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F Müller

Publications and source records attributed to F Müller.

At least 325 records · Page 18Linked to original sources
PubMed1988

Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography.

A new method is described for the large-scale reversible dissociation of flavoproteins into apoprotein and prosthetic group using hydrophobic-interaction chromatography. Lipoamide dehydrogenase from Azotobacter vinelandii and butyryl-CoA dehydrogenase from Megasphaera elsdenii are selected to demonstrate the usefulness of the method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained. The apoproteins show high reconstitutability. The holoenzymes are bound to phenyl-Sepharose CL-4B at neutral pH in the presence of ammonium sulfate. FAD is subsequently removed at pH 3.5-4.0 by addition of high concentrations of KBr. Large amounts of apoenzymes (200-500 mg), showing negligible residual activity, are eluted at neutral pH in the presence of 50% ethylene glycol. The holoenzyme of lipoamide dehydrogenase can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. In both cases the yield and degree of reconstitution of holoenzyme is more than 90% of starting material. Apo-lipoamide-dehydrogenase exists mainly as a monomer in solution and reassociates to the native dimeric structure in the presence of FAD. The apoenzyme is stable for a long period of time when kept in 50% ethylene glycol at -18 degrees C. Steady-state fluorescence-polarization measurements of protein-bound FAD indicate that reconstituted lipoamide dehydrogenase possesses a high stability which is governed by the low dissociation rate constant of the apoenzyme-FAD complex. The holoenzyme of butyryl-CoA dehydrogenase cannot be reconstituted when the apoenzyme is bound to the column. However, stable apoprotein can be isolated in the free state yielding 50-80% of starting material, depending on the immobilization conditions. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation. The apoenzyme is relatively stable when kept in 50% ethylene glycol at -18 degrees C. From kinetic and gel filtration experiments it is concluded that the reconstitution reaction of butyryl-CoA dehydrogenase is governed by both the pH-dependent hydrodynamic properties of apoenzyme and the pH-dependent stability of reconstituted enzyme. At pH 7, the apoenzyme is in equilibrium between dimeric and tetrameric forms and reassociates to a native-like tetrameric structure in the presence of FAD. The stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands as shown by fluorescence-polarization measurements. The degree of reconstitution of butyryl-CoA dehydrogenase is more than 80% of the original specific activity under certain conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Apoproteins↗
PubMed1988

Chemical modification of tyrosine-38 in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by 5'-p-fluorosulfonylbenzoyladenosine: a probe for the elucidation of the NADPH binding site? Involvement in catalysis, assignment in sequence and fitting to the tertiary structure.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens was covalently modified by the nucleotide analog 5'-(p-fluorosulfonylbenzoyl)-adenosine in the presence of 20% dimethylsulfoxide. The inactivation reaction is pH-dependent and does not obey pseudo-first-order kinetics, due to spontaneous hydrolysis of the reagent. The kinetic data further indicate that a weak, reversible enzyme-inhibitor complex is an intermediate in the inactivation reaction and that only one amino acid residue is responsible for the loss of activity. The inactivation is strongly inhibited by NADPH and 2',5'ADP. Steady-state kinetics and 2',5'ADP bioaffinity chromatography of the modified enzyme suggest that the essential residue is not directly involved in NADPH binding. Sequence studies show that Tyr-38 is the main residue protected from modification in the presence of NADPH. From crystallographic studies it is known that the hydroxyl group of Tyr-38 is 1.84 nm away from the active site. Model-building studies using computer graphics show that this distance can be accommodated when FSO2BzAdo binds in an extended conformation with the sulfonylbenzoyl portion in an orientation different from the nicotin-amide ring of NADPH.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1988

Active site-directed irreversible inhibition of glutathione S-transferases by the glutathione conjugate of tetrachloro-1,4-benzoquinone.

Purified glutathione S-transferase from rat liver cytosol are irreversibly inhibited by the glutathione conjugate of tetrachloro-1,4-benzoquinone, 2-S-glutathionyl-3,5,6-trichloro-1,4-benzoquinone. The inhibition is due to covalent binding in or near the active site, resulting in modification of a single amino acid residue/subunit, presumably a cysteine residue. The amount of inhibition is related to the molar ratio of the inhibitor and the enzyme and is independent of the enzyme concentration. A 70-80% inhibition is obtained on incubating the enzyme with a 5-fold molar excess of the conjugate. Complete 100% inhibition is never reached. The derivative bound to the enzyme still possesses a quinone structure and is able to react with thiol-containing compounds. Reduction of the enzyme-bound quinone abolishes its reactivity but does not decrease the inhibition. At 0 degrees C, the glutathione conjugate of tetrachloro-1,4-benzoquinone inhibits the glutathione S-transferases at a much higher rate than the corresponding beta-mercaptoethanol conjugate, indicating a distinct targetting effect of the glutathione moiety. However, the parent compound, tetrachloro-1,4-benzoquinone, also has a considerable affinity for the enzymes. Although it does not react as fast as the glutathione conjugate, it reacts with the same amino acid residue. Protection from inhibition by the substrate analog S-hexylglutathione also indicates an active site-directed modification. Small but significant differences exist between the different rat liver transferase isoenzymes; using a 20-fold molar excess the inhibition ranges from 78 to 98% for the conjugate, and from 72 to 93% for the quinone, with isoenzyme 1-1 being the most and isoenzyme 2-2 the least inhibited forms.

