spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online February 12, 2007
Journal of Experimental Biology 210, 733-740 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.001289
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kato, H.
Right arrow Articles by Valenzuela, J. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kato, H.
Right arrow Articles by Valenzuela, J. G.

Identification and characterization of a salivary adenosine deaminase from the sand fly Phlebotomus duboscqi, the vector of Leishmania major in sub-Saharan Africa

Hirotomo Kato1,2,*, Ryan C. Jochim1,3,*, Phillip G. Lawyer4 and Jesus G. Valenzuela1,{dagger}

1 Vector Molecular Biology Unit, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Room 2E-22C, Rockville, MD 20852, USA
2 Department of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, Japan
3 Preventive Medicine and Biometrics, Emerging Infectious Diseases, Uniformed Services University of the Health Sciences, Bethesda, MD20814
4 Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD 20852, USA


Figure 1
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 1. Amino acid alignment of the two adenosine deaminase (ADA) molecules derived from transcripts found in P. duboscqi salivary glands. Amino acids shaded black are identical and those shaded gray are similar. The secretory signal peptide is italicized.

 

Figure 2
View larger version (101K):
[in this window]
[in a new window]

  		Fig. 2. Clustal alignments of invertebrate putative salivary adenosine deaminase (ADA) of P. duboscqi (PduM73 and PduM74), Lutzomyia longipalpis, Aedes aegypti, Aedes albopictus and Culex pipiens and mammalian ADA (mouse, rat and human). Arrowheads indicate conserved amino acids located in the active site of the mammalian enzyme. Black shading indicates amino acid sequence identity, and gray regions indicate conserved amino acid substitutions.

 

Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 3. Phylogenetic tree analysis of putative adenosine deaminase (ADA). Branch lengths are proportional to genetic distance calculated by the ClustalW program. The scale bar represents 0.5% divergence.

 

Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 4. ADA activity of salivary homogenates of P. duboscqi. A cuvette containing 20 µM adenosine in PBS was scanned at 3 min intervals for 30 min following addition of salivary homogenate equivalent to 0.2 pairs of salivary gland from P. duboscqi (A) and P. papatasi (B). (C) Differential spectra of the data in (A) obtained by subtracting each scan from the scan at time zero. The arrows indicate the direction of change of the spectrum over time.

 

Figure 5
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 5. Salivary adenosine deaminase (ADA) activity from salivary gland homogenate (SGH) of unfed and blood-fed sand flies and from punctured salivary glands. A cuvette containing 20 µmol l–1 adenosine in PBS was scanned at 1.5 min intervals for 15 min following addition of salivary homogenate equivalent to 0.2 pairs of salivary gland.

 

Figure 6
View larger version (83K):
[in this window]
[in a new window]

  		Fig. 6. Three-dimensional chromatographic display of photodiode array data obtained from MS-HPLC of P. papatasi and P. duboscqi salivary gland homogenate (SGH). The three-dimensional data show retention time on the x-axis, UV absorbance on the y-axis (in milli absorbance units) and the UV absorbance spectra on the z-axis (from 200 nm to 320 nm). Adenosine and AMP standards show retention times of 18.5 and 14.5 min, respectively.

 

Figure 7
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 7. Expression of recombinant P. duboscqi salivary adenosine deaminase (ADA). cDNAs encoding P. duboscqi salivary ADA (PduM73 and PduM74) were cloned into PCRT7NT-TOPO vector, and recombinant proteins were expressed in E. coli. The affinity column-purified proteins (lane 1, PduM73; lane2, PduM74; lane 3, empty plasmid vector) were analyzed by SDS-PAGE and then subjected to (A) western blotting and (B) Coomassie blue staining.

 

Figure 8
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 8. Enzymatic activity of recombinant P. duboscqi salivary adenosine deaminase (ADA). A cuvette containing 20 µmol l–1 adenosine in PBS was scanned at 1.5 min intervals for 15 min following addition of recombinant ADA (A, PduM73; B, PduM74) or supernatant of bacteria transformed with control plasmid (C).

 




© The Company of Biologists Ltd 2007