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F Pattus

Publications and source records attributed to F Pattus.

At least 91 records · Page 5Linked to original sources
PubMed1986

Crystallization of the C-terminal domain of colicin A carrying the voltage-dependent pore activity of the protein.

The C-terminal fragment (Mr, 21,800) of colicin A (a bacterial toxin that kills sensitive Escherichia coli cells) has been crystallized. This fragment, which possesses the pore-forming activity of the toxin, resulted from thermolysin digestion of the entire molecule. The crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2) with a = b = 72.8 A, c = 170.4 A. They contain a dimer in the asymmetric unit and diffract to 2.7 A.

Colicins↗
PubMed1986

A molecular, genetic and immunological approach to the functioning of colicin A, a pore-forming protein.

We have constructed, by recombinant DNA techniques, one hybrid protein, colicin A-beta-lactamase (P24), and two modified colicin As, one (P44) lacking a large central domain and the other (PX-345) with a different C-terminal region. The regulation of synthesis, the release into the medium and the properties of these proteins were studied. Only P44 was released into the medium. This suggests that both ends of the colicin A polypeptide chain might be required for colicin release. None of the three proteins was active on sensitive cells in an assay in vivo. However, P44 was able to form voltage-dependent channels in phospholipid planar bilayers. Its lack of activity in vivo is therefore probably caused by the inability to bind to the receptor in the outer membrane. PX-345 is a colicin in which the last 43 amino acids of colicin A have been replaced by 27 amino acids encoded by another reading frame in the same region of the colicin A structural gene; it was totally unable to form pores in planar bilayers at neutral pH but showed a very slight activity at acidic pH. These results confirm that the C-terminal domain of colicin A is involved in pore formation and indicate that at least the 43 C-terminal amino acid residues of this domain play a significant role in pore formation or pore function. Fifteen monoclonal antibodies directed against colicin A have been isolated by using conventional techniques. Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A. In addition, the altered forms of the colicin A polypeptide were used to map the epitopes of ten monoclonal antibodies reacting specifically with colicin A. Some of the antibodies did not bind to colicin A when it was pre-incubated at acidic pH suggesting that colicin A undergoes conformational change at about pH 4. The effects of monoclonal antibodies on activity in vivo of colicin A were investigated. The degree of inhibition observed was related to the location of the epitopes, with monoclonal antibodies reacting with the N terminus giving greater inhibition. The monoclonal antibodies directed against the C-terminal region promoted an apparent activation of colicin activity in vivo.

Antibodies, Monoclonal↗
PubMed1986

The selectivity filter of voltage-dependent channels formed by phosphoporin (PhoE protein) from E. coli.

Phosphoporin, an Escherichia coli outer membrane-spanning protein re-incorporated in phospholipid planar bilayers generates aqueous channels similar to those of matrix porin. One phosphoporin trimer contains three pores which are induced simultaneously but fluctuate separately between open and closed states. Membrane potential shifts this two-state equilibrium in favour of closed channels. This negative resistance occurs at lower potentials than with matrix porin channels. The phosphoporin channel is poorly anion selective for small solutes. Polyphosphates and other phosphorylated molecules specifically inhibit phosphoporin pore conductance to small ions, a property which is specific to phosphoporin. There is an excellent correlation between the effect of such solutes measured in planar bilayers and their inhibitory effect on beta-lactam antibiotic uptake in vivo by phosphoporin. It is concluded that the phosphoporin channel contains a selectivity filter which is only efficient for larger molecules, most probably through basic residues.

Adenosine Triphosphate↗
PubMed1986

A molecular genetic approach to the functioning of the immunity protein to colicin A.

A plasmid (pColAF1), derived from pColA, and lacking the region encoding Cai (colicin A immunity protein) and Cal (colicin A lysis protein) has been constructed. The strains carrying pColAF1 produce normal amounts of colicin A which remains in the cell cytoplasm and does not result in loss of viability. Similar results have also been obtained for transposon insertion mutants lacking Cai. Structure prediction analysis indicates that four peptide regions of Cai might span the cytoplasmic membrane. Since the NH2- and COOH-terminal regions are charged, this analysis suggests a topology of the 178 residues polypeptide chain in which regions 38 to 70 and 124 to 143 might be exposed at the outer side of the cytoplasmic membrane. With mutants constructed using recombinant DNA techniques, we could demonstrate that the removal of a 30 residue COOH-terminal region, and mutations altering the surface exposed loop comprised of aminoacid residues 124-143 abolish the protecting function of Cai.

Amino Acid Sequence↗
PubMed1985

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles.

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.

Animals↗
PubMed1985

Secondary structure of the pore-forming colicin A and its C-terminal fragment. Experimental fact and structure prediction.

