Crotalus adamanteus

The venoms of poisonous snakes contain (among other things) a class of enzymes known as phospholipases, enzymes that cause the breakdown of phospholipids. For example, the venoms of the eastern diamondback rattlesnake (Crotalus adamanteus) and the Indian cobra Naja naja) both contain phospholipase Ag, which catalyzes the hydrolysis of fatty acids at the C-2 position of glyc-erophospholipids. 

Since the acetone precipitation step is not applicable to all venoms, the decision of which method to use depends on the availability of the starting venom Crotalus adamanteus (35), Bothrops atrox (44), Heina-chatus haemachates (43), and Vipera lebetina (48). 

All three of the fully-unblocked dodecamers underwent complete digestion to give their monomeric components when they were treated with Crotalus adamanteus snake venom and spleen phosphodiesterases. Their structures were further confirmed in the usual way. 

A very satisfactory source of phospholipase A2 is the venom of the snake, Crotalus adamanteus (Eastern diamondback rattlesnake). This venom can be obtained in lyophilized form from commercial suppliers such as Miami Ser-pentarium (Miami, FL). Of importance, the lyophilization process does not alter the chemical, physical, or enzymatic characteristics of the original venom obtained from this snake. 

Partial (Semi) Synthesis. A blend of two methodologies can be used quite effectively to prepare mixed acid phosphatidylcholines, which are of prime importance in elucidating the structure of naturally occurring phospho-glyceride and phospholipase A2 activity. Perhaps the best scenario would be to illustrate the activity of the phospholipase A2 (from Crotalus adamanteus snake venom) toward a racemic phosphatidylcholine sample and toward individual sn-1 and sn-3 enantiomers. In each case the same result would be found, and so only the racemic mixture reaction is depicted in Figure 4-8. 

Phospholipase A2 Action. Incubation of phosphatidylserine with phospholipase A2 obtained from Crotalus adamanteus or Naja Naja snake venom will show that the serine-containing phosphoglyceride was smoothly and completely converted to a lysophosphatidylserine with liberation of 1 mol of fatty acid per mole of lipid P. The experimental procedure was the same as the one described before in this and in the previous chapter. The products of the reaction can be recovered by thin-layer chromatography on Whatman K6 plates in a solvent system of chloroform-acetone-methanol-acetic acid-water (4.5 2 1 1.3 0.5, v/v). 

Phospholipase A2 isolated from snake venom (Crotalus adamanteus) can attack cardiolipin with liberation of 2 mol of fatty acid per mol of phosphorus. This result argues for an sn-3 configuration for the native cardiolipin molecule. 

Saito, K. and Hanahan, D. J. (1962) A study of the purification and properties of the phospholipase A2 of Crotalus adamanteus venom, Biochemistry 1, 521-532. 

Snake Venom Protease Family Crotalus adamanteus, HAG HiSa 3 HiSa 5 His H2O ... 

Brock, 0. G., 1980, Predatory Behavior of Eastern Diamondback Rattlesnakes (Crotalus adamanteus) Field Enclosure and Y-maze Laboratory Studies, Emphasizing Prey Trailing Behavior, unpublished doctoral dissertation, Florida State University. 

The highest L-amino acid oxidase activity is found in the venoms of a variety of snakes, the enzyme being present to the extent of up to 3% of dried, whole venom in some species. Crystalline enzymes (both flavoproteins) have been isolated from the venoms of water moccasin Ancistrodonpiscivorus) and rattle snake Crotalus adamanteus) and shown to be similar with respect to molecular weight (130,000) and flavin content (2 moles FAD/mole protein). The enzymes are not substrate-specific but are more active with the L-isomers of methionine, leucine, tryptophan, phenylalanine and tyrosine than with other L-amino acids. The reactions catalysed by L-amino acid oxidase are formally the same as those discussed in section V.A (reactions 23-26) but, as is the case for the D-amino acid oxidase, the mechanism of... 

