Murein biosynthesis in ether permeabilized Escherichia coli starting from early peptidoglycan precursors

ETB, ether treated bacteria, from E. coli and other Gram-negative strains, contain in a cell-free system all enzymes necessary for murein biosynthesis. Starting with a variety of combinations of peptidoglycan precursors, high yields of sodium dodecylsulfate (SDS, 4%) insoluble murein or murein like material were synthesized. The amount of newly synthesized SDS insoluble material (NSM) was dependent upon the growing phase at which cells had been harvested for preparation of ETB. This data may provide some insight into the regulation of peptidoglycan biosynthesis.Starting from early peptidoglycan precursors, the cell-free synthesis of NSM was inhibited by specific inhibitors of murein synthesis, such as D-cycloserine, D-fluoroalanine, 2-amino-ethylphosphonate, analogues of D-alanyl-D-alanine and -lactam antibiotics at appropriate concentrations. Some D-alanyl-D-alanine analogues and 4-chlorodiaminopimelic acid were incorporated into NSM in place of their corresponding natural substrates.Abbreviations ETB ether treated bacteria (E. coli) - NSM newly synthesized SDS insoluble material - SDS sodium dodecylsulfate - UDP-MAG UDP-MurNAc-dipeptide, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate - UDP-MAGD UDP-MurNAc-tripeptide, UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimelate - UDP-MAGDAA UDP-MurNAc-pentapeptide, UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimeloyl-D-alanyl-D-alanine - GINAc N-Acetylglucosamine Definitions Murein highly cross-linked bagshaped peptidoglycan (Weidel and Pelzer 1964)     

Purification and characterization of a newly screened microbial peptide amidase

A microbial peptide amidase was found in a limited screening and purified about 500-fold from Stenotrophomonas maltophilia. The native enzyme has a molecular mass of 38 kDa (gel filtration). The sequence of the first 16 amino acids was determined by Edman degradation. The isoelectric point was found to be around 5.8. The peptide amidase exhibited a pH optimum of 6.0 and a temperature optimum of about 39–45°C. The enzyme is stable in 50 mM TRIS/HCl, pH 7.5, at 30°C, and the residual activity was found to be above 90% after 1 week of incubation. The biocatalyst is not inhibited by potential inhibitors like Hg²⁺, EDTA, d-cycloserine or dithiothreitol and only weakly influenced by inhibitors of serine proteases. The peptide amidase deamidates selectively C-terminal amide groups in peptide amides without hydrolysing internal peptide bonds or amide functions in the side-chain of glutamine or asparagine. Unprotected amino acid amides are not hydrolysed. The enzyme is stereoselective with regard to l-enantiomers in the C-terminal position.     

Chemical composition of Eubacterium alactolyticum cell wall peptidoglycan

The mechanism of lysis of Eubacterium alactolyticum cell walls by Streptomyces albus G enzyme was studied. The analysis of the peptide terminal groups and peptide subunits isolated from the cell wall digest, released during solubilization of the cell walls, revealed that lytic action of S. albus G enzyme was mainly due to D-alanyl-A₂pm endopeptidase, N-acetylmuramyl-L-alanine amidase, N-acetylmuramidase and N-acetylglucosaminidase. E. alactolyticum cell wall peptidoglycan is composed mainly of glucosamine, muramic acid, D-glutamic acid, L- and D-alanine, meso-diaminopimelic acid and glycine. The peptide subunit consists of L-alanyl-D-glutamyl-meso-A₂pm-D-alanine. D-Alanine is connected directly with the amino group of the meso-A₂pm residue of another peptide subunit. All of the L-amino groups of meso-diaminopimelic acid are involved in cross-linking.The possible structure of the peptide moiety of E. alactolyticum cell wall peptidoglycan is presented.     

A simple method for following the fate of alanine-containing components in murein synthesis inEscherichia coli

The fate of the alanine-containing components in murein synthesis was followed by incorporation of¹⁴C-l-alanine inE. coli under conditions allowing cell-wall synthesis while preventing protein synthesis. The components were separated by chromatography and detected by autoradiography.Spots containing murein, cell-wall precursors, alanine andd-alanyl-d-alanine were identified. A further component was probably identical to pyruvic acid. Two unidentified spots were found in the region where lipid-intermediates in cell-wall synthesis are usually found. However, the absence of turnover of these two components was at variance with the proposed properties of the lipidintermediates. d-Alanyl-d-alanine and the component which is probably identical to pyruvic acid were excreted into the medium, whereas murein and cell-wall precursors were found in the cellular fraction.The influence of the concentration of alanine, and of the number of cells per ml, on the acid-precipitable activity were studied. The latter increased during, at least, the first two hours and represented mainly lysozyme-degradable material.Significant turnover of murein could be detected neither in the presence nor in the absence of protein synthesis.A time course of the activity of the radioactive components is provided. The influence of a number of antibiotics inhibiting cell-wall synthesis on the acid-precipitable activity and on the activity of the main intermediates in murein synthesis was studied.We thank Mrs. Arna van Schijndel-van Dam and Mr. A. A. G. Verweij for excellent assistance. We thank Dr. P. E. Reynolds (University of Cambridge) for teaching one of us (E J. J. L.) several techniques in the field of bacterial cell walls, and Dr. H. J. W. Wijsman for stimulating discussions.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-alanine by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli W was genetically engineered to produce l-alanine as the primary fermentation product from sugars by replacing the native d-lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native d-lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of l-alanine to d-alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l⁻¹ glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g⁻¹ glucose) with a chiral purity greater than 99.5% l-alanine. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase.

