Atroxase

ADENINE NUCLEOTIDE TRANSLOCASE ATROPISOMERS ATROXASE Attachment,... 

Willis, T. W., and Tu, A. T. (1988). Purification and biochemical characterization of atroxase, a nonhemorrhagic fibrinolytic protease from western diamondback rattlesnake venom. Biochemistry 27 4769-4777. 

ATROXASE-A FIBRINOLYTIC ENZYME ISOLATED FROM THE VENOM OF WESTERN DIAMONDBACK RATTLESNAKE... 

Atroxase was isolated according to the method of Willis and Tu (25). Homogeneity was established by two independent methods SDS-polyacrylamide gel electrophoresis, and reverse phase(RP) HPLC. Crotalus atrox venom was purchased in lyophilized form from Miami Serpentarium Laboratories, lot 16 AZ. RP HPLC was performed using Beckman System Gold Chromatography connected to binary pump model 26 and model 167 detector through a Vydac analytical reverse phase column (C g with 300 A pore) Samples were eluted with a linear gradient from 10% solvent A, (0.1% TFA in water) to 90% solvent B(0.1% TFA in acetonitrile) at a flow of 1 ml/min. Fractions were monitored at 280 and 220 nm. 

The polymerase chain reaction (PCR) was used to amplify gene fragments unique to atroxase using a combination of the synthetic degenerate oligonucleotide probes as upstream and downstream primers. 

The P labeled PCR fragments were radiolabeled with 32 P and used to probe the cDNA library for cDNA atroxase. Positive colonies were isolated and sequenced as described above. Recombinant plasmids containing PCR fragment inserts and positive cDNA clones were sequenced using USB Sequenase Version 2.0 sequencing kit. Sequence manipulations, translations and comparisons were performed using PCGENE software. 

Isolation of cDNA Atroxase. The PCR derived cDNA fragments were radiolabeled and used to probe the cDNA library for a full length cDNA encoding atroxase via colony... 

Figure 2. Translated amino acid sequence of atroxase.

Figure 3. Nucleotide bases obtained from cDNA via colony hybridization. Translated region of atroxase is also included.

Atroxase has 205 amino acids obtained by translation of a truncated cDNA clone of atroxase isolated from a cDNA library constructed from activated rattlesnake venom glands. 

However, three hemorrhagic toxins also fall into this category of sequence similarity to atroxase. So a direct comparison of the primary structure of the zinc bnding region does not give any clues as to why there is a difference in proteolytic activities between hemorrhagic and nonhemorrhagic zinc metalloproteinases. 

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