Development of a high-performance liquid chromatography-tandem mass spectrometry method for the determination of flurogestone acetate in ovine plasma.

Flurogestone (FGA) is a synthetic progesterone, with a progestational action higher than that of progesterone itself. It is intended for vaginal use in large animals to induce oestrus synchronization. A quantitative method for the analysis of flurogestone acetate (FGA) in ovine plasma by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been developed. After the incorporation of megestrol acetate (MGA) as internal standard (IS) and followed by a liquid-liquid extraction from plasma, FGA and MGA were chromatographed using a reverse-phase HPLC column and detected by tandem mass spectrometry with a TurboIonSpray source. Multiple reaction-monitoring (MRM) mode was used for the quantitative determination of FGA in ovine plasma. The precursor ions [M+H](+) at m/z 407.2 and 385.1 for FGA and MGA, respectively, produced product ions at m/z 267.1/285.1 for FGA and m/z 267.1/224.0 for MGA. The validated concentration range was 0.2-5.0 ng/ml based on 500 microl plasma aliquots. The lower limit of quantitation was 0.2 ng/ml. Fully validated selectivity, accuracy, precision and reproducibility criteria for routine use in pharmacokinetic studies were demonstrated.

Macrophage caldesmon is an actin bundling protein.

A rapid purification procedure was developed for the isolation of caldesmon (CaD) from rabbit alveolar macrophage. The purified protein migrated with an apparent M(r) of 74,000 +/- 4000 on SDS-PAGE and cross-reacted with anti-gizzard CaD antibodies. A higher M(r) isoform was isolated from chicken gizzard. Their actin-binding parameters and effects on actomyosin-ATPase activity were investigated under identical experimental conditions. Electron microscope studies revealed that macrophage CaD was able to cross-link actin filaments into both networks and bundles. Compact F-actin bundles were predominantly or exclusively seen at cross-linker to actin molar ratios in the 1:20 to 1:10 range. Apparent K(a) at extrapolated saturation of the CaD-binding sites on F-actin was 1.2 x 10(6) M(-1) for macrophage CaD and 1.6 x 10(6) M(-1) for chicken gizzard CaD. CaD from either source was able to stimulate the actin-activated ATPase activity of macrophage myosin. Unexpectedly, chicken gizzard CaD also increased the ATPase activity of gizzard myosin. The degree of stimulation was approximately doubled in the presence of a large excess of Ca(2+)-calmodulin but was unaffected by the presence of macrophage tropomyosin. However, macrophage CaD did not behave as a Ca(2+)- and calmodulin-regulated actin-binding protein. These results, together with published data on other well-characterized actin bundling proteins, suggest that nonmuscle CaD could be essentially involved in the formation and organization of actin bundles at adhesion sites and cell surface projections. However, they afforded no evidence that the macrophage isoform might play a specific role in the Ca(2+)-dependent regulation of actin and myosin II interactions.

Routine determination of flumequine in kidney tissue of pig using automated liquid chromatography.

A high-performance liquid chromatographic assay is described as a routine analytical method for the determination of flumequine (FLU) and its hydroxylated metabolite (OH-FLU) in pig kidney tissue. Kidney samples (2 g) containing FLU and OH-FLU were extracted by liquid-liquid extraction with ethyl acetate (10 ml). Analytical separations were performed by reversed-phase HPLC with fluorometric detection at 252 nm excitation and 356 nm emission under gradient conditions. The mobile phase was acetonitrile-2.7.10(-3) M oxalic acid in water (pH 2.5). The assay is specific and reproducible within the flumequine range of 0.050-2.5 micrograms/g and recovery at 0.050 microgram/g was 94.8%.

Purification and further characterization of macrophage 70-kDa protein, a calcium-regulated, actin-binding protein identical to L-plastin.

