Purification of a 24 kD parasitism-specific hemolymph protein from pharate pupae of the Caribbean fruit fly, Anastrepha suspensa.

We report purification of a 24 kD parasitism-specific protein (24 kD PSP) from pharate pupal hemolymph of the Caribbean fruit fly, Anastrepha suspensa, after parasitization by the braconid wasp, Diachasmimorpha (= Biosteres) longicaudata. We previously utilized isoelectric focusing (IEF) and two-dimensional (2-D) gel electrophoresis to demonstrate that the 24 kD PSP consists of two variants with pl 6.7 (more abundant) and pl 6.3. Purification of the more abundant 24 kD PSP variant was accomplished by Concanavalin A (Con A) sepharose B affinity chromatography followed by DEAE column chromatography. A second protocol, utilizing wheat germ agglutinin (WGA) sepharose 6MB affinity chromatography between the ConA and DEAE chromatographic steps, resulted in the purification of a partially deglycosylated form of the 24 kD PSP which retained its immunoreactivity with anti-PSP serum but which exhibited a greater relative migration in sodium dodecyl sulfate (SDS)-PAGE than the pl 6.7 24 kD PSP variant. For structural studies both 24 kD PSP variants were purified from whole hemolymph by flat bed IEF followed by SDS-PAGE. Peptide cleavage profiles in 1-D SDS-PAGE after treatment with BNPS-skatole, CNBr, and endproteinases Lys-C and Asp-N were identical for both 24 kD PSP variants. Primary N-terminus sequences of at least the first 20 amino acid residues of both variants were identical. A secondary sequence of five amino acids residues was detected in both variants at Thr, the seventh amino acid residue from the N-terminus of the primary sequence. These data indicate that both 24 kD PSPs are glycoforms of a branched, apparently homogeneous polypeptide.

Kinetic comparison of cytokinin nucleosidase activity isolated from normally ripening and mutant tomato varieties.

Kinetic parameters for cytokinin nucleosidase activity which catalyzes the deribosylation of N(6)(Delta(2)-isopentenyl)adenosine (I(6)Ado) to produce the more "active" free base N(5)(Delta(2)-isopenetyl)adenine (I(6)Ade) were compared for a normally ripening tomato (Lycopersicon esculentum L.) cultivar Rutgers, and two mutant tomato varieties (Nor and Rin). K(m) for nucleosidase activity in Rutgers was lower (K(m) = 0.1 millimolar) than that in either Nor (K(m) = 0.14 millimolar) or Rin (K(m) = 0.13 millimolar).