Summary A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27 513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M _r value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M _r value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.     

Production of l-alanine from ammonium fumarate using two immobilized microorganisms

Summary For the efficient production of l-alanine from ammonium fumarate using the aspartase activity of immobilized Escherichia coli cells and l-aspartate -decarboxylase activity of immobilized Pseudomonas dacunhae cells, alanine racemase and fumarase activities should be eliminated. We investigated various procedures to eliminate these side reactions, and found that both activities of intact E. coli cells could be eliminated by treating the culture broth at pH 5.0 and 45° C for 1 h, and those of intact P. dacunhae cells could be eliminated by treating the culture broth at pH 4.75 and 30° C for 1 h. Further, it was confirmed that l-alanine was efficiently produced using these two immobilized pH-treated microorganisms.     

Alanine dehydrogenase of the β-lactam antibiotic producer Streptomyces clavuligerus

l-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptomyces clavuligerus. The enzyme was induced by ammonia, and the level of induction was dependend on the extracellular concentration. l-Alanine was the only amino acid able to induce alanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (K _m =1.1 mM), ammonia (K _m =20 mM) and NADH (K _m =0.14 mM) were required for the reductive amination, and l-alanine (K _m =9.1 mM) and NAD (K _m =0.5 mM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (K _i =1.6 mM) and noncompetitively with respect to ammonia (K _i =2.0 mM) and pyruvate (K _i =3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6–7%) substitute pyruvate. Alanine dehydrogenase from S. clavuligerus differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.Abbreviation Alanine-DH l-alanine:NAD oxidoreductase     

Zur chemischen Zusammensetzung der Zellwand der Streptokokken

Zusammenfassung Das Murein (Peptidoglycan) eines aus Faeces isolierten Streptococcus, der in den wichtigsten Merkmalen mit Peptostreptococcus evolutus (Prevot) Smith übereinstimmt, weist folgende Molverhältnisse auf (aufgerundete bzw. abgerundete Zahlen): Mur:GlcNH₂:Ala:Glu:Lys:Gly=1:1:3:1:1:1. Das Verhältnis l-Alanin:d-Alanin=2,15:1. Die Glutaminsäure liegt in der d-Konfiguration und als Amid vor.Durch die Partialhydrolyse der Zellwände und die anschließende Isolierung und Identifizierung der Peptide konnte die Aminosäuresequenz des Mureins geklärt werden. Das Tetrapeptid stimmt mit der üblichen Sequenz l-Ala-d-Glu-NH₂-l-Lys-d-Ala der meisten übrigen Bakterien überein. Die Quervernetzung des Mureins wird durch das Peptid Glycyl-l-Alanin hergestellt, wobei l-Alanin an die -Aminogruppe des Lysins gebunden ist. Die Dinitrophenylierung der Zellwand ergab, daß 35% des Glycins und 6% des Lysins eine freie Aminogruppe aufweisen. Die Quervernetzung ist demnach nur zu höchstens 60% durchgeführt.

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The chemical composition of the cell walls of Streptococci III. The amino acid sequence of a glycine containing murein from Peptostreptococcus evolutus (Prevot) Smith

Summary Peptostreptococcus evolutus was isolated from feces. Its murein containes muramic acid, glucosamine, alanine, d-glutamic acid, lysine and glycine at a molar ratio of about 1:1:3:1:1:1. The ratio of l-alanine: d-alanine is 2,15:1. Glutamic acid is present as an amide.By acid partial hydrolysis of the cell walls and subsequent isolation and identification of the peptides the amino acid sequence of the murein was elucidated. The tetrapeptide is identical with that of most bacteria (l-Ala-d-Glu-NH₂-l-Lys-d-Ala). The crosslinking of the murein is performed by the peptide glycyl-l-alanine. l-alanine is attached to the -amino group of lysine while the amino group of glycine is bound to the carboxyl group of the c-terminal d-alanine of an adjacent tetrapeptide. About 35% glycine and 6% lysine of the murein are dinitrophenylisable indicating that maximally 60% of the possible cross-linkages are realized.

     

The dipeptidyl carboxypeptidase of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue: overproduction and molecular characterization of the recombinant enzyme

To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His₆-tagged EcDCP (His₆-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni²⁺-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His₆-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His₆-EcDCP was stimulated by 1.5 fold. The K _M and k _cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s⁻¹, respectively. His₆-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His₆-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.     

