Detection of phospholipid peroxides in biological samples

Peroxidation of membrane lipids has been hypothesized to play a key role in various types of tissue degeneration and pathology. Lipid peroxides are formed when oxygen reacts with an unsaturated fatty acid chain. Virtually all of the unsaturated fatty acids in biological systems are bound by ester linkages in phospholipids or triglycerides. Phospholipid and triglyceride peroxides are primary products of lipid peroxidation and have rarely been measured. Most of the commonly used methods for detection of lipid peroxidation are based on detection of malondialdehyde or other chemical species that are derived from oxidized fatty acids. This review presents an overview of recently developed methods aimed at identifying and measuring oxidized phospholipids and triglycerides which are direct evidence of the occurrence of lipid peroxidation in vivo.     

Characterization of three-subunit chloroplast coupling factor

The delta- and epsilon-polypeptides were removed from chloroplast coupling factor 1 (CF1). The resulting enzyme, CF1(-delta, epsilon), is a stable active ATPase containing only alpha-, beta-, and gamma-polypeptides. The dependence of the steady-state kinetics of ATP hydrolysis catalyzed by CF1(-delta, epsilon) on the concentrations of ATP and ADP was found to be essentially the same as by activated CF1. Nucleotide binding studies with CF1(-delta, epsilon) revealed three binding sites: a nondissociable ADP site (site 1), a tight MgATP binding site (site 2), and a site that binds ADP and ATP with a dissociation constant in the micromolar range (site 3). Similar results have been obtained with CF1. For both CF1 and CF1(-delta, epsilon), the binding of MgATP at site 2 is tight only in the presence of Mg2+. Fluorescence resonance energy transfer was used to map distances between the gamma-sulfhydryl ("dark" site) and gamma-disulfide and between the gamma-sulfhydryl and the three nucleotide sites. These distances are within 5% of the corresponding distances on CF1. These results indicate that removal of the delta- and epsilon-polypeptides from CF1 does not cause significant changes in the structure, kinetics, and nucleotide binding sites of the enzyme.     

Correlation of enzymatic activities and aggregation state in chicken liver fatty acid synthase

The relationships between the aggregation state and the enzymatic activities of chicken liver fatty acid synthase have been explored by monitoring the changes in light scattering, fluorescence, and the overall, beta-ketoacyl synthase, beta-ketoacyl reductase and enoyl reductase activities during dissociation and reassociation of the enzyme. The data obtained indicate that the enzyme dissociates at low temperature in both 0.1 M potassium phosphate (pH 7.0), 1 mM EDTA, and 5 mM Tris(hydroxymethyl)aminomethane, 35 mM glycine (pH 8.3) and 1 mM EDTA, but the extent of dissociation is less in the phosphate buffer. The assay conditions influence the assessment of the degree of dissociation and association: high temperatures, phosphate (high salt), NADPH and acetoacetyl-coenzyme A promote association of the monomeric enzyme, whereas dilution in the Tris-glycine buffer (low salt) and low temperature promote dissociation. Both the rate and extent of association and dissociation are altered by substrates. The monomeric enzyme does not possess beta-ketoacyl synthase and beta-ketoacyl reductase activities. Results obtained with the 1,3-dibromo-2-propanone cross-linked enzyme, which lacks beta-ketoacyl synthase activity, indicate that the NADPH-binding site of beta-ketoacyl reductase is disrupted at low ionic strength. In contrast, changes in ionic strength have little effect on the enoyl reductase activity. The dimer is stabilized by both electrostatic and hydrophobic interactions, with the former being of special importance for maintenance of the beta-ketoacyl reductase active site. site.     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: beta-ketoacyl reductase and enoyl reductase

The following reactions catalyzed by chicken liver fatty acid synthase have been studied with the stopped-flow method in 0.1 M potassium phosphate (pH 7.0) and 1 mM ethylenediaminetetraacetic acid at 25 degrees C by monitoring the change in NADPH fluorescence: the transfer of acetoacetyl from acetoacetyl coenzyme A to the enzyme, reduction of the enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase), and reduction of enzyme-bound D-hydroxybutyryl/crotonyl by NADPH (enoyl reductase). The first two reactions were studied by mixing enzyme-NADPH with acetoacetyl-CoA under conditions where the kinetics can be analyzed as two consecutive pseudo-first-order processes: a mechanism consistent with the aceto-acetyl-CoA dependence of the pseudo-first-order rate constant associated with formation of the aceto-acetyl-enzyme is a relatively rapid binding of substrate to the enzyme, with a dissociation constant of 650 microM, followed by formation of covalently bound acetoacetyl, with a rate constant of 10.2 s-1. The aceto-acetyl-enzyme is reduced by enzyme-bound NADPH with a rate constant of 20 s-1, and the NADPH binding is characterized by a dissociation constant of 5.3 microM. Reduction of the D-hydroxybutyryl-/crotonyl-enzyme was studied by mixing NADPH with enzyme that was equilibrated with D-hydroxybutyryl-CoA or crotonyl-CoA; the rate constant for reduction of an equilibrium mixture of D-hydroxybutyryl- and crotonyl-enzyme is 36.6 s-1. Steady-state kinetic studies of the reduction of acetoacetyl-CoA and crotonyl-CoA by NADPH also have been carried out.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison between dopamine-stimulated adenylate cyclase and 3H-SCH 23390 binding in rat striatum

