Derivatised lipids in membranes. Physico-chemical aspects of N-biotinyl phosphatidylethanolamines, N-acyl phosphatidylethanolamines and N-acyl ethanolamines

The physical properties of N-biotinyl phosphatidylethanolamines, N-acyl phosphatidylethanolamines and of N-acyl ethanolamines, in aqueous dispersions, are reviewed. Emphasis is placed on the calorimetric (i.e. chain melting) properties, the thermotropic phase behaviour, certain aspects of the structure and dynamics, and the miscibility with other membrane lipids. In the case of N-biotinyl phosphatidylethanolamines, the specific binding of avidin, and in the case of N-acyl ethanolamines, the function of the third chain, is also considered. All of these properties are relevant to the role of these rather unusual lipids in membranes.     

Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis.

gamma-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalton subunits. Enzymatic activity of the protein was detectable only in assays containing highly purified gamma-glutamyl phosphate reductase, the second enzyme of the proline pathway. Plots of gamma-glutamyl kinase activity as a function of glutamate concentration were sigmoidal, with a half-saturation value for glutamate of 33 mM, whereas plots of enzyme activity as a function of ATP concentration displayed typical Michaelis-Menten kinetics with a Km for ATP of 4 X 10(-4) M. Enzyme activity was insensitive to the glutamate analog L-methionine-DL-sulfoximine, but ADP was a potent competitive inhibitor. Characteristics of the enzyme were compared with those of a gamma-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E. coli. These results indicated that the only major difference was that the enzyme from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline.     

N-acyl migration in ceramides

Upon exposure of truncated ceramides, such as N-acetyl-sphingenine, and long-chain ceramides to moderate acidic conditions, three derivatives are formed. Two of them turned out to be O-acylated sphingenine, 1-O- and 3-O-acyl-sphingenine, and the other was identified as sphingenine. Truncated dihydroceramides (e.g., N-acetyl- and N-hexanoyl-sphinganine) also show this type of rearrangement, which is therefore not related to the presence of the allylic hydroxy group or to the length of the N-acyl chain. Of particular concern is the fact that the O-acylated compounds, which can be considered sphingoid base analogs, can be formed in chloroform or chloroform-methanol mixtures upon storage. For long-term storage, methanol or dichloromethane is the preferred solvent. A procedure to document the presence/formation of such O-acylated sphingoid bases in ceramide solutions in the picomole range, based on reversed-phase chromatography after derivatization of their amino group with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, is presented.     

Abnormal behaviour of proline in the isothiocyanate degradation.

It has been observed that proline residues often initiate overlaps during sequenator analysis. The cause has been shown to be an abnormally slow cleavage reaction. The kinetics of the cleavage reaction has been studied and found to obey pseudo-first-order kinetics. There are considerable differences in reaction rates depending on the position of proline in the sequence, as demonstrated for the four prolines in the N-terminal section of the H2B histone from chicken.     

Physiological characteristics of motor units in the brachioradialis muscle across fatiguing low-level isometric contractions.

The purpose of this study was to determine (i) if decomposition-based quantitative electromyography (DQEMG) could detect changes in motor unit potential (MUP) morphology and motor unit (MU) firing pattern statistics associated with muscle fatigue, (ii) if any detected changes are correlated with surface electromyographic (SEMG) signs of fatigue, and (iii) if significant fatigue-dependent changes are repeatable within individuals. Mean MU firing rates and the morphology of MUPs detected using needle and surface electrodes during constant-torque isometric contractions held until exhaustion were investigated in the brachioradialis (BR) muscle in 10 healthy volunteers (mean age=28.6 yr, SD+/-3.9). Time dependant changes were investigated using an analysis of variance with normalized time as a main effect. Partial correlation coefficients were computed using a repeated measures analysis of covariance to determine if changes in MU firing rates, needle-detected MUPs and surface-detected MUPs (SMUPs) were related to changes in SEMG signal amplitude and frequency parameters. Intraclass correlation coefficients (ICCs) were used to determine the within-subject repeatability of changes in MU firing rates, and MUP and SMUP parameters. Significant decreases in mean MU firing rates were found along with significant increases in various duration and area related parameters in both MUPs and SMUPs across the fatiguing contraction. The SEMG signal demonstrated the expected changes with fatigue: an increase in amplitude and a decrease in frequency content. SEMG amplitude was significantly positively correlated with SMUP peak-to-peak voltage (r=0.85, p<0.05), and SMUP area (r=0.86, p<0.05). Mean power frequency was significantly negatively correlated with SMUP negative peak duration (r=-0.74, p<0.05). The significant time-dependent changes were reliably observed (ICCs were 0.94 for MUP peak to peak amplitude, 0.97 for MUP area and 0.95 for MUP area to amplitude ratio, 0.95 for SMUP peak-to-peak voltage, 0.83 for SMUP area, 0.99 for SMUP negative peak amplitude and 0.88 for SMUP negative peak area). The decreases in mean MU firing rates measured along with the increases in amplitude, duration and area parameters of MUPs and SMUPs and their partial correlation with SEMG amplitude during submaximal fatiguing contractions of the BR, suggest that recruitment is a main cause of increased SEMG amplitude parameters with fatigue. We conclude that DQEMG can be effectively and reliably used to detect changes in physiological characteristics of MUs that accompany fatigue.     