Membrane preparations from Nannochloropsis were provided with the specified [l4c]-acyl-CoA (0.03 uCi), 100 mM Tris-HCl pH 8.5, 1 mM CoA, 2 mM ATP and 2 mM MgCl2 for 4 to 10 minutes at 30 C. Rates of acyl-CoA incorporation into lipids were linear and proportional to protein concentration in the ranges used. Lipids were separated by silica gel TLC using chloroform.methanol water (65 25 4) and scraped into tul s for scintillation counting. For positional analyses, lipids were eluted after separation on TLC, and digested with phospholipase A2 from Crotalus adamanteus in the presence of 100 p g of dioleoyl-PC or dioleoyl-PE carrier in 0.9 M borate buffer, pH 8.0 and ether. The digested lipids were separated by TLC as above and scraped into tubes for scintillation counting. 

To test exonuclease resistance, the oligonucleotides were incubated with venom exonuclease phosphodiesterase I from Crotalus adamanteus (0.2 mU) in a total volume of 180 pL for 15 min. To test serum stability, oligonucleotides were incubated in 10% bovine serum for 30 min... 

To achieve kinetic resolution of mexiletine (di-o-methyl-phenoxyisopropylamine), lyoph-ilized E.coli cells containing Chromobacterium violaceum (S)-selective o>TA (100 mg, prepared as described in Ref. [75]) are resuspended in phosphate buffer (20 mL, 100 mM, 0.9 mM sodium pyruvate, 1 mM PLP). Crotalus adamanteus i-amino acid oxidase (30 mg, 9 U, Sigma) is rehydrated in the buffer. (R/S)-mexiletine (100 mg, 23 mM)... 

Flafner et al first reported an oxidase-catalyzed deracemization method using amino acids as the substrate and pkDAAOx or LAAOx from Crotalus adamanteus together with sodium borohydride as the chemical reductant in 1971 [42]. A procedure for the successful deracemization of amino acids was previously reported by Soda et al. [43]. They focused on proline and pipecolic acid as substrates for the production of L-enantiomer by deracemization because these substrates formed stable imines rather than unfavorable keto acids in water by DAAOx. However, the enzyme was denatured by the chemical reaction with sodium borohydride. Turner et al developed an effective production method for (R)- or (S)-amino acids and (S)-amines by a deracemization method using milder chemical reducing reagents such as sodium cyanoborohydride and artificial transfer hydrogenase [44,45]. 

Treatment of labelled a-aminoadipic acid sjmthesized by the Cephalosporium sp. in the presence of acetate-l- C with L-amino acid oxidase from Crotalus adamanteus indicated that more than 98% of this amino acid was the L-isomer (Warren et al., 1967). Hence, the apparent concentration of endogenous D-a-aminoadipic acid in the intracellular pool appeared to be too low to cause substantial dilution of the radioactivity of D-a-aminoadipic acid- C which entered the pool from the extracellular fluid and which remained largely unchanged in the cell. The relatively high dilution of radioactivity which accompanied the incorporation of from the labelled D-a-aminoadipic acid into penicillin N and cephalosporin C suggested that either free D-a-aminoadipic acid was not involved... 

Figure 9, Gel filtration of Crotalus adamanteus venom on a Sephadex G-lOO column.

Recently, we also purified and determined the amino acid sequence of Crotalus adamanteus NGF (Horie, et al, 1994). 

The purified Crotalus adamanteus NGF was found to be a glycoprotein whose apparent molecular mass was estimated to be about 16.5 kDa by SDS-PAGE. 

As shown in Fig. 12, Crotalus adamanteus NGF was completely sequenced and found to be composed of 117 amino acid residues. The N-glycosylation site, Asn-23, was at the same position as that for Vipera russelli russelli NGF. 

Figure 12. Summary of the sequence studies on Crotalus adamanteus NGF.

---