An Escherichia colz clone was constructed to overproduce endoglucanase C (CelCCC) from Clostridium cellulolyticum. This construction made it easier to isolate the enzyme but, as observed in the case of endoglucanase A (CelCCA) from the same organism, the purification led to the isolation of two forms of the cellulase differing in their molecular masses, 48 kDa and 41 kDa. Nterminal sequence analysis of both purified enzymes showed that the shorter form was probably the result of partial proteolysis near the COOH-extremity. The difference in mass indicated that the shorter protein lacks the C-terminal reiterated domains (20-24-amino-acid twice-repeated sequences). These particular domains are characteristic of clostridial cellulases acting on cellulose by the mean of cellulosomal particles. Biochemical and enzymic studies were performed on each form of CelCCC, and revealed that their temperature and pH optima were identical, but their catalytic parameters were quite different. Furthermore, the differences of enzymic behavior observed between the two forms of CelCCC are almost identical to those already noted in the case of the two forms of CelCCA. The stereoselectivity of the reaction catalysed by CelCCC and CelCCA was determined using proton NMR spectroscopy ; CelCCC acts by configuration inversion, whereas CelCCA acts by configuration retention. The degradation patterns on cellodextrins (ranging from cellotriose to cellohexaose) and chromophoric cellodextrins (from p-nitrophenyl-cellobiose to p-nitrophenyl-cellopentaose) were also investigated in both forms of CelCCC and CelCCA. It emerged that the natural cellodextrins degradation patterns of CelCCC and CelCCA were very similar but the utilization of p-nitrophenyl-cellodextrins showed the existence of considerable differences between these two endoglucanases in terms of cleavage-site position and catalytic parameters. CelCCC and CelCCA were found not to act synergistically on the tested substrates.Clostridiurn cellulolyticum is a mesophilic Clostridium which is able to completely degrade crystalline cellulose. The four known cellulase sequences of this organism [l-31 exhibit reiterated domains. These particular sequences are thought to be characteristic of enzymes integrated into the cellulosome structure [4], and yet they are observed with cellulolytic systems of Clostridium thermocellum [4, 51 and Clostridium cellulovorans [6]. These organisms secrete highmolecular-mass particles named cellulosomes which are active against cellulose, and they possess an assembly factor, CbpA in C. cellulovorans [7] and CipA in C. thermocellum [8, 91. Four genes, celCCA, celCCC, coding for enzymes of the cellulolytic system of C. cellulolyticum have been completely sequenced and the four corresponding proteins are members of three different fami-Correspondence to H

Porcine pancreatic phospholipase A2 interacts with micelles of the substrate analogue n-octadecylphosphocholine to form a specific complex over considerably wide concentration ranges of both lipid and protein. UV absorption difference spectroscopy measurements indicate that the ratio of lipid to protein molecules in the complex is approximately 50. This number is confirmed by using other techniques to study the composition of the complex, namely, ultracentrifugation experiments and light scattering. The latter techniques furthermore demonstrate that the lipid--protein complex consists of 100 lipid and 2 enzyme molecules. Thus, the number of lipid molecules in the free micelle (200) is halved when the complex with phospholipase is being formed. The consequences of the results are discussed in relation to a theoretical model of the lipid--protein interaction.

The aim of our study was to define the mechanism by which cholesterol uptake is inhibited by lecithin but not by lysolecithin. The work compared the cholesterol uptake by everted rat jejunal sacs from bile salt-lecithin-cholesterol or bile salt-lysolecithin-cholesterol micelles. The micellar size and the cholesterol saturation were measured. The size or molecular weight increases when the lecithin concentration rises, and the cholesterol uptake decreases and leads to zero when the micelles contain more than 30% lecithin. The size of bile salt-lysolecithin-cholesterol micelles is smaller than that of lecithin micelles in comparable molar ratios. Consistent with this result is the fact that, for a given phospholipid concentration, cholesterol uptake is greater in the presence of lysolecithin than in the presence of lecithin. The diffusion rate of the micelles through the unstirred water layer decreases when micellar size increases. However, the comparison of uptakes from lecithin or lysolecithin micelles similar in size and in cholesterol saturation showed that the cholesterol uptake is still lower for lecithin micelles. This shows that with larger micelles some factor other than micellar size and cholesterol content of the micelles is important. We observe that lysolecithin absorption is 15-fold greater than lecithin absorption. We suggest that lysolecithin absorption results in a rapid supersaturation with cholesterol leading to cholesterol absorption.

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The interaction of the 20-kDa pore-forming domain of colicin A with phospholipid vesicles was investigated by gel permeation chromatography, analytical centrifugation, and electron microscopy. Under the experimental conditions of this study, this peptide was found to interact only with vesicles containing negatively charged phospholipids. It forms a well-defined disklike complex with phosphatidylglycerols with a preference for those containing 12-14 atoms of carbon in their fatty acid chain. This complex has a diameter of 120 A and is about one bilayer thick. It contains nine molecules of peptide and is formed both at acidic pH (pH 5.0) and at neutral pH (pH 7.2).