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Biomedical subjects

H Scheer

Publications and source records attributed to H Scheer.

At least 55 records · Page 3Linked to original sources
PubMed1993

Derivation of the formyl-group oxygen of chlorophyll b from molecular oxygen in greening leaves of a higher plant (Zea mays).

Using mass spectroscopy, we demonstrate as much as 93% enrichment of the 7-formyl group oxygen of chlorophyll b when dark-grown, etiolated maize leaves are greened under white light in the presence of 18O2. This suggests that a mono-oxygenase is involved in the oxidation of its methyl group precursor. The concomitant enrichment of about 75% of the 13(1)-oxygen confirms the well-documented finding that this oxo group, in both chlorophyll a and b, also arises from O2. High 18O enrichment into the 7-formyl oxygen relative to the substrate 18O2 was achieved by optimization of the greening conditions in combination with a reductive extraction procedure. It indicates not only a single pathway for Chl b formyl group formation, but also unequivocally demonstrates that molecular oxygen is the sole precursor of the 7-formyl oxygen.

Chlorophyll↗
PubMed1992

ATP-induced Ca2+ influx is regulated via a pertussis toxin-sensitive mechanism in a PC12 cell clone.

A PC12 cell clone that responds to ATP with polyphosphoinositide hydrolysis and with a marked, biphasic intracellular free Ca2+ concentration ([Ca2+]i) response (composed by release from intracellular stores accompanied by stimulated influx from the medium), was pretreated with pertussis toxin. In the pretreated cells the responses induced by ATP were differently modified. Polyphosphoinositide hydrolysis and Ca2+ release were moderately inhibited whereas Ca2+ influx was enhanced. Pharmacological experiments revealed the influx enhancement to be sustained by neither voltage-gated nor second messenger-operated Ca2+ channels. Rather, a channel of the receptor-operated type activated by ATP (P2w receptor) appears to work under the negative control of a pertussis toxin-sensitive G protein, acting presumably by direct interaction with the channel in the plane of the plasma membrane.

Adenosine Triphosphate↗
PubMed1992

Primary electron transfer kinetics in bacterial reaction centers with modified bacteriochlorophylls at the monomeric sites BA,B.

The primary electron transfer has been investigated by femtosecond time-resolved absorption spectroscopy in two chemically modified reaction centers (RC) of Rhodobacter sphaeroides, in which the monomeric bacteriochlorophylls BA and BB have both been exchanged by 13(2)-hydroxybacteriochlorophyll a or [3-vinyl]-13(2)-hydroxybacteriochlorophyll a. The kinetics of the primary electron transfer are not influenced by the 13(2)-hydroxy modification. In RCs containing [3-vinyl]-13(2)-hydroxybacteriochlorophyll a the primary rate is reduced by a factor of 10.

Bacteriochlorophylls↗
PubMed1992

The functional role of protein in the organization of bacteriochlorophyll c in chlorosomes of Chloroflexus aurantiacus.

The preparation of five different fractions containing bacteriochlorophyll (Bchl) c and their absorption and circular dichroic properties have been described. The fractions investigated were purified chlorosomes, proteolytically modified chlorosomes, chlorosomes treated with lithium dodecyl sulfate (LDS) which were subsequently subjected to size-exclusion chromatography, in vitro Bchl c aggregates and, additionally, the so-called GEF chlorosomes [prepared according to Griebenow and Holzwarth (1989) Biochim. Biophys. Acta 973, 235-240]. Proteolysis of chlorosomes caused a 35-40% decrease in absorption intensity, a 6-8 nm blue shift of the 740-nm peak and, in particular, a drastic increase of rotational strength as revealed by CD spectroscopy. Although oligomeric Bchl c aggregates and LDS-treated chlorosomes had absorption characteristics similar to Bchl c in vivo, the data clearly indicated that protein, perhaps the chlorosome-specific Mr-3700 polypeptide, was involved in the organization of Bchl c in chlorosomes from C. aurantiacus. Furthermore, the results showed that the LDS-treated chlorosome fraction was most likely comprised of a micellar complex of Bchl c with LDS which represented an entity entirely different from chlorosomes.

Bacteria↗
PubMed1992

Receptor-activated Ca2+ influx. Two independently regulated mechanisms of influx stimulation coexist in neurosecretory PC12 cells.

