Beta-alanine transaminase activity in black and suppressor of black mutations of Drosophila melanogaster.

An assay for beta-alanine transaminase activity in extracts of Drosophila melanogaster has been developed. By use of this assay, the levels of beta-alanine transaminase activity in several strains of flies has been examined as a function of developmental age. The black mutation shows elevated levels of activity compared to wild type, while suppressor of black strains show decreased levels compared to wild type.

Calcium-dependent and -independent interfacial binding and catalysis of cytosolic group IV phospholipase A2.

Cytosolic group IV phospholipase A2 (cPLA2) plays a role in liberating arachidonic acid from the sn-2 position of mammalian cellular phospholipids. The enzyme consists of a catalytic domain joined to an N-terminal calcium-dependent, membrane binding domain (C2 domain). The interfacial binding properties of the full-length, nonphosphorylated enzyme and its C2 domain to phospholipid vesicles were studied as a function of vesicle phospholipid composition and calcium concentration. The binding of cPLA2 to phosphatidylcholine vesicles is mostly governed by its C2 domain; binding is relatively weak, and calcium enhances binding and interfacial catalysis by about 10-fold. Catalytically productive interfacial binding was measured by monitoring the increase in the rate of cPLA2-catalyzed hydrolysis of a fluorimetric substrate present in vesicles as a function of bulk vesicle concentration. Enzyme-vesicle binding was also measured by fluorescence as was enzyme-calcium binding. Compared to zwitterionic vesicles, cPLA2 binding to anionic phosphatidylmethanol vesicles is of higher affinity and calcium-independent, although calcium is required for the binding of the C2 domain to these anionic vesicles. cPLA2 is fully catalytically active on phosphatidylmethanol vesicles in the absence of calcium. Phosphatidylserine is not a good replacement for phosphatidylmethanol for inducing high-affinity, calcium-independent binding of cPLA2. These results reveal two modes of catalytically productive interfacial binding of cPLA2: calcium-dependent anchoring via the C2 domain and a calcium-independent component involving a phosphatidylmethanol recognition element in the catalytic domain. They also show that membrane binding of cPLA2 is not, in general, predicted by the interfacial binding properties of its C2 domain.

Interfacial recognition by bee venom phospholipase A2: insights into nonelectrostatic molecular determinants by charge reversal mutagenesis.

The basis for tight binding of bee venom phospholipase A2 (bvPLA2) to anionic versus zwitterionic phospholipid interfaces is explored by charge reversal mutagenesis of basic residues (lysines/arginines to glutamates) on the putative membrane binding surface. Single-site mutants and, surprisingly, multisite mutants (2-5 of the 6 basic residues mutated) are fully functional on anionic vesicles. Mutants bind tightly to anionic vesicles, and active-site substrate and Ca2+ binding are not impaired. Multisite mutants undergo intervesicle exchange slightly faster than wild type, especially in the presence of salt. It is estimated that electrostatic contribution to interfacial binding is modest, perhaps 2-3 kcal/mol of the estimated 15 kcal/mol. Elution properties of bvPLA2 from HPLC columns containing solid phases of tightly packed monolayers of phosphocholine amphiphiles suggest that ionic effects provide a modest portion of the interfacial binding energy and that this contribution decreases as the number of cationic residues mutated is increased. These results are consistent with the observation that Gila monster venom PLA2 (Pa2), which is homologous to bvPLA2, has high activity on anionic vesicles despite the fact that it has only a single basic residue on its putative interfacial recognition face. Results with bvPLA2 mutants show that manoalogue and 12-epi-scalaradial inactivate bvPLA2 by modification of K94. Also, deletion of the large beta-loop (residues 99-118) is without consequence for interfacial binding and catalysis of bvPLA2. All together, the preferential binding of bvPLA2 to anionic vesicles versus phosphatidylcholine vesicles is mainly due to factors other than electrostatics. Therefore hydrogen-bonding and hydrophobic interactions must provide a major portion of the interfacial binding energy, and this is consistent with recent spectroscopic studies.