Conformational investigations, using circular dichroism, on the pore-forming protein, colicin A (Mr 60 000), and a C-terminal bromelain fragment (Mr 20 000) were undertaken to estimate their secondary structure and to search for pH-dependent conformational changes. Colicin A and the bromelain peptide are mainly alpha-helical with an enrichment of the alpha-helical content in the C-terminal domain carrying the ionophoric activity. The non-negligible beta-sheet structure in the C-terminal domain is unstable and is easily transformed into alpha-helix upon decreasing the polarity of the solvent. No evidence of pH-dependent conformational modification, correlated with modification of colicin A activity, could be obtained. The secondary structure estimated on the basis of experimental data favoured a model in which the pore is built of a minimal number of six transmembrane alpha-helical segments. Search for such segments in the amino acid sequence of the C-terminal domain of colicin A was carried out by combining secondary structure prediction methods with hydrophobicity and hydrophobic movement calculations. Similar calculations on the C-terminal domains of colicin E1 and IB indicate a common structure of the pores formed by colicin A, E1 and IB. Only two or three putative transmembrane segments could be selected in the sequences of colicin A, IB or E1. As a result, it is concluded that the channel is probably not built by a single colicin molecule but more likely by an oligomer.

Chemical Phenomena↗
PubMed1985

pH-dependent membrane fusion is promoted by various colicins.

The ability of colicin A, a bacteriocin produced by some Enterobacteriaceae, to fuse phospholipid vesicles at acidic pH, was demonstrated by electron microscopy and resonance energy transfer. The fusion depends on protein concentration and on the nature of the phospholipids. Vesicles, prepared from Escherichia coli phospholipids, fused one or more rounds at pH 4.5 upon addition of stoichiometric amounts of colicin A. Fusion was not only induced by pore-forming colicins (E1, K) but also by colicins that contain nuclease activities (E2, E3). By recombinant DNA technology it is shown that the first glycine-rich 70 NH2-terminal amino acids and, most probably, the extreme COOH-terminal end of colicin A are involved in the fusion activity of the protein. The physiological relevance of this property of colicins is discussed.

Colicins↗
PubMed1984

Surface properties of bacterial sulfhydryl-activated cytolytic toxins. Interaction with monomolecular films of phosphatidylcholine and various sterols.

Sulfhydryl-activated cytolysins are a group of bacterial protein toxins which, in the reduced state, lyse eukaryotic cells by disruption of the cytoplasmic membrane. Cell surface cholesterol is thought to be the target of the toxins. In the present work, the monolayer technique was used to investigate the interaction of four SH-activated toxins (streptolysin 0, alveolysin , perfringolysin 0, pneumolysin ) with various lipid films as a model for studying toxin-induced membrane disruption. A surface pressure increase up to very high values was elicited by reduced toxins (approximately equal to 10 nM) on films of cholesterol, other toxin-binding 3 beta-hydroxy-sterols, thiocholesterol and cholesterol-phosphatidylcholine mixtures suggesting deformation or penetration of the films. The surface-active potency of the toxins was of the same order as that of melittin and snake cardiotoxins at similar concentrations. No pressure increase was observed on films made of pure phosphatidylcholine, lanosterol and other sterols lacking the 3 beta-OH group. Optimal efficiency was at cholesterol/phosphatidylcholine molar ratio of 1 to 1. The critical pressures for toxin interaction with phosphatidylcholine and cholesterol monolayers were 25 mN X m-1 and 45 mN X m-1 respectively. Toxin interaction with phosphatidylcholine [14C]-cholesterol films did not modify monolayer radioactivity, indicating no cholesterol desorption. No pressure increase was elicited by toxins inactivated by SH-group reagents, heating or neutralization with antibody. Toxin effect was dependent temperature and pH. The overall potency of the four toxins tested was streptolysin 0 greater than alveolysin approximately equal to perfringolysin 0 greater than pneumolysin . The monolayer system mimicked in several respects toxin interaction with eukaryotic cells.

Bacterial Proteins↗
PubMed1983

Voltage and time dependence of the conductance of planar lipid bilayers doped with colicin A.

Voltage jump studies of planar bilayers doped with colicin A revealed two classes of time and voltage dependent conductance induced by colicin A. The first type of conductance, asymmetrically voltage dependent, measured as a time dependent process, was in agreement with previously described conductances of bilayers doped with colicin A or colicin E1. The second class of conductance appeared as a voltage dependent instantaneous conductance. The significance of this new type of conductance is discussed.

Colicins↗
PubMed1983

Isolation, molecular and functional properties of the C-terminal domain of colicin A.

Partial proteolytic digestion of colicin A with bromelain allowed the isolation of a 20-kd fragment. This fragment has been purified to homogeneity and its molecular properties have been studied. The sequence of the 54 N-terminal amino acid residues has been determined by automated Edman degradation. This sequence is identical to that of the predicted amino acid sequence of the 20-kd C-terminal part of the colicin A polypeptide deduced from the nucleotide sequence of the caa gene. This polypeptide can produce channels in phospholipid planar bilayers of the same size as those formed by colicin A. However, the voltage-dependence for opening and closing was drastically altered in the peptide fragment channels. The latter, in contrast to colicin A channels, remained open over a wide range of voltage. Large negative potentials were required to close the peptide fragment channels although opening took place in the same voltage range as for colicin A ionic pores.