Methods for measuring 3H-SCH 23390 binding and dopamine (DA) stimulated adenylate cyclase (AC) were established in identical tissue preparations and under similar experimental conditions. Pharmacological characterization revealed that both assays involved interaction with the D1 receptor or closely associated sites. In order to investigate whether the binding sites for 3H-SCH 23390 and DA in fact are identical, the antagonistic effects of a variety of pharmacologically active compounds were examined. Surprisingly, the Ki-values obtained from Schild-plot analysis of the antagonism of DA-stimulated AC, were 80-240 times higher than the Ki-values obtained from competition curves of 3H-SCH 23390 binding. Since both assays were performed under identical conditions, the differences in Ki-values indicate the possibility of different binding sites for DA and 3H-SCH 23390 or, that DA and 3H-SCH 23390 label different states of the same receptor.     

Biosynthesis of peptidoglycan in Gaffkya homari: role of the peptide subunit of uridine diphosphate-N-acetylmuramyl-pentapeptide

The incorporation of N-acetylmuramyl (MurNAc)-peptides from nucleotide-activated precursors (reference: uridine diphosphate [UDP]MurNAc-Ala(1)-dGlu(2)-Lys(3)- dAla(4)-dAla(5)) with incomplete or modified peptide subunits into peptidoglycan was studied with membrane preparations from Gaffkya homari. The effectiveness of their utilization at low and high concentrations was compared on the basis of the values of V(max)/K(m) and V(max), respectively. At low concentration, replacement of alanine by glycine in position 5 has a small effect on the activity of the peptidoglycan synthesizing system, whereas it has a significantly larger effect in positions 1 and 4. The importance of d-alanine in position 4 at low substrate concentrations is also observed with the incomplete UDP-MurNAc-peptides. For UDP-MurNAc-tripeptide and -tetrapeptide, V(max)/K(m) is 0.06 and 0.55, respectively, of the value for the -pentapeptide. At high substrate concentration, replacement of d-alanine by glycine in either position 1 or 5 decreases the activity to 0.37 of the value for the reference nucleotide, whereas replacement in position 4 has a smaller effect (0.74). The profiles established from V(max) and V(max)/K(m) with UDP-MurNAc-tripeptide, -tetrapeptide, and -pentapeptide show good correlation. At low concentration the specificity profiles of phospho-MurNAc-pentapeptide translocase, catalyzing the initial membrane reaction, are similar to those for the peptidoglycan synthesizing system; at high concentration, however, the profiles differ. The translocase appears to provide a primary specificity barrier at high substrate concentration for UDP-MurNAc-Ala-dGlu-Lys-dAla-dAla and UDP-MurNAc-Ala-dGlu-Lys-Gly-dAla, and at low concentration for UDP-MurNAc-Ala-dGlu-Lys and UDP-MurNAc-Ala-dGlu-Lys-Gly-dAla. Moreover, it is suggested that an additional specificity barrier exists in the peptidoglycan synthesizing system for certain nucleotides. Thus, the cytoplasmic enzymes and the membrane-associated enzyme(s) cooperate to insure the formation of functioning peptidoglycan in this organism.     

On the specificity of the uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: Diamino acid ligase of Bifidobacterium globosum

The peptidoglycan of Bifidobacterium globosum contains ornithine and lysine alternately in the same position of the peptide subunit. The uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: diamino acid ligase of this organism was purified 700-fold. Since the activities for the incorporation of ornithine and lysine into uridine diphospho-N-acetylmuramyl-tripeptide did not separate during purification and since the incorporation of ornithine is competitively inhibited by lysine and vice versa, both ornithine and lysine are assumed to be incorporated by one single enzyme. Studies on the specificity of the ligase toward analogs of ornithine have shown that the enzyme requires a diamino, monocarboxylic acid with 4–6 carbon atoms. Methylation of the -amino group or hydroxylation of the -carbon atom of lysine decreases the competitive properties of the analog, whereas the substitution of the -methylen group by sulfur (S-2-aminoethyl cysteine) results in a highly competitive compound.Abbreviations BSA bovine serum albumine - MurNAc N-acetyl-muramyl - DA diamino acid - Ala-DGlu--L-DA-DAla-D-Ala pentapeptide - Ala-DGlu--LDA tripeptide - Ala-DGlu dipeptide - DSM Deutsche Sammlung von Mikroorganismen - CEM clostridial enrichment medium