A novel finding that Streptomyces clavuligerus can produce the antibiotic clavulanic acid using olive oil as a sole carbon source.

AIMS: This study aims to establish whether commercially available food oils can be used by Streptomyces clavuligerus as sole carbon sources for growth and clavulanic acid production. METHODS AND RESULTS: Batch cultures in bioreactors showed that Strep. clavuligerus growth and clavulanic acid yields in a P-limited medium containing 0.6% (v/v) olive oil were respectively 2.5- and 2.6-fold higher than in a glycerol-containing medium used as control. Glycerol- and olive oil-grown cells present different macromolecular composition, particularly lipid and protein content. CONCLUSIONS: Streptomyces clavuligerus uses olive oil as the sole carbon and energy source for growth and clavulanic acid production. Yields and production rates in olive oil are comparable to those reported for oil-containing complex media. Differences in yields and in the macromolecular composition indicate that different metabolic pathways convert substrate into product. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of oils being used as the sole carbon source by Strep. clavuligerus. Apart from economic benefits, interesting questions are raised about Strep. clavuligerus physiology. Defined culture media allow physiological studies to be performed in the absence of interference by other compounds. Understanding how Strep. clavuligerus catabolises oils may have an economic impact in clavulanic acid production.

Collision-mediated transfer of long-chain fatty acids by neural tissue fatty acid-binding proteins (FABP): studies with fluorescent analogs.

Mammalian fatty acid-binding proteins (FABP) are a family of intracellular proteins (approx 15 kDa) that bind long-chain fatty acids (FA) with high affinity. They are believed to serve as cytoplasmic transporters of FA and to target FA to specific cellular sites of utilization. Several different FABPs are expressed in neural tissue, including brain FABP (B-FABP), myelin FABP (M-FABP), and heart FABP (H-FABP). We have previously shown that H-FABP transfers FAvia direct collisional interactions with acceptor model membranes. In the present studies, we use a fluorescence resonance energy transfer (FRET) assay to examine the rate and mechanism of transfer of a fluorescent long-chain fatty acid from B-FABP to phospholipid vesicles. The rate of transfer is shown to be independent of buffer ionic strength and dramatically enhanced by the presence of specific anionic phospholipids. These results are consistent with a mechanism by which FA are transferred from B-FABP to phospholipid membranes by a transient collision-based mechanism.

The fatty acid transport function of fatty acid-binding proteins.

The intracellular fatty acid-binding proteins (FABPs) comprise a family of 14-15 kDa proteins which bind long-chain fatty acids. A role for FABPs in fatty acid transport has been hypothesized for several decades, and the accumulated indirect and correlative evidence is largely supportive of this proposed function. In recent years, a number of experimental approaches which more directly examine the transport function of FABPs have been taken. These include molecular level in vitro modeling of fatty acid transfer mechanisms, whole cell studies of fatty acid uptake and intracellular transfer following genetic manipulation of FABP type and amount, and an examination of cells and tissues from animals engineered to lack expression of specific FABPs. Collectively, data from these studies have provided strong support for defining the FABPs as fatty acid transport proteins. Further studies are necessary to elucidate the fundamental mechanisms by which cellular fatty acid trafficking is modulated by the FABPs.

Liver and intestinal fatty acid-binding proteins obtain fatty acids from phospholipid membranes by different mechanisms.

Intestinal enterocytes contain high concentrations of two cytosolic fatty acid-binding proteins (FABP), liver FABP (L-FABP) and intestinal FABP (I-FABP), which are hypothesized to play a role in cellular fatty acid trafficking. The mechanism(s) by which fatty acids move from membranes to each of these proteins is not known. Here we demonstrate that fluorescent anthroyloxy fatty acid analogues (AOFA) are transferred from phospholipid vesicles to L-FABP versus I-FABP by different mechanisms. For L-FABP a diffusion-mediated transfer process is demonstrated. The AOFA transfer rate from phosphatidylcholine-containing vesicles (POPC) to L-FABP is similar to that observed with another diffusional process, namely inter-membrane AOFA transfer. Furthermore, the AOFA transfer rate was modulated by buffer ionic strength and AOFA solubility, while the transfer rate remained relatively unchanged by the presence of anionic phospholipids in vesicles. In contrast, the data for I-FABP suggest that a transient collisional interaction of I-FABP with the phospholipid membrane occurs during AOFA extraction from the vesicles by the protein. In particular, the presence of the anionic phospholipid cardiolipin in donor vesicles increased the rate of AOFA transfer to I-FABP by 15-fold compared with transfer to POPC vesicles. The effects of ionic strength on transfer suggest that the interaction of I-FABP with cardiolipin-containing vesicles is likely to contain an electrostatic component. Finally, based on the regulation of AOFA transfer to I-FABP compared with transfer from I-FABP, it is hypothesized that apo- and holo-I-FABPs adopt conformations which may differentially promote I-FABP-membrane interactions. In summary, the results suggest that I-FABP, but not L-FABP, can directly extract fatty acids from membranes, supporting the concept that I-FABP may increase the cytosolic flux of fatty acids via intermembrane transfer.