Animals↗
PubMed1988

Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The oxidation-reduction potential of p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and Müller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1988

Differential induction of rat hepatic glutathione S-transferase isoenzymes by hexachlorobenzene and benzyl isothiocyanate. Comparison with induction by phenobarbital and 3-methylcholanthrene.

Male Wistar rats were treated with hexachlorobenzene, benzyl isothiocyanate, phenobarbital or 3-methylcholanthrene. Hepatic cytosolic glutathione S-transferase (GST) activity was determined with the substrates 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, ethacrynic acid and trans-4-phenyl-3-buten-2-one. Cytosolic glutathione peroxidase activity was measured with cumene hydroperoxide. GST activity toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene and ethacrynic acid was enhanced by all compounds, hexachlorobenzene and 3-methylcholanthrene causing the largest and the smallest increase respectively. Trans-4-phenyl-3-buten-2-one-conjugating activity exhibited only small changes, while peroxidase activity with cumeme hydroperoxide was not changed by any of the inducing agents. GST isoenzymes were purified on S-hexylglutathione Sepharose 6B and separated by means of FPLC-chromatofocusing, to evaluate effects on the GST isoenzyme pattern. Hexachlorobenzene and phenobarbital both caused an increase in the relative amounts of subunits 1 and 3 when compared with subunits 2 and 4 respectively. For 3-methylcholanthrene only induction of subunit 1 was observed, possibly due to the relatively low induction levels of total GST activity. In benzyl isothiocyanate-treated animals, an induction of subunit 3 was found as well as an increase in the relative amount of subunit 2. Thus, benzyl isothiocyanate behaves differently from hexachlorobenzene, phenobarbital and 3-methylcholanthrene as an inducing agent of rat hepatic glutathione S-transferases.

Animals↗
PubMed1988

Murine genomic DNA sequences replicating autonomously in mouse L cells.

Plasmids that replicate autonomously in mouse L cells were constructed by inserting random genomic DNA fragments from Ltk- cells into a plasmid containing the HSV-1 thymidine kinase gene with a truncated low-efficiency promoter. HAT resistance was used as a selective marker. The presence of free plasmids in the DNA of transformants was demonstrated by hybridization with a specific plasmid probe, by electron microscopic visualization of circular DNA, and by recovering these plasmids by E. coli transformation. Nineteen different DNA fragments were isolated. They were characterized as murine autonomously replicating sequences by Mbol restriction endonuclease sensitivity, by bromodeoxyuridine substitution, by copy number determination, and by segregation analysis. Sequence analysis of the inserts of nine plasmids revealed a conserved element of 12 bp (CTCATGAGAGGCCAA) in five out of nine autonomously replicating sequences.

Acetyltransferases↗
PubMed1988

Computer ranking of the sequence of appearance of 40 features of the brain and related structures in staged human embryos during the seventh week of development.

The sequence of events in the development of the brain in human embryos, already published for stages 8-17, is here continued for stages 18 and 19. With the aid of a computerized bubble-sort algorithm, 58 individual embryos were ranked in ascending order of the features present. The increasing structural complexity provided 40 new features in these two stages. The chief characteristics of stage 18 (approximately 44 postovulatory days) are rapidly growing basal nuclei; appearance of the extraventricular bulge of the cerebellum (flocculus), of the superior cerebellar peduncle, and of follicles in the epiphysis cerebri; and the presence of vomeronasal organ and ganglion, of the bucconasal membrane, and of isolated semicircular ducts. The main features of stage 19 (approximately 48 days) are the cochlear nuclei, the ganglion of the nervus terminalis, nuclei of the prosencephalic septum, the appearance of the subcommissural organ, the presence of villi in the choroid plexuses of the fourth and lateral ventricles, and the stria medullaris thalami.

Brain↗
PubMed1988

The development of the human brain, including the longitudinal zoning in the diencephalon at stage 15.

