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Plasmodium falciparum chitinase (PfCHT1) |
 | | | | Plasmodium falciparum chitinase (PfCHT1) | | | |
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Prices are in US dollars and valid only for US orders.
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Description:
Plasmodium falciparum chitinase (PfCHT1) is thought to have a role in parasite transmission by acting on the chitin-containing peritrophic membrane of the mosquito vector's bloodmeal to release the ookinetes (1, 2). Inhibition of chitinase activity in the mosquito midgut with allosamidin, a chitinase inhibitor, blocks parasitic transmission (3).
Source:
Isolated from E. coli cells carrying a plasmid with the PfCHT1 gene cloned from Plasmodium falciparum (kindly provided by J. Vinetz) (1).
Enzyme Properties
Heat Inactivation:
100 units at 65°C for 20 minutes
Molecular Weight:
Theoretical: 40 kDa and Apparent: 40 kDa
Reaction & Storage Conditions
Unit Definition:
One unit is defined as the amount of enzyme required to release the equivalent fluorescence produced by 1 pmol 4-methylumbelliferone from the substrate 4-methylumbelliferyl-N,N',N"-triacetyl-β-chitotrioside in a total reaction volume of 100 μl in 1 minute at 25°C in 200 mM NaCl with 20 mM NaPO4 (pH 6.0), 1 mM EDTA, 20 µM 4-methylumbelliferyl-N,N',N"-triacetyl-β-chitotrioside.
Concentration:
2,000 units/ml
Storage Conditions:
20 mM Tris-HCl
200 mM NaCl
1 mM EDTA
50% Glycerol
0.1% Triton X-100
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Chitin Binding Assay::
100 units of PfCHT1 were added to 100 µl of chitin resin in binding buffer (200 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA) mixed and left on ice for 15 minutes. After centrifugation to settle the resin, the supernatant was collected containing the unbound protein sample. The resin was then washed twice with 1 ml of binding buffer, centrifuged and the wash samples collected. The chitinase activity of the initial load, the unbound sample, and the wash samples were determined. Protein samples of the load, unbound protein, wash and resin were also run on an SDS-PAGE gel.
Results: No chitinase activity was detected in the unbound or wash samples. Also, SDS-PAGE gel shows that the 65 kDa band corresponding to the PfCHT1 was not present in the unbound or wash samples but was present in the chitin resin sample. Therefore, the PfCHT1 was bound to the chitin resin.
Assay Conditions::
200 mM NaCl, 20 mM NaPO4 (pH 6.0), 1 mM EDTA, 20 µM 4-methylumbelliferyl-N,N',N"-triacetyl-β-chitotrioside.
[3H]-chitin Assay::
160 units of PfCHT1 were incubated with 10,000 cpm [3H]-chitin in 200 mM NaCl, 20 mM NaPO4 (pH 6.0), 1 mM EDTA, 500 µg/ml BSA in 200 µl at 37°C. Aliquots were removed at various time points up to 24 hours, mixed with unlabelled chitin and after centrifugation, the total soluble cpm for each time point was determined.
Results: 160 units of PfCHT1 released 30% of the total soluble cpm in 24 hours.
References
- Vinetz, J.M. et al. (1999) Proc. Natl. Acad. Sci. USA, 96, 14061-14066.
- Tsai, Y.L. et al. (2001) Infect. Immun., 69, 4048-4054.
- Shahabuddin, M. et al. (1993) Proc. Natl. Acad. Sci. USA, 90, 4266-4270.
Legal
Research Use Assurance:
This product is sold for NON-COMMERCIAL RESEARCH USE ONLY. A license for commercial use thereof, including screening for therapeutics, diagnostics applications or clinical applications may be obtained from the University Texas Medical Branch by contacting the Office of Technology Management at (409) 772-0567.
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