Binding of recombinant rat liver fatty acid-binding protein to small anionic phospholipid vesicles results in ligand release: a model for interfacial binding and fatty acid targeting.

A number of intracellular proteins bind to negatively charged phospholipid membranes, and this interfacial binding results in a conformational change that modulates the activity of the protein. Using a fluorescent fatty acid analogue, 11-[5-(dimethylamino)naphthalenesulfonyl]undecanoic acid (DAUDA), it is possible to demonstrate the release of this ligand from recombinant rat liver FABP in the presence of phospholipid vesicles that contain a significant proportion of anionic phospholipids. The ligand release that is observed with anionic phospholipids is sensitive to the ionic strength of the assay conditions and the anionic charge density of the phospholipid at the interface, indicating that nonspecific electrostatic interactions play an important role in the process. The stoichiometric relationship between anionic phospholipid and liver FABP suggests that the liver FABP coats the surface of the phospholipid vesicle. The most likely explanation for ligand release is that interaction of FABP with an anionic membrane interface induces a rapid conformational change, resulting in a reduced affinity of DAUDA for the protein. The nature of this interaction involves both electrostatic and nonpolar interactions as maximal release of liver FABP from phospholipid vesicles with recovery of ligand binding cannot be achieved with high salt and requires the presence of a nonionic detergent. The precise interfacial mechanism that results in the rapid release of ligand from L-FABP remains to be determined, but studies with two mutants, F3W and F18W, suggest the possible involvement of the amino-terminal region of the protein in the process. The conformational change linked to interfacial binding of this protein could provide a mechanism for fatty acid targeting within the cell.

Monoacylglycerol binding to human serum albumin: evidence that monooleoylglycerol binds at the dansylsarcosine site.

The binding of monoacylglycerides of long-chain fatty acids to human serum albumin has been examined using monooleoylglycerol as the ligand. Binding was investigated using changes in tryptophan fluorescence and also the displacement of a variety of well-studied fluorescent ligands from human serum albumin (HSA). Monooleoylglycerol caused a decrease in fluorescence from tryptophan-214 when measured at 350 nm while oleic acid had no effect on fluorescence at this wavelength and did not compete with monooleoylglycerol. In contrast, oleic acid caused an increase in fluorescence at 330 nm whereas monooleoylglycerol did not affect fluorescence intensity at this wavelength. These results suggest that these two ligands do not bind to the same site on HSA. From competition studies using dansylglycine, dansylsarcosine, 11-(dansylamino)-undecanoic acid and 1-anilino-8-naphthalenesulfonic acid it was proposed that monooleoylglycerol binds at the dansylsarcosine site (site II) of HSA. Monooleoylglycerol was a competitive inhibitor of dansylsarcosine binding with a Kd of about 2.5 microM whereas oleic acid was not competitive with dansylsarcosine binding.

A fluorescence displacement assay for the measurement of arachidonoyl ethanolamide (anandamide) and oleoyl amide (octadecenoamide) hydrolysis.

We describe a simple fluorescence displacement assay to measure hydrolysis of arachidonoyl ethanolamide and oleoyl amide, two important pharmacological compounds. Hydrolysis at the amide linkage of these ligands releases a fatty acid as one of the products. The displacement of a fluorescent fatty acid analogue from rat liver fatty acid-binding protein by the released fatty acid can thus be measured as a decrease in fluorescence. This process is time- and concentration-dependent and shows hyperbolic enzyme kinetics. Electrospray ionisation mass spectrometry was used to validate the assay.

The binding of cholesterol and bile salts to recombinant rat liver fatty acid-binding protein.

The physiological role of liver fatty acid-binding protein (L-FABP) has yet to be clarified. An important feature of this member of the family of intracellular lipid-binding proteins is the wide range of compounds that have been identified as potential physiological ligands. By using recombinant L-FABP, the binding of cholesterol, bile salts and their derivatives has been investigated under conditions that allow a direct comparison of the binding affinities of these ligands for fatty acids. The results demonstrate an inability of L-FABP to bind cholesterol, although the anionic derivative, cholesteryl sulphate, will bind under similar assay conditions. Of the bile salts examined, lithocholate and taurolithocholate sulphate showed the greatest binding to L-FABP. It is proposed that an important function of L-FABP is to bind certain physiological amphipathic anions, thus preventing the "free' concentrations of these compounds from exceeding their critical micelle concentration, which could result in cell damage.