Positional preference of proline in alpha-helices.

Conformational free energy calculations have been carried out for proline-containing alanine-based pentadecapeptides with the sequence Ac-(Ala)n-Pro-(Ala)m-NHMe, where n + m = 14, to figure out the positional preference of proline in alpha-helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the alpha-helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N-terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N' (Ncap - 1), and C' (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X-ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing alpha-helical conformations, which, however, provides the favored hydration of the corresponding N-H and C=O groups. The highest preference of proline at the beginning of alpha-helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of alpha-helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of alpha-helix by proline. The average free energy change for the substitution of Ala by Pro in a alpha-helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of approximately 4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme.     

Synthesis of nucleotide antibiotics having N-acyl phosphoramidate linkages.

This paper reports the synthesis of nucleotide antibiotics having N-acyl phosphoramidate linkages. The key reaction, the construction of the N-acyl phosphoramidate linkage was achieved by the reaction of nucleoside 5'-phosphoramidite derivatives with carboxamide derivatives in the presence of 5-(3,5-dinitrophenyl)-1H-tetrazole as a very effective activator. By use of this activator, Phosmidosine was synthesized by condensation of an appropriately protected 8-oxoadenosine 5'-O-phosphoramidite derivative with an N-protected prolinamide derivative. In the case of Agrocin 84, the two P-N bonds were constructed progressively. The N-acyl phosphoramidate linkage at the 5'-position of the ribose moiety was similarly synthesized. After phosphorylation of the amino group of the adenine moiety, a fully protected Agrocin 84 derivative, which would be converted to Agrocin 84, was successfully synthesized.     

Genetics and physiology of proline utilization in Saccharomyces cerevisiae: enzyme induction by proline.

Proline is converted to glutamate in the yeast Saccharomyces cerevisiae by the sequential action of two enzymes, proline oxidase and delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase. The levels of these enzymes appear to be controlled by the amount of proline in the cell. The capacity to transport proline is greatest when the cell is grown on poor nitrogen sources, such as proline or urea. Mutants have been isolated which can no longer utilize proline as the sole source of nitrogen. Mutants in put1 are deficient in proline oxidase, and those in put2 lack P5C dehydrogenase. The put1 and put2 mutations are recessive, segregate 2:2 in tetrads, and appear to be unlinked to one another. Proline induces both proline oxidase and P5C dehydrogenase. The arginine-degradative pathway intersects the proline-degradative pathway at P5C. The P5C formed from the breakdown of arginine or ornithine can induce both proline-degradative enzymes by virtue of its conversion to proline.     

Submitochondrial location and electron transport characteristics of enzymes involved in proline oxidation

Isolated corn mitochondria (Zea mays cv. B73 × Mo17) were fractionated and the fragments were separated on a 20-45% (weight/weight) continuous sucrose gradient. Soluble enzymes remained at the top of the gradient overlapping with the outer membranes, while inner membrane vesicles and intact inner membranes were distributed farther down the gradient. Proline oxidase and Δ¹-pyrroline-5-carboxylic acid dehydrogenase activities were associated only with the inner mitochondrial membrane. Glutamate dehydrogenase was confirmed as a matrix enzyme.     

Effect of the proline analogue baikiain on proline metabolism in Salmonella typhimurium

A proline analogue, 4,5-dehydro-l-pipecolic acid (baikiain) induces the formation in Salmonella typhimurium of the two enzymes catalyzing the degradation of proline, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid (P5C) dehydrogenase. The level of induction by 20 mm baikiain is about 10% of the maximum level induced by proline. Since the analogue is a substrate of proline oxidase the first enzyme of the proline catabolic pathway, the oxidation derivative rather than baikiain itself might be the actual effector. Baikiain is also an inducer of proline oxidase in Escherichia coli K-12 and E. coli W. An additional effect of this analogue on proline degradation in S. typhimurium is inhibition of P5C dehydrogenase. At a concentration of 5 x 10(-4)m, baikiain inhibits completely the growth of strains constitutive for proline oxidase. This inhibition, which can be overcome by proline, occurs in the presence or absence of P5C dehydrogenase activity. Three spontaneously occurring mutants resistant to baikiain were isolated from constitutive strains. All are pleiotropic-negative for the proline-degrading enzymes. The sites of these mutations are linked to the put region. Although the mechanism of toxicity has not been determined, baikiain provides a simple and direct selection for obtaining mutants unable to degrade proline. In addition, it allows selection for strains with an inducible rather than constitutive phenotype.     

A general high-performance liquid chromatography-based assay for the hydrolysis of N-acyl glutamates.

