Transbilayer flipping of glycerophospholipids in the endoplasmic reticulum (ER) is a key feature of membrane biogenesis. Flipping appears to be an ATP-independent, bidirectional process facilitated by specific proteins or flippases. Although a phospholipid flippase has yet to be identified, evidence supporting the existence of dedicated flippases was recently obtained through biochemical reconstitution studies showing that certain chromatographically resolved fractions of detergent-solubilized ER proteins were enriched in flippase activity, whereas others were inactive. We now extend these studies by describing two convenient assays of flippase activity utilizing fluorescent phospholipid analogues as transport reporters. We use these assays to show that (i) proteoliposomes generated from a flippase-enriched Triton X-100 extract of ER can flip analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; (ii) flipping of all three phospholipids is likely due to the same flippase(s) rather than distinct, phospholipid-specific transport proteins; (iii) functional flippases represent approximately 1% (w/w) of ER membrane proteins in the Triton extract; and (iv) glycerophospholipid flippase activity in the ER can be attributed to two functionally distinct proteins (or classes of proteins) defined by their sensitivity to the cysteine and histidine modification reagents N-ethylmaleimide and diethylpyrocarbonate, respectively. Analyses of the N-ethylmaleimide-sensitive class of flippase activity revealed that the functionally critical sulfhydryl group in the flippase protein is buried in a hydrophobic environment in the membrane but becomes reactive on extraction of the protein into Triton X-100. This observation holds considerable promise for future attempts to isolate the flippase via an affinity approach.

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Hydrolysis specificities of lipase from Rhizomucor miehei were compared for various fatty acyl ethyl esters. Activity yields of immobilized lipases, measured with 1 mM substrate, were more than 100%. Differences in hydrolysis rate and affinity for the substrates between lipase preparations were also typically higher during hydrolysis of substrates at 100 mM than at 1 mM, indicating better mass transfer effects for 1-mM substrates. The native lipase showed higher affinity for polyunsaturated fatty acid substrates at 1 mM than at 100 mM. Hydrolysis rates for 1-mM substrates were observed with immobilized lipases, fixed on anion exchange resin with glutaraldehyde and on cation exchange carrier with carbodiimide, and suggested some modification of the basic amino acid related to the lid of R. miehei lipase. Activation with these bifunctional reagents was not observed for 100-mM substrates, indicating that interfacial activation always occurred because of aggregation of 100-mM substrates. These results show that lipase from R. miehei recognizes molecular aggregation of lipids, and that various changes occur in the hydrolysis specificities for fatty acids. JAOCS 74, 1395-1399 (1997) KEY WORDS: Aggregation, fatty acid, immobilized lipase, interfacial activation, lipase, polyunsaturated fatty acid, specificity.The aggregate structure of polyunsaturated lipids under aqueous conditions is of great interest. Investigation of the oxidative stability of 1 mM polyunsaturated fatty acids (PUFA) under aqueous conditions showed that the highest stability was achieved by docosahexaenoic acid (22:6n-3, DHA), followed by eicosapentaenoic (20:5n-3), arachidonic (20:4n-6), α-linolenic (18:3n-3), γ-linolenic (18:3n-6) and linoleic (18:2n-6) acids, indicating that stability increases with increasing degree of unsaturation (1). However, most data indicate that the oxidative stability of these PUFA is inversely proportional to the number of bis-allylic hydrogens in the molecule. Therefore, DHA is susceptible to lipid peroxidation in the air. The structural patterns of 1 mM DHA under aqueous conditions may stabilize the molecule, but little is known about the structure of PUFA in aqueous solution.Lipase attacks the ester bonds that bind hydrophobic and hydrophilic residues in amphipathic lipids. The amphipathic nature of lipids is the origin of many substrate aggregates, such as monolayers, micelles, liposomes, and membranes. Aggregate formation depends on the lipid concentration, temperature, the nature of the solute, and lipid structure (2). Porcine pancreatic lipase activity is greatly increased at the water/lipid interface, suggesting that interfacial activation of lipase occurs (3). The catalytic triad Ser-144, His-203, Asp-257 of Rhizomucor miehei is buried completely beneath a short helical segment (residues 82-96), or lid (4). X-ray crystallographic analysis of the lipase-inhibitor complex revealed that interfacial activation is achieved by the displacement of the lid structure, which exposes the catalytic groups and creates a h...