that the protease occurred on the surface of sporozoites, but was not associated with oocyst walls, rhoptries, or micronemes.
Division of Biology, Kansas State University, Manhattan 66506, USA.
M V
MV
Nesterenko
M
M
Tilley
S J
SJ
Upton
D000818:Animals; D015153:Blotting, Western; D002364:Caseins / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D016785:Cryptosporidium parvum / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005780:Gelatin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism; D016622:Silver Staining
D002364:Caseins; D011480:Protease Inhibitors; C026018:azocasein; D012710:Serum Albumin, Bovine; D005780:Gelatin; D003546:Cysteine Endopeptidases; D008666:Metalloendopeptidases