Twenty-six embryos (6-11 mm) of stage 15 (approximately 33 days) were studied in detail and graphic reconstructions of three of them were prepared. Characteristic features of this stage include closed lens vesicles, presence of nasal pits, and retinal pigment. The neuromeric pattern is still visible. Each cerebral hemisphere is limited by the torus hemisphericus internally and by the di-telencephalic sulcus externally. The medial (diencephalic) eminence of the basal nuclei (previously misinterpreted by others as the lateral) had appeared in stage 14, and the lateral eminence, which is telencephalic, is now distinguishable. The amygdaloid body in stages 14 and 15 is derived from the medial eminence. The hippocampal thickening is identifiable in the dorsomedial part of the cerebral hemisphere. Medial and basal forebrain bundles are developing. The olfactory eminence is visible. Future olfactory bulb and tubercle possess an intermediate layer. The wall of the diencephalon presents five longitudinal zones: epithalamus, dorsal thalamus, ventral thalamus, subthalamus, and hypothalamus. The primordium of the epiphysis cerebri is beginning in the more advanced embryos. The sulcus limitans ends rostrally at the midbrain (M1) and is not continuous with the hypothalamic sulcus. Hence the alar/basal distinction does not arise in the forebrain. In the roof of the midbrain (M2) the mesencephalic evagination already noticed at stage 14 is characteristic. It is suggested that it may function as a temporary circumventricular organ. The precursors of some new tracts are identifiable: habenulo-interpeduncular, medial tectobulbar, and mamillotegmental fibres. Commissures include the supramamillary, that of the superior colliculi, and (in some embryos) the first fibres of the posterior commissure. Nuclei include the habenular, mamillary, and probably subthalamic. The cerebellum, the beginning of which was already noted at stages 13 and 14, consists of (1) a rostral part that arises from the alar plate of the isthmic segment and will form the superior medullary velum and part of the corpus cerebelli; and (2) a caudal part that develops from rhombomere 1. The involvement of the isthmic segment, first elucidated with stage 14, has not been observed in previous reports. All cranial nerves except the olfactory and optic are present in the more advanced embryos.

Brain↗
PubMed1988

The first appearance of the future cerebral hemispheres in the human embryo at stage 14.

Thirty-five embryos of stage 14 (32 days) were studied in detail and graphic reconstructions of four of them were prepared. Characteristic features of this stage include the beginning formation of the future cerebral hemispheres and the cerebellar plates. The ventral boundary between telencephalon medium and diencephalon is the preoptic recess. Although a velum transversum is not yet distinguishable as a dorsal boundary, its site is indicated by a change in the thickness of the roof of the forebrain. As the cerebral vesicles (future hemispheres) begin to evaginate, a di-telencephalic sulcus and a corresponding lateral ventricle and ventricular ridge (torus hemisphericus) develop. The telencephalic wall is mainly ventricular layer but three areas show advanced differentiation: olfactory area, future amygdaloid body (which lies at first mainly in the diencephalon), and primordium of the hippocampus. The telencephalon is growing in length, and the forebrain now occupies almost one quarter of the total length of the brain. The two neuromeres of the diencephalon are no longer as clearly delineated. The floor of D1 presents a thickened chiasmatic plate; that of D2 includes the infundibulum, which is closely related to the adenohypophysial pouch. The ventricular surface of D1 presents elevations for the dorsal and ventral thalami, separated by the sulcus medius. Other features of the diencephalon include the ventricular eminence (medial ventricular ridge) of the basal nuclei and the hypothalamic cell cord, from which the preopticohypothalamotegmental tract arises. The roof of D2 contains the evaginating part of the synencephalon. The mesencephalic angle continues to diminish. Two neuromeres, M1 and M2, are still distinguishable. The oculomotor nucleus emits nerve fibres, as does also the trochlear nucleus, which lies in the isthmic segment. Some extracerebral oculomotor fibres are present, but decussating and extracerebral trochlear fibres have not yet appeared. In the region of the tectum, two nuclei are discernible, and will form the medial tectobulbar tract and the mesencephalic root of the trigeminal nerve, respectively. The medial longitudinal fasciculus is present. A "median ventricular formation" is sometimes found in the mesencephalic roof. The cerebellum is the widest part of the brain. Two neuromeres (isthmic segment and Rh1) are involved in its formation. Most of the cerebellar plate has differentiated an intermediate layer, and the future rhombic lip is discernible. Indications of an efferent fibre system are present. In addition to the cerebellum, the rhombencephalon includes Rh1 to Rh7, and RhD.(ABSTRACT TRUNCATED AT 400 WORDS)

Amygdala↗
PubMed1988

Intrathecal synthesis of specific IgG in syphilitic patients with human immunodeficiency virus 1 infection.