Receptor-activated Ca2+ influx was investigated in PC12 cells clones loaded with fura-2. Cells were stimulated in a Ca(2+)-free medium and studied after reintroduction of the cation or addition of Mn2+ into the medium. A first influx component, independent of receptor activation and sustained by depletion of the intracellular inositol 1,4,5-trisphosphate sensitive Ca2+ store (store-dependent Ca2+ influx, SDCI), was identified by experiments with carbachol followed by atropine and with agents that induce store discharge without polyphosphoinositide hydrolysis: thapsigargin, an inhibitor of Ca(2+)-ATPase activity; ryanodine and caffeine, activators of the ryanodine receptor. A second component of Ca2+ influx, induced by carbachol and rapidly blocked by atropine, relies on receptor-effector coupling via G protein(s) different from that (those) involved in phospholipase C activation. SDCI and receptor-coupled influx are similar in their voltage dependence and insensitivity to forskolin and phorbol esters but they differ with respect to their Mn2+ permeability and their sensitivity to the SC 38249 imidazole blocker. The two components might play different roles. SDCI might act as a safety device to prevent Ca2+ store depletion whereas receptor-dependent influx might control physiological functions such as secretion and growth.

Animals↗
PubMed1990

Infrared spectroscopy of phytochrome and model pigments.

Fourier-transform infrared difference spectra between the red-absorbing and far-red-absorbing forms of oat phytochrome have been measured in H2O and 2H2O. The difference spectra are compared with infrared spectra of model compounds, i.e. the (5Z,10Z,15Z)- and (5Z,10Z,15E)-isomers of 2,3,7,8,12,13,17,18-octaethyl-bilindion (Et8-bilindion), 2,3-dihydro-2,3,7,8,12,13,17,18-octaethyl-bilindion (H2Et8-bilindion), and protonated H2Et8-bilindion in various solvents. The spectra of the model compounds show that only for the protonated forms can clear differences between the two isomers be detected. Since considerable differences are present between the spectra of Et8-bilindion and H2Et8-bilindion, it is concluded that only the latter compound can serve as a model system of phytochrome. The 2H2O effect on the difference spectrum of phytochrome supports the view that the chromophore in red-absorbing phytochrome is protonated and suggests, in addition, that it is also protonated in far-red-absorbing phytochrome. The spectra show that protonated carboxyl groups are influenced. The small amplitudes in the difference spectra exclude major changes of protein secondary structure.

Fourier Analysis↗
PubMed1990

Modified reaction centers from Rhodobacter sphaeroides R26. 2: Bacteriochlorophylls with modified C-3 substituents at sites BA and BB.

Monomeric bacteriochlorophylls BA and BB in photosynthetic reaction centers from Rhodobacter sphaeroides R26 were exchanged with (13(2)-hydroxy-)bacteriochlorophylls containing a 3-vinyl- or 3-(alpha-hydroxyethyl)-substituent instead of the 3-acetyl group. The corresponding binding sites must be tolerant to the introduction of the polar residue at C-13(2) and modifications of the 3-acetyl group. According to HPLC analysis, the exchange with both pigments amounts to less than or equal to 50% of the total BChl contained in the complex, corresponding to less than or equal to 100% of the monomeric BChl alpha BA,B. The absorption spectra show significant changes in the QX and QY-region of the monomeric bacteriochlorophylls. By contrast, the absorption of the primary donor (P870) and reversible photobleaching is retained. The circular dichroism is also unchanged in the 870 nm region. The positive cd band located at around 800 nm in native reaction centers, shifts with the (blue-shifted) QY absorption(s) of BA and/or BB, whereas the position of the negative one remains nearly unaffected. The data indicate that the latter is the upper excitonic band of the primary donor, and that there is little interaction of the monomeric BA/BB with the primary donor.

Bacteriochlorophylls↗
PubMed1990

Initial electron-transfer in the reaction center from Rhodobacter sphaeroides.

The initial electron transfer steps in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides have been investigated by femtosecond time-resolved spectroscopy. The experimental data taken at various wavelengths demonstrate the existence of at least four intermediate states within the first nanosecond. The difference spectra of the intermediates and transient photodichroism data are fully consistent with a sequential four-step model of the primary electron transfer: Light absorption by the special pair P leads to the state P*. From the excited primary donor P*, the electron is transferred within 3.5 +/- 0.4 ps to the accessory bacteriochlorophyll B. State P+B- decays with a time constant of 0.9 +/- 0.3 ps passing the electron to the bacteriopheophytin H. Finally, the electron is transferred from H- to the quinone QA within 220 +/- 40 ps.

Journal Article↗
PubMed1990

Fourier-transform Raman spectroscopy applied to photobiological systems.

