Purification and characterization of recombinant pyruvate phosphate dikinase from Giardia.

The gene encoding pyruvate phosphate dikinase (PPDK) from Giardia duodenalis was expressed using a baculovirus system. The recombinant enzyme was purified to homogeneity and its enzymological and solution structure properties characterized. The catalytic constant for the pyruvate-producing reaction was about twice as high (1560 min(-1) at 30 degrees C) as that for the reverse reaction (700 min(-1)) and the k(cat)/Km for PPi was about two orders of magnitude higher than k(cat)/Km for Pi, indicating that the pyruvate-forming reaction is much more efficient than the reverse, phosphoenolpyruvate (PEP)-forming process. The endogenous substrate levels found for PEP (0.5 mM) and pyruvate (< 80 microM) support the assumption that, under physiological conditions, the enzyme primarily performs a catabolic function. The molecular mass of the purified recombinant PPDK was analyzed by analytical ultracentrifugation and size exclusion chromatography using different assay conditions that have been reported to affect the quaternary structure of PPDKs in other organisms. Both methods clearly indicated a dimeric structure for giardial PPDK with a molecular mass of about 197 kDa (monomer mass 97.6 kDa). Several compounds, primarily structural analogs of PPi, were tested for their ability to inhibit PPDK activity. Most of the bisphosphonates examined showed either no, or only a moderate, inhibitory effect on the enzyme. Imidodiphosphate was the only competitive inhibitor with respect to PPi (Kic = 0.55 mM), whereas the bisphosphonates produced a mixed type of inhibition. The most active compound in inhibiting PPDK activity was oxalate, with a Kic value of less than 1 microM with respect to PEP.

The release of the variant surface protein of Giardia to its soluble isoform is mediated by the selective cleavage of the conserved carboxy-terminal domain.

The trophozoites of Giardia duodenalis are covered by a coat composed of an apparently single species of a group of novel, cysteine-rich proteins. These variant-specific surface proteins (VSPs) can be changed by sequential expression of different VSP genes, a process for which a gradual exchange of VSP molecules appears to be required. In the present study, we have examined the in vitro release of one of these VSPs (VSP4A1, formerly named CRISP-90) expressed by a sheep-derived variant Giardia clone. During in vitro incubation of the cloned trophozoites, the membrane-associated form of VSP4A1 (mVSP4A1) was specifically converted to a water-soluble isoform that was continuously released into the culture medium. The time required for mVSP4A1 to decline to half of the initial amount was 7.8 h. Analysis of the two purified protein species by mass spectrometry revealed molecular mass values of 68,991 Da for mVSP4A1 and of 65,425 Da for its soluble counterpart. Amino-terminal sequencing and metabolic labeling experiments indicated that the release of mVSP4A1 was associated with the cleavage of a carboxy-terminal peptide carrying the palmitic acid recently demonstrated to be attached to mVSP4A1. Calculations using the molecular mass and predicted amino acid sequence data indicated that fragmentation of the protein possibly occurs at a site located between the lysine and serine residues of the highly conserved NKSGLS motif directly preceding the hydrophobic sequence previously postulated to serve as a membrane anchoring domain of other VSP molecules. The observed processing of the membrane-associated VSP to its soluble isoform is assumed to be an essential requirement for the ability of the parasite to undergo surface antigenic variation and thus for its establishment and survival within the vertebrate host.

Design, synthesis, and characterization of HIV-1 enhancer-binding polypeptides derived from bacteriophage 434 repressor.

We have designed and synthesized HIV-1 enhancer-binding polypeptides that were derived from bacteriophage 434 repressor. These peptides were 39-54 residues long and contained either the recognition helix or the entire helix-turn-helix motif of the DNA-binding domain of 434 repressor. The dissociation constant of the complex formed between the standard peptide (R42) and a synthetic 70-bp HIV enhancer DNA was ca. 10(-8) M. The specificity of the interaction of R42 with the two HIV enhancers was demonstrated by competitive band shift assays, stepwise displacement of the p50 subunit of transcription factor NF-kappa B from its two HIV enhancer binding sites, and DNase I footprinting; R42 seemed to protect best the two TTTCC sequences of the HIV enhancers against digestion by DNase I. R42 analogues with mutated recognition helix had lower DNA binding specificity. It remains to be investigated whether our artificial HIV enhancer-binding polypeptides are active in vivo.