We have previously identified a macrophage 70-kDa, actin-bundling protein as a constituent of actin-based cytoplasmic gel and showed that its association with or dissociation from cytoplasmic gels was remarkably affected by submicromolar calcium. In this study, we purified the 70-kDa protein from soluble cytosolic extracts and carried out a more detailed characterization. The amino acid sequences of four peptidic fragments, obtained from the purified protein by enzymatic or chemical cleavage, were completely or nearly identical to those of L-plastin, a protein initially identified in transformed cells from solid tumors (Goldstein & Leavitt, 1985). By Western blot analysis of normal cells and tissues using specific anti-70-kDa protein antibodies, the 70-kDa molecule was detected only in hematopoietic cells. The 70-kDa protein bound to actin with apparent Kd values of 1.8 and 5.5 microM in the absence and presence of 20 microM free calcium, respectively. Cross-linking activity measured by falling-ball viscosimetry was optimal at free calcium lower than 0.15 microM but was progressively inhibited at higher calcium concentrations, within the physiological range. Half-maximal inhibition occurred at 1.6 microM free calcium. No severing of actin filaments by the 70-kDa protein was observed in any of these assays or previously (Pacaud & Harricane, 1987). Major conformational changes of the protein, as measured by the fluorescence emission intensity of tyrosine residues, occurred at free calcium concentration ranging between 0.15 and 1.5 microM. Magnesium did not mimic the calcium effect. The results suggest that the 70-kDa protein possesses both high-affinity sites and selectivity for calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage alpha-actinin is not a calcium-modulated actin-binding protein.

alpha-Actinin was purified from rabbit macrophages to apparent homogeneity by a procedure designed to remove other actin-binding proteins. Large bundles of filaments were formed when 1 molecule of alpha-actinin interacted with 10-12 actin monomers. This process involved the successive occupancy of two classes of actin-binding sites with different affinities. The apparent Kd of alpha-actinin for F-actin was unaffected by the addition of 25 microM free Ca2+. Analysis of the influence of increasing Ca2+ concentrations on alpha-actinin-F-actin interactions by low-speed sedimentation assays, low-shear viscosity, and electron microscopy indicated that Ca2+ had a small inhibitory effect in the approximate range of 50-1000 microM. Furthermore, the ability of alpha-actinin to assemble actin filaments into bundles was apparently inhibited only at Ca2+ concentrations which also affected the physical properties of F-actin alone. alpha-Actinin immobilized on a nitrocellulose membrane did not bind detectable amounts of Ca2+. Nevertheless, Ca2+ or Mg2+ binding to alpha-actinin induced small decreases in the fluorescence emission intensity of tryptophan and tyrosine residues. The maximal change induced by Mg2+ was smaller than that observed with Ca2+, but Ca2+ and Mg2+ effects were abolished by the addition of 140 mM KCl. Under near-physicological ionic conditions, Ca(2+)-binding sites with an apparent Kd higher than 80-100 microM could not be detected. The results on the functional and physical properties of alpha-actinin are consistent with the hypothesis that Ca2+ decreases alpha-actinin--F-actin interactions by acting both on actin filaments and on cross-linking molecules. Although this conclusion is in contradiction with the generally accepted idea that alpha A is a Ca(2+)-regulated actin-binding protein, it could be predicted from the primary sequence of the two EF-hand-like motifs in the alpha-actinin monomer [Arimura et al. (1988) Eur. J. Biochem. 177, 649-655] based on the crucial role of some Ca(2+)-binding residues as recently demonstrated by point mutations in Ca(2+)-binding sites of calmodulin [Haiech et al. (1991) J. Biol. Chem. 266, 3427-3431]. It is also in agreement with our previous finding that Ca2+ does not affect the behavior of alpha-actinin in actin gel networks from macrophage cytosolic extracts [Pacaud & Harricane (1987) J. Cell Sci. 88, 81-94].

Induction of differentiation of the human histiocytic lymphoma cell line U937 in the absence of vimentin expression.

We have studied the expression of vimentin in the human histiocytic lymphoma cell line U937, induced to differentiate along the monocyte/macrophage pathway. Normal monocytes possess a network of vimentin intermediate filaments (IFs) at all stages of maturation. The undifferentiated U937 leukemia cells contain very low amounts of vimentin, but express a conspicuous IF network when exposed to phorbol myristate acetate. In parallel, they acquire functional properties typical of cells of the monocyte lineage. These concomitant variations suggest that vimentin IFs could play a role in the process of differentiation. However, we observed that all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 confer monocyte-like properties upon U937 cells without inducing vimentin expression. We obtained increased phenotypic changes, yet in the absence of a vimentin network, by combining the effects of both inducers. These results show that vimentin expression is not crucial for the acquisition of some of the functions characteristic of the monocyte/macrophage lineage.

Calcium control of macrophage cytoplasmic gelation: evidence for the involvement of the 70,000 Mr actin-bundling protein.

