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An Asp49 PLA2 (ACL-I PLA2) was purified from the venom of Agkistrodon contortrix laticinctus by gel filtration and cation-exchange chromatography. It has a relative molecular mass of 14,000, and its N-terminal sequence has more than 65% of identity with other snake venom PLA2s. ACL-I PLA2 injected into the Tibialis anterior muscle of rats and mice at doses of 0.3 and 1.6 mg/kg, respectively, induced muscle fiber necrosis, cellular infiltration and edema 3 and 48 h after injection. The effect of the purified enzyme on water permeability was tested in the isolated toad urinary bladder. Water flow through the membrane was measured gravimetrically in bag preparations of the bladder. ACL-I PLA2 (20 nM) did not significantly alter the water permeability in the bladder preparations, whereas ACL myotoxin (ACLMT), a Lys49 PLA2 isolated from the same venom, at similar concentration significantly increased (81%) the water permeability. However, both toxins inhibited the AVP-stimulated water permeability. These results strongly suggest that PLA2 activity is not involved in the ACLMT effect on water transport and the effect of ACL-I PLA2 myotoxin on membrane permeability is mediated by mechanisms that are different in comparison to ACLMT.

ACL myotoxin (ACLMT) is a K49 phospholipase A 2 -like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded copperhead) that induces necrosis of skeletal muscle. We have previously cloned and sequenced the cDNA coding for ACLMT from a venom gland cDNA library. In order to perform structure and function studies, we have developed an expression system for production of ACLMT as a fusion protein with maltose binding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expression vector. The cDNA coding for the mature toxin without the signal peptide was amplified by PCR and subcloned into the pMAL-p2 vector. The new plasmid (pMAL-MT) was used to transform BL21(DE3) E. coli cells. Culture of transformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusion protein was purified from the bacterial periplasm by affinity chromatography in an amylose column and by gel filtration. The purified fusion protein (MBP-rACLMT) was able to induce necrosis of skeletal muscle of mice very similar to that caused by the native myotoxin.

Protein crystallography has significantly contributed to the development of many areas of biochemical research, particularly in the understanding of phenomena related to molecular recognition. Examples include the formation of enzyme–substrate complexes (and their subsequent catalysis), host cell invasion by viruses, antigen neutralization and peptide display by proteins of the immune system and many others. More recently, protein crystallography has also proved to be of great value in unraveling the molecular basis of many diseases as well as in the development of new drugs for their treatment. The X-ray diffraction technique in the elucidation of macromolecular structures is situated at the interface between the traditional research fields of biology, biochemistry, chemistry and physics where researchers are united by a common interest in the detailed understanding of macromolecule function and its relationship to three-dimensional structure. The purpose of this review is to describe, without resort to mathematical detail, all of the necessary steps for the complete determination of a three-dimensional structure by X-ray diffraction techniques. The basic procedures used for protein isolation and crystallization, crystallographic data collection and analysis and, finally, structure determination and refinement are all briefly reviewed. As such our efforts are not directed towards the specialist. Rather, it is our hope that the information presented will aid interested readers from other fields in the understanding of more specialized literature and who may wish to employ the information contained therein in the planning of their biological research. We hope that in so doing we will make clear both the power and limitations of the technique.