Mutations of recombinant rat liver fatty acid-binding protein at residues 102 and 122 alter its structural integrity and affinity for physiological ligands.

Rat liver fatty acid-binding protein (FABP) is able to accommodate a wide range of non-polar anions in addition to long-chain fatty acids. This property means that the liver protein is functionally different from other FABPs from intestine, muscle and adipose tissue that have a more restricted ligand specificity and stoichiometry. The availability of crystal structures for the latter proteins has highlighted the importance of two arginine residues that are involved in the binding of the fatty acid carboxylate. Only one of these arginine residues, arginine-122, is conserved in liver FABP, whereas the other arginine, at position 102, is replaced by a threonine. In order to gain further insight into the nature of ligand interactions with liver FABP these key residues (102 and 122) have been changed by site-directed mutagenesis. The results with an R122Q mutant highlight the critical role of this arginine in determining ligand affinity, while similar but less dramatic effects were observed with the T102Q mutant. The double mutant T102Q/R122Q was expressed but had lost the ability to bind fluorescent ligands. It is concluded that Arg-122 plays a role in accommodating the carboxylate group of at least one fatty acid. It is proposed that physiological ligands with more bulky headgroups, such as lysophospholipids, acyl-CoA and mono-olein, bind with the headgroups in a solvent-exposed location near the portal region of the protein. The portal region is suggested to be more flexible in the mutants (R122Q and to a lesser extent T102Q). The net result is that the ligand specificity of the R122Q mutant changes to that of a protein with enhanced affinity for acyl-CoA, lysophospholipids and mono-olein.

The binding of natural and fluorescent lysophospholipids to wild-type and mutant rat liver fatty acid-binding protein and albumin.

Rat liver fatty acid-binding protein (FABP) is able to bind a wide range of non-polar anionic ligands, including lysophospholipids. In order to understand the nature of lysophospholipid interactions with liver FABP, the binding of natural lysophospholipids and two fluorescent analogues, N-(5-dimethylaminonaphthalenesulphonyl)-1-palmitoyl-sn-glycero-3- phosphoethanolamine (dansyl lysoPE) and 1-(O-[11-(5-dimethylaminonaphthalene-sulphonyl)amino]undecyl)-sn-glycero -3- phosphocholine (dansyl-C11-lysoPAF), has been investigated. The results confirmed the ability of liver FABP to bind lysophospholipids with KD values in the range of 1-2 microM, and a 1:1 binding stoichiometry was indicated. Binding of fluorescent lysophospholipids was enhanced with the FABP mutant, R122Q, possibly due to increased flexibility of the binding cavity as a result of reduced hydrogen-bonding constraints. The fluorescent lysophospholipids also bound to albumin, with KD values in the range 0.1-1.0 microM, and could be displaced by oleic acid. The fluorescence characteristics of the dansyl lysophospholipid analogue dansyl-C11-lyso-PAF suggested that this probe binds to the same site(s) on albumin as the fluorescent fatty acid probe 11-(5-dimethylaminonaphthalene-sulphonylamino)-undecanoic acid (DAUDA).

The binding of lysophospholipids to rat liver fatty acid-binding protein and albumin.

The binding of lysophospholipids to rat liver fatty acid-binding protein (FABP) and to BSA and human serum albumin was investigated by using competitive displacement fluorescence assays by monitoring the displacement of the fluorescent fatty acid probe 11-(dansylamino)undecanoic acid (DAUDA). In addition, direct binding assays using changes in tryptophan fluorescence were possible with albumin. Liver FABP was able to bind a range of lysophospholipids, oleoyl-lysophosphatidic acid (lysoPA), oleoyl-lysophosphatidylcholine (lysoPC), oleoyl-lysophosphatidylethanolamine (lysoPE) and oleoyl-lysophosphatidylglycerol, with similar affinity and a Kd of about 1 microM. Liver FABP was also able to bind lysophospholipids generated by the action of phospholipase A2 or phospholipase A1 (triacylglycerol lipase) on phospholipid vesicles. A possible physiological role for liver FABP in lysophospholipid metabolism within the cell is discussed. Albumin was shown to bind lysoPA with higher affinity than either lysoPC or lysoPE, and the initial minimal DAUDA displacement by lysoPA indicated that lysoPA was binding to the primary high-affinity fatty acid-binding sites on albumin and that, like oleic acid, about 3 mol of ligand/mol was bound to these sites. Kd values in the microM range were indicated for lysoPC and lysoPE, whereas, by comparison with oleic acid, the Kd for lysoPA was significantly lower and high-affinity binding in the nM range was indicated. Overall, the data suggest that, because of structural similarity, lysoPA binds to albumin in a similar manner to long-chain fatty acids.