Under appropriate conditions macrophage cytosolic extracts can form a three-dimensional gel network of cross-linked actin filaments. These cytoplasmic gels are mainly composed of actin, filamin, alpha-actinin, and two new proteins of about 70,000 and 55,000 Mr (70 and 55 K). The behaviour of 70 K protein was found to be remarkably affected by Ca2+. Ca2+ treatment of isolated cytoplasmic gels led to the selective solubilization of the 70 K protein along with a 17 K polypeptide. Half-maximal recovery in the supernatant fraction was obtained from about 0.15 microM free Ca2+. The cytoplasmic gel constituents solubilized in high ionic strength buffer were able to re-assemble into an insoluble actin network when returned to near physiological ionic conditions. However, the inclusion of micromolar Ca2+ prevented the re-association of 70 K protein with actin in these complexes. As compared to the 70 K protein, alpha-actinin was fully resistant to any variations in Ca2+ concentrations. On the other hand, purified 70 K protein displayed the property of assembling actin filaments into bundles at low Ca2+ concentrations (less than 0.15 microM). However, the bundling activity decreased progressively at higher Ca2+, as detected by co-sedimentation and electron microscopy of the 70 K protein-actin mixtures. Half-maximal inhibition was observed at about 0.3 microM free Ca2+. Re-assembly of actin filaments into bundles occurred after chelation of Ca2+ by EGTA, indicating that the inhibitory effect of Ca2+ was reversible. Severing of actin filaments by 70 K protein was not observed in any of the solution conditions used. The Ca2+-dependent inhibition of the ability of 70 K protein to interact with actin networks resulted in a marked distortion of the overall organization of actin filaments, as revealed by thin-section electron microscopy of cytoplasmic gels formed in the presence and absence of Ca2+. Large zones of oriented bundles of filaments were replaced by a looser mesh. When the actin gel constituents were re-assembled in the presence of Ca2+ and exogenous gelsolin, it was also observed that the filament bundles (essentially generated by alpha-actinin) collapsed into dense aggregates. Furthermore, gelsolin did not significantly affect the ability of actin to re-combine with other proteins. The data presented here and previously led us to suspect that the Ca2+ control of the functional state of 70 K protein might be one of the prime factors in the triggering of rapid assembly and disassembly of microfilaments within macrophages.

Cytoplasmic gels from macrophages. Evidence for the involvement of four proteins in the calcium-dependent solation of actin networks.

A method has been devised to study the influence of Ca2+ on the in vitro formation of actin gel networks. Under appropriate conditions low-Ca2+ cytosolic extracts (less than 1 nM) from macrophages rapidly formed a macromolecular complex composed of actin, filamin, alpha-actinin and two new proteins of 70 kDa and 55 kDa. [Pacaud, M. (1986) Eur. J. Biochem. 156, 521-530]. Increasing concentrations of free Ca2+ to 1-2 microM resulted in complete inhibition of the association of 70-kDa protein, a protein which associates actin filaments into parallel arrays. Concentrations of Ca2+ greater than 3 microM caused incorporation of two additional proteins, gelsolin and a 18-kDa polypeptide, with no change in either the actin or alpha-actinin content of the cytoskeletal structures. Use of a polyacrylamide gel overlay technique with 125I-calmodulin revealed that a high-Mr calmodulin-binding protein analogous to spectrin was also associated with these structures when micromolar Ca2+ was present. Similar assays with 45CaCl2 indicated that the 70-kDa protein binds Ca2+ with high affinity. It is thus suggested that Ca2+ might regulate the dynamic assembly of microfilaments through several target proteins, gelsolin, the 70-kDa protein and calmodulin.

Separation and identification of the major constituents of cytoplasmic gels from macrophages.

Proteins which bind to actin filaments in macrophages were investigated by developing a procedure for the isolation of cytoplasmic gels. The gels were found to consist of five major constituents: actin, filamin and the 105-kDa, 70-kDa and 55-kDa components. Prolonged exposure of this macromolecular complex to high-ionic-strength buffer solubilized almost all the proteins, leaving behind the 55-kDa component along with a large amount of actin. Gel filtration of the solubilized extract led to the isolation of five constituents comprising actin, filamin, the 105-kDa and 70-kDa polypeptides, plus a molecular species which eluted at the position of a 280-kDa globular protein. The biochemical and immunological properties of the 105-kDa component were analogous to those of alpha-actinin. Although several attempts were made to correlate the three other constituents (280-kDa, 70-kDa and 55-kDa) with known cytoskeletal proteins, their identity remains to be established. alpha-Actinin, and the 280-kDa and 70-kDa species all exhibited the ability to co-sediment with F-actin and to pack actin filaments into bundles. The bundling activity of the 70-kDa protein was significantly decreased in the presence of micromolar concentrations of calcium, while the activity of the 280-kDa protein was not. Such a Ca2+-sensitive protein could be very important in controlling the local cytoplasmic viscosity.