Intrathecal synthesis of immunoglobulin G (IgG) specific to Treponema pallidum, ssp. pallidum, or to the human immunodeficiency virus 1 (HIV-1) was investigated in patients with contemporary treponemal and HIV-1 infections. Using a T. pallidum and an HIV-1 IgG enzyme-linked immunosorbent assay, specific antibody units per milligram of total IgG were estimated in serum and cerebrospinal fluid (CSF) of 13 patients infected with both agents. Synthesis of anti-treponemal IgG in the central nervous system (CNS) was shown in three and of anti-HIV-1 IgG in nine patients with neurological disorders. It is concluded that a differentiation of local IgG immune response in the CNS to one or both infectious agents is possible.

AIDS-Related Complex↗
PubMed1988

The development of the human brain from a closed neural tube at stage 13.

Twenty-five embryos of stage 13 (28 days) were studied in detail and graphic reconstructions of seven of them were prepared. Thirty or more somitic pairs are present, and the maximum is possibly 39. The notochord is almost entirely separated from the neural tube and the alimentary epithelium, and its rostral tip is closely related to the adenohypophysial pocket. Caudal to the cloacal membrane, the caudal eminence is the site of secondary neurulation. The eminence, which usually contains isolated somites, is the area where new notochord, hindgut, and neural tube are forming. The neural cord develops into neural tube without the intermediate phase of a neural plate (secondary neurulation). Canalization is regular and the lumen is continuous with the central canal. The neural tube is now a closed system, filled with what may be termed "ependymal fluid." The brain is widening in a dorsoventral direction. Neuromeres are still detectable. The following features are distinguishable: infundibular area of D2, chiasmatic plate of D1, "adult" lamina terminalis, and commissural plate (at levels of nasal plates). The beginning of the synencephalon of D2 can be discerned. The retinal and lens discs are being defined. The mesencephalic flexure continues to diminish. The midbrain possesses a sulcus limitans, and the tegmentum may show the medial longitudinal fasciculus. The isthmic segment is clearly separated from rhombomere 1. Lateral and ventral longitudinal fasciculi are usually present in the hindbrain, and the common afferent tract is beginning. Somatic and visceral efferent fibres are seen in certain nerves: 6, 12; 5, 7, 9-11. The first indication of the cerebellum may be visible in the alar lamina of rhombomere 1. The terminal-vomeronasal crest appears. Various cranial ganglia (e.g., vestibular, superior ganglia of 9, 10) are forming. The trigeminal ganglion may show its three major divisions. Epipharyngeal placodes of pharyngeal arches 2 to 5 contribute to cranial ganglia 7, 9, and 10. The spinal neural crest is becoming segregated, and the spinal ganglia are in series with the somites. Ventral spinal roots are beginning to develop.

Brain↗
PubMed1988

The oxidation of tetrachloro-1,4-hydroquinone by microsomes and purified cytochrome P-450b. Implications for covalent binding to protein and involvement of reactive oxygen species.

The enzymatic oxidation of tetrachloro-1,4-hydroquinone (1,4-TCHQ), resulting in covalent binding to protein of tetrachloro-1,4-benzoquinone (1,4-TCBQ), was investigated, with special attention to the involvement of cytochrome P-450 and reactive oxygen species. 1,4-TCBQ itself reacted very rapidly and extensively with protein (58% of the 10 nmol added to 2 mg of protein, in a 5-min incubation). Ascorbic acid and glutathione prevented covalent binding of 1,4-TCBQ to protein, both when added directly and when formed from 1,4-TCHQ by microsomes. In microsomal incubations as well as in a reconstituted system containing purified cytochrome P-450b, 1,4-TCHQ oxidation and subsequent protein binding was shown to be completely dependent on NADPH. The reaction was to a large extent, but not completely, dependent on oxygen (83% decrease in binding under anaerobic conditions). Inhibition of cytochrome P-450 by metyrapone, which is also known to block the P-450-mediated formation of reactive oxygen species, gave a 80% decrease in binding, while the addition of superoxide dismutase prevented 75% of the covalent binding, almost the same amount as found in anerobic incubations. A large part of the conversion of 1,4-TCHQ to 1,4-TCBQ is apparently not catalyzed by cytochrome P-450 itself, but is mediated by superoxide anion formed by this enzyme. The involvement of this radical anion is also demonstrated by microsomal incubations without NADPH but including the xantine/xantine oxidase superoxide anion generating system. These incubations resulted in a 1.6-fold binding as compared to the binding in incubations with NADPH but without xantine/xantine oxidase. 1,4-TCHQ was shown to stimulate the oxidase activity of microsomal cytochrome P-450. It is thus not unlikely that 1,4-TCHQ enhances its own microsomal oxidation.