Characterization of binding and structural properties of rat liver fatty-acid-binding protein using tryptophan mutants.

Rat liver fatty-acid-binding protein (FABP) does not contain tryptophan. Three mutant proteins have been produced in which a single tryptophan residue has been inserted by site-directed mutagenesis at positions 3 (F3W), 18 (F18W) and 69 (C69W). These tryptophans have been strategically located in order to provide fluorescent reporter groups to study the binding and structural characteristics of rat liver FABP. Two fluorescent fatty acid analogues, DAUDA (11-[(5-dimethylaminonaphthalene-1- sulphonyl)amino]undecanoic acid) and 3-[p-(6-phenyl)-hexa-1,3,5-trienyl]phenylpropionic acid, showed no significant difference in binding affinities for the different mutant proteins, although maximum fluorescence values were decreased for F3W and increased for C69W. These findings were confirmed by studies of DAUDA displacement by oleate. Protein-denaturation studies in the presence of urea indicated subtle differences for the three mutants which could be explained by multiple unfolding pathways. Fatty acid binding increased tryptophan fluorescence emission in the case of the F18W protein, but had no effect on the F3W and C69W proteins. Fluorescence quenching studies with 2-bromopalmitate showed that a fatty acid carboxylate is close to the tryptophan in the F18W protein. Energy-transfer studies showed that the fluorescent moiety of DAUDA is equidistant from the three mutated amino acids and is bound within the beta-clam solvent cavity of liver FABP. This interpretation of the fluorescence quenching and energy-transfer data supports the difference in ligand orientation between intestinal and liver FABP observed in previous studies.

Haem peptide/protein interaction. Part 6: The kinetic mechanisms of the interactions with, and inhibition of enzymic activity of the human erythrocyte glutathione S-transferase isoenzyme rho (p), by haem octa-, nona-, and undecapeptides MP-8/-9/-11.

The binding of the cytochrome-c derived haem peptides microperoxidase-8, -9, and -11 (MP-8, -9, and -11) to the human erythrocyte glutathione S-transferase rho (GST-p) enzyme is demonstrated. Inhibition by the haem peptides of the enzymic conjugation of glutathione (GSH) with the electrophilic cosubstrate 1-chloro-2, 4-dinitrobenzene (CDNB) is mixed-type with respect to CDNB, and Ki, the inhibition constant, increases with increasing length of the peptide chain. The results obtained here for the GST-p are compared to those published recently for the previously-supposed identical isoenzyme human placental GST-pi.

Effect on ligand binding of arginine mutations in recombinant rat liver fatty acid-binding protein.

Rat liver fatty acid-binding protein is able to accommodate a wide range of non-polar anions in addition to long-chain fatty acids. The two arginine residues of rat liver fatty acid-binding protein, Arg122 and Arg126, have been mutated and the effect of mutation on ligand binding investigated. No significant decrease in affinity for the fluorescent fatty acid analogue, 11-(5-dimethylaminonaphthalenesulphonyl amino)undecanoic acid, or oleate was observed. However, the apparent affinity for oleoyl-CoA was slightly increased with the mutations Ala122 and Gln122 such that oleoyl-CoA rather than oleate became the preferred ligand for these mutants. Small changes in protein stability were observed with the Arg122 mutations. The lack of notable ionic involvement of the conserved internal residue Arg122 in ligand binding is consistent with the hypothesis that the mode of ligand binding in liver fatty acid-binding protein is markedly different from that of other members of this lipid-binding protein family.

Kinetic mechanism of human erythrocyte acidic isoenzyme rho.

The kinetic mechanism has been determined for human glutathione S-transferase rho (rho), an isoenzyme related to the human pi (pi) isoenzyme. The kinetic mechanism was investigated by both non-linear regression studies and the analysis of primary and secondary plots, utilizing initial rate and product inhibition data. It was concluded that human isoenzyme rho obeys a random sequential Bi-Bi rapid equilibrium mechanism with the formation of an enzyme-substrate-product (enzyme-CDNB-conjugate) dead-end complex. The values of KCDNB, KGSH and Kconjugate were 0.70 +/- 0.11, 0.12 +/- 0.02 and 0.016 +/- 0.004 mM, respectively. Comparison of the kinetic mechanism and kinetic parameters obtained for glutathione S-transferase isoenzyme rho with other class pi isoenzymes showed similarities at the primary kinetic level.