Fluorescence and initiation of photoreactions are problems frequently encountered with resonance Raman spectroscopy of photobiological systems. These problems can be circumvented with Fourier-transform Raman spectroscopy by using the 1064-nm wavelength of a continuous wave neodymium-yttrium/aluminum-garnet laser as the probing beam. This wavelength is far from the absorption band of most pigments. Yet, the spectra of the investigated systems--bacteriorhodopsin, rhodopsin, and phycocyanin--show that these systems are still dominated by the chromophore, or that preresonant Raman scattering is still prevalent. Only for rhodopsin were contributions of the protein and the membrane discernible. The spectra of phycocyanin differ considerably from those obtained by excitation into the UV-absorption band. The results show the usefulness of this method and its wide applicability. In addition, analysis of the relative preresonant scattering cross sections may provide a detailed insight into the scattering mechanism.

Journal Article↗
PubMed1986

Reconstitution of the purified alpha-latrotoxin receptor in liposomes and planar lipid membranes. Clues to the mechanism of toxin action.

The receptor of alpha-latrotoxin (the major toxin of the black widow spider venom), purified from bovine synaptosomal membranes, was reconstituted into small unilamellar liposomes. These (but not control) liposomes exhibited high-affinity, specific binding of [125I]alpha-latrotoxin. In the receptor-bearing liposomes alpha-latrotoxin induced depolarization and stimulated 45Ca efflux. These responses to alpha-latrotoxin, that were observed only in the presence of external divalent cations, resembled those previously demonstrated in mammalian brain synaptosomes. The alpha-latrotoxin-activated ion fluxes are therefore, at least in part, the result of the direct interaction of the toxin with its receptor. When control and receptor-bearing liposomes were pre-incubated with alpha-latrotoxin and then added to a solution bathing a planar lipid bilayer membrane, single channel cationic conductances were observed. In the presence of the receptor, the conductances induced by alpha-latrotoxin were markedly different from those observed without the receptor, but not identical to those observed without the receptor, but not identical to those recently characterized by patch clamping in the cells of a line (PC12) sensitive to alpha-latrotoxin. These results demonstrate that the reconstituted receptor is functional, and suggest that the cationic channel activated by the toxin-receptor interaction is modulated by additional component(s) in the membrane of synapses and cells.

Animals↗
PubMed1985

Purification of the putative alpha-latrotoxin receptor from bovine synaptosomal membranes in an active binding form.

alpha-Latrotoxin (alpha-LTx, apparent mol. wt. 130 000) is a presynaptically active neurotoxin purified from the venom of the black widow spider that causes massive exocytotic release of neurotransmitters, presumably via binding to presynaptic membrane protein(s). Solubilization and purification experiments were undertaken to identify and characterize this membrane component. An immunoaffinity matrix was prepared by sequentially binding anti-alpha-LTx antibodies and alpha-LTx to Protein A-Sepharose CL-4B. Beads were irreversibly cross-linked with dimethyl pimelimidate. These beads were capable of extracting alpha-LTx binding activity from Triton X-100 solubilized bovine synaptosomal membranes. Following extensive washing, bound material was eluted with 6 M urea. Analysis of silver stained and radiolabel-containing gels revealed one major band (apparent mol. wt. 200 000) under non-reducing conditions and two major bands (apparent mol. wts. 66 000 and 54 000) under reducing conditions. The purified material was still capable of specifically binding alpha-LTx as determined by solid phase assays on microtiter plates. The affinity for alpha-LTx of the purified preparation was similar to that of the native membrane (KA approximately 10(10) M). It is concluded that a putative alpha-LTx receptor protein can be purified from synaptosomal membranes using an immunoaffinity matrix in a form that retains its defined biological property (alpha-LTx binding).

Animals↗
PubMed1984

Alpha latrotoxin of black widow spider venom: an interesting neurotoxin and a tool for investigating the process of neurotransmitter release.

Alpha latrotoxin, purified from the venom of the black widow spider, is a high Mr (130000) protein devoid of detectable enzymatic activity. When applied to vertebrate nerve terminals (of the central as well as peripheral nervous systems) the toxin elicits massive release of neurotransmitters by stimulating the fusion of synaptic vesicles with the presynaptic membrane (exocytosis). Among non-neuronal systems, only the neurosecretory cell line PC12 is sensitive to alpha latrotoxin; all others investigated so far are insensitive. In order to act, alpha latrotoxin requires the presence of divalent cations in the medium. Ca2+ can be substituted by other divalent cations as Sr2+, Ba2+, Mn2+, Mg2+. However, with the last two the catecholamine release response is reduced in PC12 cells and synaptosomes. A specific, high affinity receptor of alpha latrotoxin exists in preparation sensitive to the toxin. This receptor has been purified and found to be a high Mr, integral membrane protein. In the frog neuromuscular junction the receptor is localized exclusively in the presynaptic membrane. Binding of alpha latrotoxin to the receptor in a Ca2+-containing incubation medium induces membrane depolarization (insensitive to tetrodotoxin), stimulation of Ca2+ influx (insensitive to verapamil) with consequent increase in the cytoplasmic free Ca2+ concentration and stimulation of phosphoinositide breakdown. In Ca2+ free medium depolarization is maintained, but free Ca2+ concentration does not rise after toxin application. In conclusion, alpha latrotoxin seems to act through a dual mechanism. The Ca2+-independent part of this mechanism may be mediated by the activation of protein kinase C, triggered by phosphoinositide metabolism. The relevance of these findings for presynaptic physiology is discussed.

