The Pi z HOH exchange reaction of oxidative phosphorylation is considerably less sensitive to uncouplers than the Pi T ATP and ATP ;± HOH exchanges. The uncoupler-insensitive Pi 2. HOH Previous findings have shown that the Pi T HOH exchange is less sensitive to 2,4-dinitrophenol than the Pi T. ATP exchange or the capacity for net oxidative l)hosphorylation (10)(11)(12). However, the significance or the source of this exchange has not been known. The possibility exists that it might reflect activities of enzymes such as alkaline phosphatase or pyrophosphatase known to catalyze a Pi ± HOH exchange (7), perhaps activated in some manner by 2,4-dinitrophenol. In addition, whether such behavior is limited to 2,4-dinitrol)henol or might be shown by more potent uncouplers of oxidative phosphorylation has not been shown. Results with other uncouplers and with oligomycin inhibition reported here cover these points.The effects of increasing concentrations of the potent uncoupler 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13) (13) on the exchanges catalyzed by mitochondria are shown ill Fig. 1. In the absence of uncoupler, the relative rates of the reactions Pi 2. HOH, ATP ¢± HOH, and Pi T ATP are about 12:6: 1, respectively, under the conditions used. At low concentrations of uncoupler, the Pi ± ATP and the ATP z HOH exchanges are much more sensitive to the uncoupler than the Pi z HOH exchange. At a concentration of S-13 sufficient to inhibit the Pi T ATP and ATP 2 HOH exchange by about 50%, the Pi T HOH exchange is inhibited by less than 5%. At a concentration S-13 that gives a near zero value for the Pi T ATP and ATP T HOH exchanges and a maximum value for the uncoupler-stimulated ATPase activity, the Pi T HOH exchange is still rapid and inhibited by only 35%. Responses similar to those reported in Fig. 1 for S-13 with mitochondria are also observed with 2,4-dinitrophenol and m-chlorocarbonvlcvanide phenylhydrazone.Important for the present considerations are the demonstrations, not given in detail here, of the effects of oligomycin.This antibiotic is a potent inhibitor of oxidative phosphorylation and inhibits the Pi T HOH exchange (14). In the absence of uncouplers and under conditions like those described with Fig. 1 Abbreviation: S-13, 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide.

Lipids containing the dimethyl BODIPY fluorophore are used in cell biology because their fluorescence properties change with fluorophore concentration (C.-S. Chen, O. C. Martin, and R. E. Pagano. 1997. Biophys J. 72:37-50). The miscibility and steady-state fluorescence behavior of one such lipid, 1-palmitoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (PBPC), have been characterized in mixtures with 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). PBPC packs similarly to phosphatidylcholines having a cis-unsaturated acyl chain and mixes nearly ideally with SOPC, apparently without fluorophore-fluorophore aggregation. Increasing PBPC mole fraction from 0.0 to 1.0 in SOPC membranes changes the emission characteristics of the probe in a continuous manner. Analysis of these changes shows that emission from the excited dimethyl BODIPY monomer self quenches with a critical radius of 25.9 A. Fluorophores sufficiently close (< or =13.7 A) at the time of excitation can form an excited dimer, emission from which depends strongly on total lipid packing density. Overall, the data show that PBPC is a reasonable physical substitute for other phosphatidylcholines in fluid membranes. Knowledge of PBPC fluorescence in lipid monolayers has been exploited to determine the two-dimensional concentration of SOPC in unilamellar, bilayer membranes.

Oral administration of -polylysine to rats reduced the peak plasma triacylglycerol concentration. In vitro, -polylysine and polylysine strongly inhibited the hydrolysis, by either pancreatic lipase or carboxylester lipase, of trioleoylglycerol (TO) emulsified with phosphatidylcholine (PC) and taurocholate. The -polylysine concentration required for complete inhibition of pancreatic lipase, 10 g/ml, is 1,000 times lower than that of BSA required for the same effect. Inhibition requires the presence of bile salt and, unlike inhibition of lipase by other proteins, is not reversed by supramicellar concentrations of bile salt. Inhibition increases with the degree of polylysine polymerization, is independent of lipase concentration, is independent of pH between 5.0 and 9.5, and is accompanied by an inhibition of lipase binding to TO-PC emulsion particles. However, -polylysine did not inhibit the hydrolysis by pancreatic lipase of TO emulsions prepared using anionic surfactants, TO hydrolysis catalyzed by lingual lipase, or the hydrolysis of a water-soluble substrate. In the presence of taurocholate, -polylysine becomes surface active and adsorbs to TO-PC monomolecular films. These results are consistent with -polylysine and taurocholate forming a surface-active complex that binds to emulsion particles, thereby retarding lipase adsorption and triacylglycerol hydrolysis both in vivo and in vitro. -Tsujita, T

Pancreatic colipase and its precursor, procolipase, facilitate interfacial lipid hydrolysis catalyzed by pancreatic lipase. To better understand how procolipase functions, its interactions with mixed-lipid monolayers at the argon-buffer interface have been characterized. The lipid mixtures consisted of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and either 1,3-dioleoylglycerol, a model lipase substrate, or 13,16-cis,cis-docosadienoic acid, a model lipase product. Analysis of the lipid composition dependence of procolipase-induced surface pressure increases shows thermodynamically that procolipase interacts strongly and preferentially with the lipase substrate or product. This finding was confirmed by fluorescence measurements of procolipase interaction with pyrene lipid analogs. Analysis of the quantity of procolipase adsorbed to the lipid monolayers shows that interfacial packing obeys a simple, geometric model. The partial molecular areas obtained for procolipase (708 A2) and the phosphatidylcholine (70 A2) agree with their known cross-sectional areas. However, the areas for the fatty acid (14 A2) and diacylglycerol (18 A2) are less than half the expected values, indicating the formation of substrate multilayers. Overall, the results indicate a previously unrecognized role for procolipase, recruiting substrate laterally to its vicinity and, hence, to pancreatic lipase with which procolipase forms a 1:1 interfacial complex. Accompanying this preferential interaction of procolipase with lipase substrates is their rearrangement normal to the interface. These previously unrecognized properties of this lipase cofactor should have relevance for the regulation of other lipases, like lipoprotein lipase, which are regulated by cofactor proteins.

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In the presence of bulk water, the lipase‐catalyzed synthesis and hydrolysis of insoluble lipid esters occur at the lipid‐water interface. For water‐soluble lipases, a necessary step in this process is the partitioning of enzyme from the bulk aqueous phase to the surface phase. In surface phases of phospholipids and the substrates and products of lipolysis, physical studies have demonstrated the formation of preferred packing arrays or lipid‐lipid “complexes.” Such interactions involve changes in both lipid molecular area and hydration. Binding of pancreatic carboxylester lipase (cholesterol esterase) and colipase to monomolecular films of a phosphatidylcholine and its complexes with fatty acid or diglyceride is negligible. In contrast, saturation of film of pure fatty acid or diglyceride correlates with formation of a protein monolayer. With mixtures of complex and uncomplexed fatty acid or diglyceride, binding to the uncomplexed lipid occurs, but only with colipase can saturation of available sites be achieved. The lower affinity of carboxylester lipase for surfaces containing complexes can be qualitatively explained by differences in the size of lipid and protein molecules. Because it involves no direct interaction between enzyme and complex, such “proxinhibition” of enzyme binding is potentially an important regulation of lipid‐protein interactions.

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