Haem-peptide-protein interactions: Part 5. The haem undecapeptide microperoxidase-11 (Fe3+MP-11)/human serum albumin (HSA) reaction in aqueous methanolic solution. A simple system demonstrating the effect of hydrophobicity on ligand release from a ligand-protein complex.

Previous studies of the interaction of the haem undecapeptide (MP-11) with lipidated human serum albumin in aqueous solution have been extended to a range of MeOH/H2O solution compositions. It is demonstrated that the kinetic mechanism for the interaction does not change from a simple second- and first-order reversible scheme as XMeOH is increased, however while k1--the association rate constant is essentially invariant with XMeOH, K-1 increases some 600-fold over the range studied. The result is interpreted in terms of increased solvational stabilization of MP-11 and transition state as XMeOH increases and it is noted that the system provides a simple demonstration of the effect of hydrophobicity on facilitating transported ligand release from ligand/carrier protein molecules.

Reversible inhibition of rat hepatic glutathione S-transferase 1-2 by bilirubin.

The inhibition of rat hepatic glutathione (GSH) S-transferase 1-2 by bilirubin exhibited pseudo first-order kinetics with k(obs) values of 0.0214 +/- 0.0005 and 0.040 +/- 0.008 sec-1 at 4 and 8 microM bilirubin, when followed to 72 and 84% completion respectively. These correspond to calculated second-order rate constants of 5.3 +/- 0.1 x 10(3) and 5.0 +/- 1.0 x 10(3)/M.sec. The extent of inhibition of the transferase increased with bilirubin concentration, with half-maximal inhibition at 4 microM bilirubin. Inhibition was reversed by 10-fold dilution of bilirubin or by increasing the pH from 6.0 to 7.4. Premixing 0.2 to 0.5 microM albumin, hemoglobin or aldolase with bilirubin prevented inhibition of GSH S-transferase 1-2. Protection by these proteins occurred at a selected high concentration (0.2 to 0.4 microM) at which they reduced free bilirubin to concentrations (less than 0.5 microM) that did not inhibit isoenzyme 1-2 significantly. No protection was afforded by a selected low protein concentration (0.001 to 0.01 microM) which did not strikingly reduce bilirubin levels in solution. We conclude that bilirubin inhibition of GSH S-transferase 1-2 appears to be a second-order process; the reaction is clearly first-order with respect to GSH S-transferase and appears also to be first-order with respect to bilirubin. It is proposed that (a) inhibition of GSH S-transferase 1-2 results from slow, reversible bilirubin binding, and (b) added proteins appear to prevent GSH S-transferase inhibition by binding high molar ratios of bilirubin.

Kinetics of heme octapeptide (microperoxidase-8; MP-8) formation studied by high-pressure liquid chromatography (HPLC) monitoring of the peptic and tryptic hydrolysis of horse heart cytochrome-c.

The kinetics of the sequential peptic and tryptic hydrolysis of cytochrome-c to give the heme-peptides microperoxidase-11 (MP-11) and -8 (MP-8), respectively, has been investigated by high performance liquid chromatography (HPLC), and we demonstrate that MP-8 can be prepared from cytochrome-c to the point of lyophilization within 4 hr.

Halothane: inhibition and activation of rat hepatic glutathione S-transferases.

Multiple halothane anesthesias (1.25 MAC for 1 hr on 3 alternate days) of male Long-Evans rats initially decreased by up to 30% and subsequently increased to up to 185% liver cytosolic glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene, 3,4-dichloro-1-nitrobenzene and trans-4-phenyl-3-buten-2-one and glutathione peroxidase activity. Halothane rapidly and reversibly activated hepatic cytosolic glutathione S-transferases and purified isoenzyme 1-2 but not isoenzymes 1-1 and 3-3. At high concentrations of halothane (ca. 22 mM), maximal activation was ca. 25%. Halothane, enflurane, isoflurane and methoxyflurane, but not the halothane metabolite 1-chloro-2,2-difluoroethylene, inhibited a mixture of liver cytosolic glutathione S-transferases with time (ca. 30% inhibition/15 min). The inhibition exhibited pseudo-first order kinetics (kobs = 0.13 min-1) and an I50 for halothane of greater than or equal to 15 mM. Halothane inhibited glutathione S-transferases 3-3, 3-4, and 4-4 by 50-60%, but did not affect isoenzymes 1-1 and 1-2. The ability of halothane to diminish hepatic glutathione S-transferase activity in vivo may in part reflect the time-dependent inhibition of glutathione S-transferase isoenzymes containing the 3- and 4-subunits.