Amino Acid Sequence↗
PubMed1982

Product activation of pancreatic lipase. Lipolytic enzymes as probes for lipid/water interfaces.

During the action of pancreatic lipase and colipase on racemic 1,2-didodecanoylglycerol monolayers in the absence of bile salts, biphasic kinetics was observed under conditions of high lipid packing. Similar kinetics has earlier been reported using phospholipid-emulsified triolein droplets (Borgström, B. (1980) Gastroenterology 78, 954-962). These kinetics are characterized by a lag time tau d, dependent on products accumulation at the substrate/water interface. This lag time is differentiated from the previously described enzyme concentration independent lag time tau i in systems of low lipid packing (Verger, R., Mieras, M. C. E., and de Haas, G. H. (1973) J. Biol. Chem. 248, 4023-4034). Both tau i and tau d reflect a rate-limiting step due to the slow enzyme penetration into the substrate interface. The variation of tau d under different conditions (change in pH and concentration of Ca2+, enzyme, bovine serum albumin, and lipolytic products) lead us to propose a model for the product activation during lipolysis. We will discuss the use of the pancreatic lipase-colipase system to probe the lipid packing of emulsified triglyceride particles and lipoproteins using tau d as a reference value.

Animals↗
PubMed1981

Structure, composition, enzymatic activities of human erythrocyte and sarcoplasmic reticulum membrane films.

Air/water interface films were obtained from human erythrocytes and rabbit sarcoplasmic reticulum membranes at 'zero surface pressure. according to Verger, R and Pattus, F. (Chem. Phys. Lipids (1976) 16, 285-291). The lipid and protein distribution of these membrane films suggest that the film composition is determined by the composition of the membrane and the mode of integration of its components. When kept at low surface pressure, slow film expansion occurred due to unfolding of proteins at the interface. This process can be stopped by compressing the films at a higher surface pressure than 15 dyn/cm. Acetylcholinesterase activity from human erythrocyte films is highly dependent on the condensation state of the film. Ca2+-ATPase from sarcoplasmic reticulum films was still activable by Ca2+. Freeze-fracture studies on erythrocyte membrane films suggest the such films are monolayers in which proteins are randomly distributed.

Acetylcholinesterase↗
PubMed1980

Mechanism of action of milk lipoprotein lipase at substrate interfaces: effects of apolipoproteins.

The mechanism of action of bovine milk lipoprotein lipase was studied by using a monomolecular film of 1,2-didecanoylglycerol. The apparent rate of hydrolysis of diglyceride increased with increasing surface pressures above 12 mN/m; the enzyme was inactive at pressures less than 12 mN/m. We have measured the effects of four plasma apolipoproteins (apoC-II, apoC-III, apo-I, and apoE), bovine serum albumin, porcine pancreatic colipase, heparin, and NaCl on the kinetics of lipid hydrolysis. At a surface pressure of 15 mN/m, all of the proteins, with the exception of colipase, gave increased enzyme activity compared to lipase alone; apoC-II gave maximal activation. At 25 mN/m, apoC-II at concentrations of less than 0.25 microgram/mL showed a specific activation, whereas the other proteins had no effect. Heparin activated at both high and low surface pressures; NaCl had little or no effect in this system. At a higher concentration of apoC-II (0.50 microgram/mL), the apoprotein inhibited the enzyme. The addition of apoC-III, apoA-I, or apoE (final concentration 0.25 microgram/mL), but not albumin or colipase, to apoC-II (0.25 microgram/mL) caused an increase in surface pressure of 5-6 mN/m and an apparent rate which was less than half that found for lipase alone, suggesting that all of the apoproteins inhibit the apoC-II specific activation.

Animals↗
PubMed1979

Interaction of plasma apolipoproteins with lipid monolayers.

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidy[14C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipids by apolipoprotein C-III. The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface. Although apolipoprotein A-I and arginine-rich protein gave surface pressure increases, phospholipid was only slightly removed fromthe interface by the addition of liposomes. Based on these findings, we conclude that the apolipoproteins C interact specifically with phosphatidylcholine at the interface. This interaction is important as it relates to the transfer of the apolipoproteins C and phospholipids from very low density lipoproteins to other plasma lipoproteins. The addition of human plasma high density lipoproteins or very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of phosphatidyl[14C]choline from the interface 3--4 fold. Low density lipoproteins did not affect the rate of decrease. During lipolysis of very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of with the lipid monolayer. Lipolysis experiments were performed in a monolayer trough containing a surface film of egg phosphatidyl[14C]choline and a subphase of very low density lipoproteins and bovine serum albumin. Lipolysis was initiated by the addition of purified milk lipoprotein lipase to the subphase. As a result of lipolysis, there was a decrease in surface radioactivity of phosphatidylcholine. The pre-addition of high density lipoproteins decreased the rate of decrease in surface radioactivity...

Animals↗