A rapid and simple HPLC assay has been developed to separate and quantify N-acyl glutamates and the corresponding carboxylic acids of the acyl moiety. This method was specifically developed to assay hydrolytic activity for glutamate carboxypeptidases. Although established assays for specific substrates of such enzymes exist, they may not be amenable for examining the hydrolytic activity of new substrate probes. This assay was developed to accommodate such probes.     

N-acyl phosphatidylethanolamines affect the lateral distribution of cholesterol in membranes

N-Acyl phosphatidylethanolamines are negatively charged phospholipids, which are naturally occurring albeit at low abundance. In this study, we have examined how the amide-linked acyl chain affected the membrane behavior of the N-acyl-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-POPE) or N-acyl-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-DPPE), and how the molecules interacted with cholesterol. The gel-->liquid crystalline transition temperature of sonicated N-acyl phosphatidylethanolamine vesicles in water correlated positively with the number of palmitic acyl chains in the molecules. Based on diphenylhexatriene steady state anisotropy measurements, the presence of 33 mol% cholesterol in the membranes removed the phase transition from N-oleoyl-POPE bilayers, but failed to completely remove it from N-palmitoyl-DPPE and N-palmitoyl-POPE bilayers, suggesting rather weak interaction of cholesterol with the N-saturated NAPEs. The rate of cholesterol desorption from mixed monolayers containing N-palmitoyl-DPPE and cholesterol (1:1 molar ratio) was much higher compared to cholesterol/DPPE binary monolayers, suggesting a weak cholesterol interaction with N-palmitoyl-DPPE also in monolayers. In bilayer membranes, both N-palmitoyl-POPE and N-palmitoyl-DPPE failed to form sterol-rich domains, and in fact appeared to displace sterol from sterol/N-palmitoyl-sphingomyelin domains. The present data provide new information about the effects of saturated NAPEs on the lateral distribution of cholesterol in NAPE-containing membranes. These findings may be of relevance to neural cells which accumulate NAPEs during stress and cell injury.     

Glycerophospho-(N-acyl)-ethanolamine lipids in degenerating BHK cells.

The phospholipids, which accompany semilysobisphosphatidic acid from degenerating BHK cells, were identified as a mixture of glycerophospho-(N-acyl)-ethanolamine lipids. The identification was based on infrared spectroscopy, thin-layer chromatography and ethanolamine content of the intact lipids or their partial degradation products. Sequential treatments with mild acid and alkali revealed the presence of three different derivatives: the most abundant of these was the O-(1-alkenyl) ether derivative (plasmenyl-(N-acyl)-ethanolamine), which represented 55-60% of the total glycerophospho-(N-acyl)-ethanolamine lipids; the O-alkyl derivative (plasmanyl-(N-acyl)-ethanolamine) and the di-O-acyl derivative (phosphatidyl-(N-acyl)-ethanolamine) each represented about 20% of the total.     

Growth characteristics and proline content in relation to water status in twoZea mays L. Cultivars during rehydration

Growth behaviour, proline changes and water saturation deficit (WSD) changes were studied in two maize cultivars, Agati-76, a drought resistant oultivar, and cv. Vijay, a susceptible one after stress conditions were removed. Plants of both the cultivars showed a tendency to recover during rehydration. Phenotypically the plants appeared just normal on 7th day of rehydration, although recovery was never complete. Significant positive correlation existed between free proline content and concomitant change in water saturation deficit. Significantly higher proline content in cv. Agati-76 was reoorded in cv. Vijay, immediately after the stress was released, thereafter continuous decline was observed up to 7th day of rehydration in both the cultivars. Proline changes in relation to recovery of plants from stress conditions are discussed.     

Gene-enzyme relationships in the proline biosynthetic pathway of Saccharomyces cerevisiae.

The PRO1, PRO2, and PRO3 genes were isolated by functional complementation of pro1, pro2, and pro3 (proline-requiring) strains of Saccharomyces cerevisiae. Independent clones with overlapping inserts were isolated from S. cerevisiae genomic libraries in YEp24 (2 microns) and YCp50 (CEN) plasmids. The identity of each gene was determined by gene disruption, and Southern hybridization and genetic analyses confirmed that the bona fide genes had been cloned. Plasmids containing each gene were introduced into known bacterial proline auxotrophs, and the ability to restore proline prototrophy was assessed. Interspecies complementation demonstrated that the S. cerevisiae PRO1 gene encoded gamma-glutamyl kinase, PRO2 encoded gamma-glutamyl phosphate reductase, and PRO3 encoded delta 1-pyrroline-5-carboxylate reductase. The presence of the PRO3 gene on a high-copy-number plasmid in S. cerevisiae caused a 20-fold overproduction of delta 1-pyrroline-5-carboxylate reductase. The PRO2 gene mapped on chromosome XV tightly linked to cdc66, and the PRO3 gene was located on the right arm of chromosome V between HIS1 and the centromere.