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Biomedical subjects

H Kühn

Publications and source records attributed to H Kühn.

At least 145 records · Page 8Linked to original sources
PubMed1988

Resolution of enantiomers of hydroxyeicosatetraenoate derivatives by chiral phase high-pressure liquid chromatography.

A new method was developed for analyzing the steric configuration of hydroxyeicosatetraenoates (HETEs) and other hydroxy fatty acids. Racemic HETE methyl esters were reacted with either benzoyl or naphthoyl chloride in pyridine and the resulting aromatic ester derivatives purified by reversed phase HPLC and subsequently chromatographed on a chiral stationary phase HPLC column [(R)-(-)-N-3,5-dinitrobenzoyl-alpha-phenylglycine)]. In contrast to the enantiomers of the underivatized HETE methyl esters which were only partially resolved, the enantiomers of their aromatic ester derivatives were completely separable on this chiral phase. Chiral HETEs can be retrieved from the aromatic derivatives by alkaline hydrolysis. Thus, this method has both analytical and preparative applications.

Chromatography, High Pressure Liquid↗
PubMed1988

The biological dynamics of lipoxygenase in rabbit red cells in the course of an experimental bleeding anaemia. Unexpected effects of the calcium ionophore A 23187.

The lipoxygenase activity of red cell lysates with linoleic acid as substrate, the concentration of immunologically detectable lipoxygenase protein as well as the metabolization of external [1-14C]arachidonic or -linoleic acid by intact cells were determined during bleeding anaemia and the recovery period of rabbits. All three criteria behaved in a parallel manner. Before bleeding no lipoxygenase was detectable. After the third day of strong bleeding high amounts and activities of lipoxygenase appeared in parallel to the appearance of megaloreticulocytes. As few as about a million of cells were sufficient to detect the utilization of [1-14C]polyenoic fatty acids via the lipoxygenase pathway in intact cells in the absence of ionophore A 23187. After discontinuation of strong bleeding the amount and activity of the lipoxygenase declined gradually but persisted for 2-3 months corresponding to the presumed life-span of red cells. Several types of evidence indicate the identity of the lipoxygenase of red cells during the recovery period with that of reticulocytes during strong bleeding: (1) comparable specific activity, (2) Western blot analysis, (3) identical cellular products from [1-14C]-linoleic and -arachidonic acid which were identified by means of HPLC analysis to be 13 S-hydroxyoctadecadienoic acid (13 S-HODE) and 15 S-hydroxyeicosatetraenoic acid (15 S-HETE), respectively. Use of large amounts of cells during the recovery period (5.10(9) cells) led to an apparent masking of the polyenoic fatty acid added. Haemolysis of the cells or addition of calcium and ionophore A 23187 abolished or reduced this masking. Both masking and influence of haemolysis or ionophore disappeared at sufficiently low concentrations of cells. Under these conditions the rate of the formation of lipoxygenase products in intact cells corresponded to the lipoxygenase activity measured in membrane-free cell lysates. In red cells during the recovery period but not during strong bleeding, calcium and ionophore A 23187 stimulated the secondary conversion of 15 S-HETE to more polar products after arachidonic acid had been exhausted. The implications of the results on the performance and the interpretation of the effects of the calcium ionophore A 23187 on cellular arachidonic acid metabolism are discussed.

Anemia↗
PubMed1988

[180 degree versus 360 degree rotation in SPECT of bone].

Opinions differ as to the advantages of the 180 degrees and the 360 degrees techniques concerning myocardial scintigraphy with 201Tl. Following our own encouraging experiences with the 180 degrees technique, we have looked for cases in bone scintigraphy where reconstructions from a semicircle could be advantageous over a full circle reconstruction. There were metastases and foci of osteomyelitis in spine, ribs and skull (especially in the skull base), to be seen better in reconstructions using images out of 180 degrees close to the object. In knee SPECT a closer radius of rotation can be chosen with single-head cameras so that, when using the 180 degrees technique, a better reproduction of the anatomical structures and pathological alterations is achieved.

Adult↗
PubMed1987

Formation of lipoxin B by the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate.