Animals↗
PubMed1983

Solubilization and partial characterization of rat epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase).

Epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase), the enzyme that catalyses the conversion of testosterone into the biologically active metabolite dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), is a membrane-bound enzyme found in both nuclear and microsomal subcellular fractions. In order to characterize epididymal delta 4-steroid 5 alpha-reductase, it was first necessary to solubilize the enzymic activity. Of the various treatments tested, a combination of 0.5% (w/v) Lubrol WX, 0.1 M-sodium citrate and 0.1 M-KCl maintained enzymic activity at control values and solubilized 66% of total epididymal delta 4-steroid 5 alpha-reductase activity in an active and stable form. The sedimentation coefficient of solubilized delta 4-steroid 5 alpha-reductase, as determined in continuous sucrose density gradients, was greater for the microsomal than for the nuclear enzyme (11.6S compared with 10.1S). Although the apparent Km values of the enzyme for testosterone were similar in nuclear and microsomal subcellular fractions (range 1.75 x 10(-7) - 4.52 x 10(-7)M), the apparent Km of the enzyme for NADPH was about 30-fold greater for the microsomal enzyme than for the nuclear enzyme. The apparent Km of the enzyme for either substrate was not significantly altered after solubilization. The relative capacity of steroids to inhibit the enzymic activity, the pH optima and the effects of Ca2+ and Mg2+ were similar for membrane-bound and solubilized delta 4-steroid 5 alpha-reductase in both the nuclear and the microsomal fractions. The results reported demonstrate that epididymal delta 4-steroid 5 alpha-reductase can be solubilized in an active and stable form with no significant changes in the kinetic characteristics of the enzyme after solubilization; furthermore, kinetic and molecular-size differences observed for the nuclear and the microsomal forms of the enzyme suggest that there may exist at least two forms of epididymal delta 4-steroid 5 alpha-reductase.

Animals↗
PubMed1983

Subcellular distribution of steroid delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the rat epididymis during sexual maturation.

The curve of the specific activity of rat epididymal nuclear delta 4-5 alpha-reductase is bell shaped as a function of age, whereas that of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase does not change significantly with age. The present study examines the subcellular distribution of delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the caput-corpus and cauda epididymidis during development. A 5-step discontinuous sucrose gradient was developed for fractionation of epididymal homogenates. By using enzyme markers specific for different subcellular organelles, the five different subcellular fractions obtained were shown to be of cytoplasmic, microsomal, mitochondrial, nuclear and spermatozoal origin. 3 alpha-Hydroxysteroid dehydrogenase activity was associated only with the cytoplasmic fraction. The activity of the enzyme did not change significantly with age in either the caput-corpus or cauda epididymidis. delta 4-5 alpha-Reductase activity was found in fractions containing microsomal and nuclear markers. delta 4-5 alpha-Reductase activity in the nuclear fraction of the caput-corpus epididymidis was evident in the youngest age group (Day 25), increased 4-fold and peaked in the next age group (Day 35), and declined with each successive age group: Day 45 (60% of maximum), Day 60 (20% of maximum), Day 75 (15% of maximum) and Day 105 (10% of maximum). In contrast, microsomal delta 4-5 alpha-reductase activity increased successively from Day 25 to Day 105; enzyme activity doubled between these two ages. The ratio of nuclear to microsomal delta 4-5 alpha-reductase activity from the caput-corpus epididymidis thus changed markedly with age: Day 25:1.32; Day 35:3.76; Day 45:2.44; Day 60:1.03; Day 75:0.41; and Day 105:0.21. In the cauda epididymidis nuclear delta 4-5 alpha-reductase activity was only evident at Day 35 and Day 45; in microsomal fractions, activity was first found at Day 35 and did not subsequently change with age. These results demonstrate that: 1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity is found only in the cytoplasmic fraction; 2) delta 4-5 alpha-reductase activity is found in nuclear and microsomal fractions; and 3) the subcellular distribution of delta 4-5 alpha-reductase activity changes markedly with age and epididymal section, suggesting differential regulation of nuclear and microsomal delta 4-5 alpha-reductase activities.

3-Hydroxysteroid Dehydrogenases↗