Murein structure of Acetobacterium woodii

The isolated cell walls of Acetobacterium woodii contain a murein of the crosslinkage type B. d-Orinithinyl residues function as interpeptide bridges between the -carboxyl group of d-glutamic acid and the carboxyl group of the terminal d-analyl residue of an adjacent peptide subunit. The usual l-alanyl residue in position 1 of the peptide subunit is replaced by a l-seryl residue. As yet this murein type was only found in Eubacterium limosum, an organism which was supposed to be related to Acetobacterium because of some metabolic similarities.     

l-Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family

Brevundimonas diminuta TPU 5720 produces an amidase acting l-stereoselectively on phenylalaninamide. The enzyme (LaaA_Bd) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of ≈53,000. The native molecular weight of the enzyme was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal activity at 50°C and pH 7.5. LaaA_Bd lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was largely restored by the addition of Co²⁺ ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co²⁺, suggesting that LaaA_Bd is a Co²⁺-dependent enzyme. LaaA_Bd had hydrolyzing activity toward a broad range of l-amino acid amides including l-phenylalaninamide, l-glutaminamide, l-leucinamide, l-methioninamide, l-argininamide, and l-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaA_Bd was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence of seven open-reading frames (ORFs), one of which (laaA _Bd ) encodes the amidase. LaaA_Bd is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA _Bd gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaA_Bd in cell-free extracts of the E. coli transformant was 25.9 units mg⁻¹ with l-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720.     

Morpholine-induced formation of l-alanine dehydrogenase activity in Mycobacterium strain HE5

An NAD-dependent, morpholine-stimulated l-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4.6-fold increase of l-alanine dehydrogenase activity when l-alanine was supplied at suboptimal concentration. l-Alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of l-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent K _m values for l-alanine and NAD were 1.0 and 0.2 mM, respectively. K _m values of 0.6, 0.02, and 72 mM for pyruvate, NADH, and NH₄ ⁺, respectively, were estimated at pH 8.7 for the reductive amination reaction. Received: 25 September 1998 / Accepted: 11 March 1999     

Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. Ferric iron reductase activity of 9 nmol Fe²⁺ mg^-1 protein min^-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe²⁺-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification).     

From growth to autolysis: the murein hydrolases inEscherichia coli

Murein hydrolases cleave bonds in the bacterial exoskeleton, the murein (peptidoglycan) sacculus, a covalently closed bag-shaped polymer made of glycan strands that are crosslinked by peptides. During growth and division of a bacterial cell, these enzymes are involved in the controlled metabolism of the murein sacculus. Murein hydrolases are believed to function as pacemaker enzymes for the enlargement of the murein sacculus since opening of bonds in the murein net is needed to allow the insertion of new subunits into the sacculus. Furthermore, they are responsible for splitting the septum during cell division. The murein turnover products that are released during growth are further degraded by these hydrolases to products that can be recycled by the biosynthetic enzymes. As potentially suicidal (autolytic) enzymes, murein hydrolases must be strictly controlled by the cell, Inhibition of murein synthesis, for example by penicillin, triggers an unbalanced action of murein hydrolases causing bacteriolysis. InEscherichia coli, 14 different murein hydrolases have so far been identified, includingN-acetylmuramyl-l-alanine amidases,dd-endopeptidases,dd-carboxypeptidases,ld-carboxypeptidases, andN-acetylglucosaminidases. In addition lysozyme-like enzymes, called “lytic transglycosylases,” produce (1→6)-anhydromuramic acid derivatives by an intramolecular transglycosylation reaction.     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

Modulation of Escherichia coliN-acetylmuramoyl-l-alanine amidase activity by phosphatidylglycerol

The activity of pure Escherichia coli murein (peptidoglycan) amidase ($\text{N-}\text{acetylmuramoyl-}\text{l}\text{-alanine}$ amidase, EC 3.5.1.28) was measured after preincubation with E. coli phosphatidylglycerol microdispersions in final concentration ranging over micro- and millimolarities. The enzyme activity was increased up to 160% of the control for phosphatidylglycerol concentrations increasing from 2 to 50 μM. After a plateau extending from 0.05 to 0.3 mM, higher phosphatidylglycerol concentrations inactivated the enzyme down to 15% of initial activity for concentrations of 2 mM. Positive kinetic cooperativity was observed for the activation as well as for the inactivation processes. Cardiolipin (or diphosphatidylglycerol) from the same origin and under same conditions had no significant effect. Molecular sieving experiments have shown that, when inactivated, the enzyme remained firmly bound to the phosphatidylglycerol vesicles, whereas the activated phosphatidylglycerol-enzyme complex was totally dissociable by dilution. Activated phosphatidylglycerol complexes were recovered by gel exclusion chromatography at equilibrium in 40 μM phosphatidylglycerol. Possible physiological meaning of the results is briefly discussed in the context of our work and that done previously by others.