The pure reticulocyte lipoxygenase converts 15LS-hydroxy-5,8,11,13(Z,Z,Z,E)-icosatetraenoic acid (15LS-HETE) methyl ester to a complex mixture of products containing 5DS,14LR,15LS-trihydro(pero)xy-6E,++ +8Z,10E,12E-icosatetraenoate methyl ester (lipoxin B methyl ester), 5DS,15LS-DiH(P)ETE methyl ester and four 8,15LS-DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy-icosatetraenoic acid]. After a short incubation period (15 min) 5DS,15LS-DiH(P)ETE methyl ester was found to be the main product, whereas after a 3-h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400-MHz 1H-NMR. Atmospheric oxygen was incorporated at carbon-5 and carbon-14 into the major product. 5DS,15LS-DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15LS-HETE and 5DS,15LS-DiHETE. The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.

Animals↗
PubMed1987

Analysis of the stereochemistry of lipoxygenase-derived hydroxypolyenoic fatty acids by means of chiral phase high-pressure liquid chromatography.

A chiral phase HPLC method was developed for the simultaneous determination of the positional and optical isomers of the lipoxygenase-derived hydroxypolyenoic fatty acids. With a Bakerbond chiral phase HPLC column (dinitrobenzoyl phenylglycine as chiral phase) the positional and optical isomers of the reduced dioxygenation products (by triphenylphosphine or borohydride) of linoleic acid and arachidonic acid were separated after methylation of the carboxylic groups. No cumbersome chemical derivatization such as conversion to a diastereomer was necessary. As compared with the methods used up till now chiral phase HPLC proved to be simpler and more sensitive. About 10 pmol of hydroxy fatty acids suffice for an analysis. The chiral phase HPLC can be used for the preparative separation of the optical antipodes of the lipoxygenase products. An optical purity of more than 90% can be reached in one preparative run. The method was applied to the determination of the stereochemistry of the dioxygenation products of polyenoic fatty acids formed by the lipoxygenases from soybeans, reticulocytes, pea seeds (isoenzyme I and II), tomato fruits, by the quasilipoxygenase activity of hemoglobin, and by the methylene blue-mediated photooxidation of arachidonic acid.

Animals↗
PubMed1987

Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein).

The beta-adrenergic receptor kinase is an enzyme, possibly analogous to rhodopsin kinase, that multiply phosphorylates the beta-adrenergic receptor only when it is occupied by stimulatory agonists. Since this kinase may play an important role in mediating the process of homologous, or agonist-specific, desensitization, we investigated the functional consequences of receptor phosphorylation by the kinase and possible analogies with the mechanism of action of rhodopsin kinase. Pure hamster lung beta 2-adrenergic receptor, reconstituted in phospholipid vesicles, was assessed for its ability to mediate agonist-promoted stimulation of the GTPase activity of coreconstituted stimulatory guanine nucleotide-binding regulatory protein. When the receptor was phosphorylated by partially (approximately 350-fold) purified preparations of beta-adrenergic receptor kinase, as much as 80% inactivation of its functional activity was observed. However, the use of more highly purified enzyme preparations led to a dramatic decrease in the ability of phosphorylation to inactivate the receptor such that pure enzyme preparations (approximately 20,000-fold purified) caused only minimal (approximately 1off/- 7%) inactivation. Addition of pure retinal arrestin (48-kDa protein or S antigen), which is involved in enhancing the inactivating effect of rhodopsin phosphorylation by rhodopsin kinase, led to partial restoration of the functional effect of beta-adrenergic receptor kinase-promoted phosphorylation (41 +/- 3% inactivation). These results suggest the possibility that a protein analogous to retinal arrestin may exist in other tissues and function in concert with beta-adrenergic receptor kinase to regulate the activity of adenylate cyclase-coupled receptors.

Animals↗
PubMed1987

Deactivation of photoactivated rhodopsin by rhodopsin-kinase and arrestin.