On-line pharmacokinetics of chloramphenicol in rat cortex by in vivo electrochemical detection.

In vivo electrochemical detection of chloramphenicol in rat cortex was investigated with microelectrodes after intravenous injection of chloramphenicol succinate (170 mg X kg-1). A classical pharmacokinetic study with high-performance liquid chromatography (HPLC) determination of chloramphenicol was performed. The two methods gave the same result when chloramphenicol and chloramphenicol succinate were added for the HPLC assay and compared with the voltametric assay. Clinical applications of this new method are suggested.

Identification and localization of two membrane-bound esterases from Escherichia coli.

Hydrolytic activities of isolated membrane fractions of Escherichia coli against chromogenic substrates, p-nitrophenyl ester and beta-naphthyl ester derivatives of N-substituted amino acids, were investigated by spectrophotometric and electrophoretic methods. Although detergents were absolutely necessary for the solubilization of enzymes, the amount of solubilized activities was increased by adding salt, such as NaCl or KCl. Two esterases were identified and separated by PAGE and by chromatography of the solubilized proteins in the presence of detergent. One hydrolyzed the alanine derivatives preferentially, whereas the other was mainly active on phenylalanine derivatives. Only the first was inactivated by diisopropyl fluorophosphate, a serine hydrolase inhibitor. Whereas the chymotrypsin-like enzyme was equally distributed between the inner and the outer membrane, the alanine activity was only detected in the inner membrane. They were both resistant to extraction with high salt concentrations, indicating their integral association with membranes. A study of the accessibility of these enzymes to their substrate in membrane vesicles with known polarity suggests that both alanine and phenylalanine activities are localized near the external surface of the cytoplasmic (inner) membrane. However, the phenylalanine activity (chymotrypsin-like enzyme) appears to be deeply buried inside the outer membrane. Because of its insensitivity to diisopropyl fluorophosphate, this last esterase seems to be distinct from the previously isolated periplasmic endopeptidase, protease I, which is also a chymotrypsin-like enzyme.

Protease I from Escherichia coli. Some physicochemical properties and substrate specificity.

Protease I, a periplasmic endopeptidase from Escherichia coli has been further purified by a modified procedure. While the purified protein consists of a single polypeptide chain of about 21000 daltons, its molecular weight in dilute salt solution was estimated to be near 43000, suggesting that the enzyme has a marked tendency to dimerize. It has only one disulphide bond and is very sensitive to urea. In agreement with previous evidence of a chymotrypsin-like specificity, hydrolytic assays of various p-nitrophenyl esters of N-substituted amino acids showed that phenylalanine and tyrosine derivatives are the best substrates for the enzyme. The Km(app) for N-benzoyloxycarbonyl-L-tyrosin-p-nitrophenyl ester at pH 7.5 In 100 mM sodium phosphate buffer at 25 degrees C was found to be 0.2 mM. In contrast to chymotrypsin, protease I is unable to hydrolyse N-acetyl-L-phenylalanine ethyl ester and its tyrosine analogue. Moreover, the enzyme appears devoid of amidase activity and exhibits a low activity upon polypeptides. At 37 degrees C, it cleaves the carboxymethylated B-chain of bovine insulin at four points: Phe25-Tyr26, Phe24-Phe25, Leu15-Tyr16 and Ser9-His10. From a detailed study of peptides bonds hydrolyzed, it was concluded that protease I has a stringent requirement for both residues forming the scissile bond, and appears to possess an extended hydrophobic binding site.

Purification of protease II from Escherichia coli by affinity chromatography and separation of two enzyme species from cells harvested at late log phase.

N-Acetyl-D-arginine linked to an agarose matrix has been used to purify protease II from Escherichia coli by affinity chromatography. The specific adsorption of protease II to this absorbent was achieved in 220 mM potassium phosphate buffer pH 7.6, and the enzyme was eluted with L-arginine. Enzyme preparations from cells harvested at late log phase have been resolved into two molecular species which differ in specific activity, kinetic constants and carbohydrate content. Both species appeared homogeneous by electrophoresis in conventional buffers and also in the presence of sodium dodecyl sulfate. Only one enzyme species was obtained by the same procedure using bacteria harvested at the middle of exponential growth.

Protease II from Escherichia coli. Purification and characterization.

We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.