Structural modifications induced by the binding of mitochondrial creatine kinase (mtCK) to saturated and unsaturated phospholipids were monitored by using Laurdan, a membrane probe sensitive to the polarity of the environment. The abrupt change characteristic of a phase transition of lipids alone was attenuated by addition of mtCK. Generalized polarization spectra indicated that mtCK surface binding changed the phospholipid liquid-crystalline state to a more rigid state. Infrared spectra of lipids further strengthened these results: upon mtCK binding, the phospholipid methylene chains had a more rigid conformation than that observed without mtCK at the same temperature. After mtCK binding to vesicles of perdeuterated dimyristoylphosphatidylcholine and nondeuterated dimyristoylphosphatidylglycerol, no lateral phase separation was observed, suggesting that both lipids were rigidified. Moreover, mtCK bound to liposomes exhibited an uncommon red edge excitation shift of 19 nm, while that of the soluble enzyme was only 6 nm. These results indicated that the environment of some mtCK tryptophan residues was motionally restricted. Strong stabilization of the enzyme structure against heat denaturation was observed upon lipid binding. In addition, lipids promoted a new reversible protein-protein or protein-lipid interaction, as evidenced by infrared data showing a slight modification of the beta sheet over alpha helix ratio with formation of a new 1632-cm(-)(1) beta sheet instead of the soluble protein 1636-cm(-)(1) one. Such modifications, inducing a decrease in the fluidity of the mitochondrial membranes, may play a role in vesicle aggregation; they could be implicated in the appearance of contact sites between internal and external mitochondrial membranes.
The sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 has been analysed by proton-induced X-ray emission. It contains 1 equivalent Zn2+ per mol of protein. As derived from gene cloning and sequencing, the B. sphaericus Zn peptidase I is a two-module protein. A 100-amino-acid-residue N-terminal domain consisting of two tandem segments of similar sequences, is fused to a 296-amino-acid-residue C-terminal catalytic domain. The catalytic domain belongs to the Zn carboxypeptidase A family, the closest match being observed with the Streptomyces griseus carboxypeptidase [Narahashi (1990) J. Biochem. 107, 879-886] and with the family prototype, bovine carboxypeptidase A. The catalytic domain of the B. sphaericus peptidase I possesses, distributed along the amino-acid sequence, peptide segments, a triad His162-Glu165-His307 and a dyad Tyr347-Glu366 that are equivalent to secondary structures, the zinc-binding triad His69-Glu72-His196 and the catalytic dyad Tyr248-Glu270 of bovine carboxypeptidase A respectively. The N-terminal repeats of the B. sphaericus peptidase I have similarity with the C-terminal repeats of the Enterococcus hirae muramidase 2, the Streptococcus (now Enterococcus) faecalis autolysin and the Bacillus phi PZA and phi 29 lysozymes, to which a role in the recognition of a particular moiety of the bacterial cell envelope has been tentatively assigned. Detergents enhance considerably the specific activity of the B. sphaericus peptidase I.
The walls of Nocardia kirovani are composed of three main constituents : the peptidoglycan matrix, a polysaccharide polymer and a variety of free and bound lipids. The free lipids represent 17.5O/, (dry weight) of the walls and consist for the major part of C,,-,, fatty acids and nocardic acids, and for the minor part, of nocardones, triglycerides and carotenoid pigments. The nocardic acids were identified as tri-and tetra-unsaturated, &-branched, 8-hydroxylated compounds C,,H,,,O,-C,,H,,,O,, the nocardones as tri-and tetra-unsaturated ketones C5,HIo60 -C6,H,,,0, and the main carotenoid pigment as phlei-xantophylle palmitate. Esters of glycerol with C,,, C,,, C,, fatty acids and, for some of them, with odd numbered poly-unsaturated acids containing 35 to 45 carbon atoms, were also identified. Bound lipids represent about 20°/, (dry weight) of the walls and consist mainly of nocardic acids probably ester-linked t o an arabinogalactan polymer. The peptidoglycan (about 40°/, dry weight) is composed of ~-1,4-N-acetylglucosaminyl-N-tides, where Azpm is meso-diarninopimelic acid. Crosslinking between some of the peptide units is mediated through D-Ala-(D)-A,pm linkages (peptidoglycan of chemotype I).All bacterial walls contain a peptidoglycan polymer which is basically a network of glycan strands that are interconnected through peptide chains. The glycan moiety consists of linear strands of alternating 8-1,4 linked pyranoside N-acetylglucosamine and N-acetylmuramic acid residues. The muramic acid residues, or a t least some of them, are substituted by tetrapeptide units which consistently have the general sequence L-Ala-D-Glu-L-Res-D-Ala. Most often, the L-Res residue is either a neutral amino acid or a diamino acid such as meso-diaminopimelic acid. The peptide units belonging to adjacent strands are, in turn, cross-linked through peptide bridges. Again, the composition and the location of the bridges vary. Cross-linking between two peptide units, however, always involves the C-terminal D-alanine residue of one of them. I n many Bacillaceae and gram-negative bacteria, for Abbreviations. NMR, nuclear magnetic resonance; aGln, a-glutamine; A,pm, meso-diaminopimelic acid, LL-A,pm, LL isomer of diaminopimelic acid. The notation (L) or (D) written immediate11 before A,pm specifies on which one of the two asymmetric carbons of mesodiaminopimelic acid the substituted amino groups are located. Similarly, the notation (L) or (D) immediately after A,pm distinguishes between the carboxyl-substituted groups.example, the bridging is an interpeptide bond which extends from the C-terminal D-alanine of one peptide unit to the amino group located on the D carbon of meso-diaminopimelic acid of another peptide unit.Most often, the walls of gram-positive bacteria are deprived of lipids and consist of a peptidoglycan matrix to which are covalently attached an almost endless variety of polysaccharides that are frequently negatively charged and sometimes of polyolphosphate polymers that are collectively called teichoic acid [ 11. The gr...
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