Photoactivated rhodopsin (R) catalyses, by repetitively interacting with many copies of a guanosine nucleotide binding protein (transducin), the amplified binding of GTP to transducin molecules which then activate cyclic GMP phosphodiesterase. Electrophysiologists recently have shown that cyclic GMP keeps ion channels in the plasma membrane of the rod outer segment open in darkness, and that light-induced hydrolysis of cyclic GMP leads to closure of the channels and therefore to hyperpolarization of the rod cell. Photoactivated rhodopsin interacts not only with transducin, but with two more proteins: a protein kinase that specifically phosphorylates R (in contrast to dark-adapted rhodopsin) at multiple sites; and an abundant soluble protein of 48 KDal (called 48 K-protein, S-antigen, or arrestin) that specifically binds to phosphorylated R. Phosphorylation partially suppresses the ability of R to catalyze transducin-mediated phosphodiesterase activation even in the absence of arrestin. Binding of arrestin to the phosphorylated R potentiates this inhibitory effect, most probably because arrestin competes with transducin for binding on the phosphorylated R. Phosphorylation, in conjunction with arrestin binding, therefore appears to be a mechanism that terminates the active state of the receptor, R.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1986

Purification and properties of guanylate kinase from bovine retinas and rod outer segments.

The presence of three soluble nucleotide phosphotransferases in bovine rod outer segments was demonstrated: guanylate kinase (EC 2.7.4.8), nucleoside-diphosphate kinase (EC 2.7.4.6) and adenylate kinase (EC 2.7.4.3). The enzyme guanylate kinase, which catalyzes the reaction GMP + ATP in equilibrium GDP + ADP, was purified to homogeneity from isolated bovine rod outer segments as well as from bovine retinas. The enzyme preparations obtained from both sources are identical in their chromatographic properties, molecular mass (20-23 kDa for both native enzyme and dodecylsulfate-denatured polypeptide), Km values (13 microM for GMP and 430 microM for ATP), specific activities, and nucleotide specificities. The enzyme's turnover number was estimated to be 130 s-1. The minimum amount of enzyme found in rod outer segments is about 1 copy per 800 rhodopsin molecules. The role of the enzyme in the cyclic GMP cycle in rod outer segments is discussed.

Adenosine Triphosphate↗
PubMed1986

Formation of lipoxin B by the pure reticulocyte lipoxygenase.

The pure reticulocyte lipoxygenase converts 5,15-DiHETE via a lipoxygenase reaction to 5,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid (a lipoxin B isomer) as shown by GC/MS analysis of its trimethylsilyl ether. With arachidonic acid, 15-HETE and 15-HETE methyl ester this lipoxin B isomer was also formed. The results presented here indicate that pure mammalian lipoxygenases are able to form lipoxins via sequential multiple oxygenation of arachidonic acid or its hydroxy derivatives.

Gas Chromatography-Mass Spectrometry↗
PubMed1986

Rapid affinity purification of retinal arrestin (48 kDa protein) via its light-dependent binding to phosphorylated rhodopsin.

Arrestin (also named '48 kDa protein' or 'S-antigen') is a soluble protein involved in controlling light-dependent cGMP phosphodiesterase activity in retinal rods, and is also known for its ability to induce autoimmune uveitis of the eye. We report a rapid and simple purification method based on the property of arrestin to bind specifically and reversibly to illuminated and phosphorylated rhodopsin [(1984) FEBS Lett. 176, 473-478]. This method does not require column chromatography and yields about 2-4 mg purified arrestin from 15 bovine retinas. Pure arrestin can be resolved by isoelectric focusing into at least 10 distinct bands, all of which stain with a monoclonal antibody specific for S-antigen.

Animals↗
PubMed1986

Requirement of monohydroperoxy fatty acids for the oxygenation of 15LS-HETE by reticulocyte lipoxygenase.

The lipoxygenase from reticulocytes oxygenates 15LS-HETE to 8-hydroperoxy-15-hydroxy-5,9,11,13-eicosatetraenoic acid and 5-hydroperoxy-15-hydroxy-6,8,11,13-eicosatetraenoic acid only in the presence of catalytic concentrations of monohydroperoxy fatty acids. During this reaction the hydroperoxy fatty acids are converted to more polar products including hydroxy fatty acids. From kinetic measurements of 15LS-HETE oxygenation it was calculated that 1 mol monohydroperoxy fatty acid is consumed during the oxygenation of about 9 mol 15LS-HETE.

Fatty Acids↗