Animals↗
PubMed1988

Immunoglobulin M and G antibody response in rabbits after experimental Haemophilus ducreyi infection.

The enzyme-linked immunosorbent assay and immunoblot technique were used to investigate the humoral immune response following intradermal infection of rabbits with Haemophilus ducreyi. Basically, the kinetics of IgM and IgG antibody synthesis was the same as in other bacterial infections. However, antibody titres in the enzyme-linked immunosorbent assay were remarkably low. In the immunoblot experiments, antibodies of both immunoglobulin classes were mainly directed against Haemophilus ducreyi antigens with relative molecular masses of 79,000, 62,000, 55,000, 49,000, and 26,000, suggesting that these antigens may play a dominant role in the humoral immune response after infection.

Animals↗
PubMed1988

Development of hypothalamic obesity in growing rats.

Administration of monosodium glutamate to neonate rats causes hypothalamic lesions in the region of the nucleus arcuatus and the eminentia mediana, followed by massive accumulation of triglycerides, diminished secretion of growth hormone, reduced body length and organ weights and diminished number of adipocytes (hypoplastic-hypertrophic obesity). Locomotor activity of obese animals is reduced by about 50%. Food intake is increased by about 10% during growth and development of obesity but decreased beneath the level of that in control animals in the stationary phase of obesity. Hyperinsulinemia coupled with insulin resistance develops in the stationary phase of obesity, i.e. when adipocyte diameter has reached approximately 100 microns. The effects of reduced secretion of growth hormone are considered to be a main factor of fat accumulation in this type of obesity.

Adipose Tissue↗
PubMed1988

Angiotensin II controls angiotensinogen secretion at a pretranslational level.

It has been proposed that feedback by angiotensin II, the effector peptide of the renin-angiotensin system stimulates hepatic angiotensinogen synthesis, since long-term infusion of this octapeptide in vivo induced an increase in plasma angiotensinogen concentrations. In the present study, the effects of angiotensin II (9 and 90 nmol/l) on angiotensinogen messenger (m)RNA concentrations and on angiotensinogen secretion of freshly isolated rat hepatocytes were compared with those of glucocorticoids (hydrocortisone, 10(-4) mol/l, and dexamethasone, 10(-5) mol/l). Angiotensin II and the glucocorticoids elevated angiotensinogen mRNA concentrations two- to threefold. Angiotensinogen secretion rates were correspondingly increased with a time lag of about 2 h. Differences in the time-course of changes in mRNA following onset or decay of the hormonal effect suggest that angiotensin II and glucocorticoids express their effects by different intracellular mechanisms. This view is supported by the observation that angiotensin II but not dexamethasone has a stabilizing effect on angiotensinogen mRNA, when further synthesis was blocked by actinomycin D.

Angiotensin II↗
PubMed1988

Pharmacological modulation of the rod pathway in the cat retina.

1. In the intact cat eye, the responses of ganglion cells to light stimulation were recorded extracellularly and the actions of iontophoretically applied 2-amino-4-phosphonobutyrate (APB), a potent agonist at ON-bipolars, and of strychnine, a glycine antagonist, were investigated. 2. Under light-adapted conditions, the activity of ON-center ganglion cells is decreased by APB but is increased by strychnine. APB and strychnine act independently of one another. 3. The activity of light-adapted OFF-center ganglion cells is increased by APB and by strychnine. The light response remains clearly modulated. Strychnine blocks the action of simultaneously applied APB. The results are in agreement with the action of a push-pull mechanism, according to which ON-cone-bipolars provide a glycinergic input into OFF-center ganglion cells. 4. Under dark-adapted conditions, APB blocks the light responses of both ON-center and OFF-center ganglion cells. The discharge rate of ON-center ganglion cells is completely suppressed; OFF-center ganglion cells show a high maintained discharge. 5. Strychnine blocks the scotopic light response of OFF-center ganglion cells and blocks the action of simultaneously applied APB. The light response of ON-center ganglion cells is hardly affected by strychnine. 6. The effects of strychnine on OFF-center ganglion cells are in agreement with the hypothesis that the glycinergic AII amacrine cells modulate the activity of the scotopic OFF-channel. 7. Intravitreally applied APB abolished the scotopic b-wave of the electroretinogram at concentrations of 100 microM. 8. Our data suggest that as in rabbit (10) the rod bipolars in cat retina are depolarizing (ON) bipolar cells.

Aminobutyrates↗