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.

No abstract

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).

The negatively charged detergents S-n-alka-noylthioglycol sulfates (C8, C9, and C10) are substrates for porcine pancreatic phospholipase A2 and its zymogen. At pH 6.0 and detergent concentrations up to 0.08 X critical micelle concentration (cmc), the activities of active enzyme and zymogen are similar and very low. From 0.08 X cmc to 0.12 X cmc a tremendous increase in activity is observed for phospholipase A2, but not for the zymogen. Concomitant with this increase in activity there is a sharp rise in molecular weight of the substrate-enzyme complex, from 15 000 to 95 000, and in detergent to protein molar ratio of 1:1 to about 7:1. This indicates both substrate and enzyme aggregation. Most probably a lipid-water interface is formed inside the aggregated protein particle by which the enzyme is activated. Although the zymogen also forms high molecular weight complexes with similar molar ratios, no activation is observed probably because of distortion of its lipid binding domain.

A detailed investigation by ultracentrifugation of the colipase-taurodeoxycholate system showed the formation of well-defined mixed associations with a sedimentation coefficient of about 2.2 S. The fact that these associations were only detectable above the critical micelle concentration of the salt indicated that micelles rather than monomers were bound to the cofactor.Two technical difficulties must be overcome before the weight of the associations could be measured with a reasonable accuracy. Firstly, the partial specific volume of the associations was determined using a digital microdensimeter and the interferometric system of the ultracentrifuge for concentration determinations. Secondly, due to the fact that micelle concentrations could not be equilibrated by dialysis, even after an extended period of time, an appropriate dilution of the ligand in the buffer compartment was necessary in order to compensate for its fixation by colipase in the solution. Then, the ionic strength dependence of the weight of the associations was found to vary in parallel with that of the micelles and to be in each case equal to the sum of the weights of one colipase molecule and one micelle. Therefore, colipase can be expected to contain a single high affinity site for bile salt micelle binding.During the last few years, several low molecular weight proteins designated colipase have been purified from porcine pancreas. The smallest and structurally best known, colipase 11, contains 84 amino acid residues [I]. It is composed of a central "core" crosslinked by four disulfide bridges and of two "tails" loosely bound by a single bridge [2]. Colipase I, which is the form used throughout this and the next work, differs from colipase I1 by the presence of ten additional residues at the end of the C-terminal tail [l]. Little is known so far about the structure of a third colipase characterized in porcine pancreas, except for the existence of seven N-terminal residues not present in colipases I and I1 [3].Flowing into the duodenum with pancreatic juice [4], colipase has been reported to prevent the inhibition of the lipase-catalyzed hydrolysis of dietary triglycerides by physiological concentrations of bile salts [5]. This effect has been assumed to imply in the first place a bile salt-induced dimerization of colipase and an association of the dimer with lipase [ 6 ] .It has been confirmed in this laboratory [7] that the apparent sedimentation coefficient and molecular weight of colipase are approximately doubled in the presence of sodium taurodeoxycholate. However, the present and following paper will show that this increase results from the binding of a bile salt micelle to the colipase molecule rather than from dimerization. MATERIALS AND METHODS ColipasePorcine pancreatic colipase I was purified to homogeneity by a modification of a recently described technique [7]. The starting material was a commercial pancreas powder (Choay, France) which was exhaustively delipidated in the laboratory prior to use. The extracts were submitted as ...

The hydrodynamic properties of colicin A have been studied. The molecular mass of colicin A was determined from sedimentation equilibrium centrifugation to be 63 f 1.2 kDa, in agreement with that determined from the primary amino acid sequence [Morlon et al. (1983) J . Mol. Biol. 110, 271 -2891. The sedimentation coefficient has been analyzed over a wide range of ionic strength (NaCl 0.06-0.56 M) and pH and was found to remain almost constant. However, below pH 5 an oligomerization of colicin A to tetramers occurred. The frictional coefficient value indicated that the shape of the colicin A monomer was very asymmetric. Analysis of the pH dependence of circular dichroism of colicin A and of its COOH-terminal domain indicated that a sharp transition occurred between pH 4 and 3. This transition was very much reduced for the COOH-terminal domain in the presence of a non-ionic detergent. The presence of a lipid-binding site in colicin A at neutral pH was demonstrated both by hydrodynamic studies with micelles of n-hexadecanoyl and n-octadecanoylphosphocholine and by differential sensitivity to a proteolytic enzyme in the presence or absence of detergent micelles. About 75 molecules of lipid were bound under these conditions suggesting that colicin A was bound to lipid micelles. In contrast, at acid pH, in the presence of an excess of lipid the tetramer was dissociated into monomers complexed to 20-30 lipid molecules, indicating the exposure of a high-affinity lipid-binding site.Colicins are bactericidal proteins which kill sensitive Escherichia coli cells. The major group of colicins comprises those such as colicins A, El, Ia, Ib and K which collapse the membrane potential, presumably by forming ionic channels The primary structures of colicins A, El, Ia and Ib have been deduced from the nucleotide sequence of the corresponding genes. These proteins appear to be organized into three domains that are linearly arranged along the polypeptide chain [6 -91. Secondary-structure studies carried out with colicins A [lo] and El [ll] have indicated that the COOHterminal domains contain high percentages of M. helix. These studies as well as secondary structure predictions have led to the conclusion that the channel may not be built by a single molecule but by an oligomer [lo]. Moreover, we have recently reported a possible difference in the structure of cytoplasmic and extracellular colicin A. Only the cytoplasmic colicin A can form stable dimers and features a high affinity for membranes at neutral pH [12].In the work described here the hydrodynamic properties of colicin A have been studied. We have shown that soluble purified colicin A is a monomer with an asymmetric shape. The secondary structure of this monomer is rather stable