The edema inducing activity of phospholipase A2 (PLA2) enzymes from snake venoms and porcine pancreas was investigated using mouse paw as experimental model. All ten PLA2 enzymes exhibited potent edema inducing activity. PLA2, however, is generally not the major edema inducing component of snake venom. Chemical modification studies indicated that enzymatic activity of PLA2 was required for its edema inducing activity. All PLA2 enzymes examined displayed a rapid onset edema which was suppressed by pretreatment of the mice with antihistamine. Dexamethasone pretreatment also inhibited edemas elicited by some PLA2 enzymes.
In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.
Four homologous single chain phospholipases A2 (Pa-1G, Pa-5, Pa-12C and Pa-15) were tested for neuromuscular effects on chick biventer cervicis and mouse hemidiaphragm nerve-muscle preparations. The four isozymes blocked directly elicited (mouse hemidiaphragm) and indirectly elicited (mouse and chick nerve-muscle preparations) twitch responses in concentrations of 1-30 micrograms/ml. The order of potency seen in both types of preparations was Pa-1G = Pa-5 greater than Pa-12C much greater than Pa-15. All four isozymes caused slow-onset, sustained contractures and reduction of muscle membrane potentials. In the chick preparation, responses to acetylcholine, carbachol and KCl were reduced by exposure to the toxins. It is concluded that the toxins act primarily postsynaptically to depress muscle contractility, perhaps by directly damaging muscle fibres. The order of potency agrees with their phospholipase A2 activity. Pa-1G is unusual because it is an acidic molecule, most toxic phospholipases being basic.
cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.
To investigate the geographic variations in venoms of two medically important pitvipers, we have purified and characterized the phospholipases A2 (PLA2s) from the pooled venoms of Calloselasma rhodostoma from Malaysia, Thailand, Indonesia, and Vietnam, as well as the individual venom of Trimeresurus mucrosquamatus collected from both North and South Taiwan. Enzymatic and pharmacological activities of the purified PLA2s were also investigated. The complete amino acid sequences of the purified PLA2s were determined by sequencing the corresponding cDNAs from the venom gland and shown to be consistent with their molecular weight data and the N-terminal sequences. All the geographic venom samples of C. rhodostoma contain a major noncatalytic basic PLA2-homolog and two or three acidic PLA2s in different proportions. These acidic PLA2s contain Glu6-substitutions and show distinct inhibiting specificities toward the platelets from human and rabbit. We also found that the T. mucrosquamatus venoms from North Taiwan but not those from South Taiwan contain an Arg6-PLA2 designated as TmPL-III. Its amino acid sequence is reported for the first time. This enzyme is structurally almost identical to the low- or nonexpressed Arg6-PLA2 from C. rhodostoma venom gland, and thus appears to be a regressing venom component in both of the Asian pitvipers.