Mechanism of action of heme oxygenase. A study of heme degradation to bile pigment by 18O labeling

The formation of bile pigment from heme by a reconstituted heme oxygenase system containing purified bovine spleen heme oxygenase, NADPH-cytochrome P-450 reductase, and biliverdin reductase was studied under an atmosphere containing 18,18O2. The product, bilirubin, was isolated and subjected to mass spectrometry, which revealed incorporation of 18O consistent with a two-molecule mechanism, whereby the product bile pigment contains oxygen atoms derived from two different oxygen molecules.     

Fluorescence resonance energy transfer on the voltage-dependent sodium channel. Spatial relationship and site coupling between the batrachotoxin and Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin receptors

A fluorescent N- methylanthraniloyl derivative of the potent depolarizing agent batrachotoxin has been used to probe the structural and conformational properties of the neurotoxin receptor site on the voltage-dependent sodium channel. Batrachotoxin A 20-alpha-N- methylanthranilate (BTX-NMA) retains high affinity for its receptor site on the synaptosomal sodium channel with a Kd between 78 and 91 nM and an average site capacity of 2 pmol/mg of synaptosomal protein in the presence of Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin. The fluorescence emission of BTX-NMA upon binding to synaptosomes indicates a hydrophobic environment. Toxin V from L. quinquestriatus, an allosteric activator, effects a 20-nm red shift in the spectrum of bound BTX-NMA and a 4-fold enhancement in the fluorescence quantum yield disclosing a conformational change into a hydrophilic environment. Fluorescence resonance energy transfer measurements show that the distance separating the receptor sites is 37 +/- 10 A. Thus, the binding of alpha-scorpion toxin must involve conformational changes that extend over large distances from the batrachotoxin-binding locus. This information together with the distance measurements between the tetrodotoxin and alpha-scorpion toxin receptors and the conformational transition associated with this distance upon batrachotoxin addition indicate a conformationally flexible channel with coupling of sites through the polyatomic framework of individual subunits or through extensive alterations in subunit/subunit interactions.     

Intermediates in the enzymic synthesis of tetrahydrobiopterin in Drosophila melanogaster

9 partially purified enzyme (Enzyme A) from Drosophila melanogaster Aatalyzes the conversion of 7,8- dihydroneopterin triphosphate to a compound that, from its ultraviolet absorption spectrum and other characteristics, appears to be 6- pyruvoyl -tetrahydropterin. This product can be converted to 6-lactoyl-tetrahydropterin in the presence of another partially purified enzyme (Enzyme B) and NADPH, and to 5,6,7,8-tetrahydrobiopterin in the presence of a third enzyme preparation (biopterin synthase) and NADPH. The enzymically-produced 6-lactoyl-tetrahydropterin, when exposed to air, is oxidized nonenzymically to sepiapterin (6-lactoyl-7,8- dihydropterin ). The results indicate that although 6-lactoyl-tetrahydropterin can be converted enzymically to tetrahydrobiopterin, neither it nor sepiapterin is an obligate intermediate in the conversion of 7,8- dihydroneopterin triphosphate to tetrahydrobiopterin.     

Light-induced dephosphorylation of a 33K protein in rod outer segments of rat retina

Phosphorylated proteins may play an important role in regulating the metabolism or function of rod photoreceptors. In mammalian retinas, a photoreceptor protein of 33 000 (33K) molecular weight is phosphorylated in a cyclic nucleotide dependent manner in vitro. Since light initiates the activation of a photoreceptor-specific phosphodiesterase and a rapid reduction in guanosine cyclic 3',5'-phosphate concentration, phosphorylation of the 33K protein may be modulated by light in situ. In order to test this possibility, dark-adapted rat retinas were incubated for 30 min in the dark in phosphate-free Kreb's buffer containing [32P]orthophosphate. Following incubation, rod outer segments were detached by shaking, and the 32P-labeled rod outer segment proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and quantitated by densitometric scanning. The incorporation of radioactivity (32P) into the 33K protein was higher than into any other rod outer segment protein, and the amount of 32P-labeled 33K protein in the detached rod outer segments remained unchanged during 10 additional min of darkness. The addition of isobutylmethylxanthine to the incubation medium enhanced the incorporation of 32P into 33K protein to about 400% of the original level. Exposure of freshly detached rod outer segments to room light for 90 s decreased the amount of labeled 33K protein to 45% of its original level. The dephosphorylation of labeled 33K protein continued, reaching 12% of the original dark value 10 min after the previously illuminated sample was returned to darkness. Light initiated the phosphorylation of rhodopsin, and rhodopsin phosphorylation continued during the postillumination period of darkness.     

Reversible structure transition in gap junction under Ca++ control seen by high-resolution electron microscopy.

Deoxycholate-extracted rat liver gap junction was studied by high-resolution low-dose electron microscopy. Communicating channels between two adjoining cells supposedly form along the common axis of two apposed hexameric trans-membrane protein assemblies. These double hexamers are often arranged in large plaques on an ordered hexagonal net (8-9 nm lattice constant) and seem able to undergo structural alteration as a possible permeability control mechanism. Calcium is widely reported to uncouple gap junction, and we observed this alteration on exposure to Ca++ down to 10(-4) M concentration. When EGTA was added at matching concentrations, the alteration was reversible several times over one hour, but with considerable variability. It was imaged in the absence of any negative stain to avoid ionic and other complications. The resulting lack of contrast plus low-dose "shot" noise required digital Fourier filtering and reconstruction, but no detail was recovered below 1.8 nm. In other experiments with negative stain at neutral pH, gap junction connexons were apparently locked in the "closed" configuration and no transition could be induced. However, recovery of repeating detail to nearly 1.0 nm was possible, reproducibly showing a fine connective matrix between connexons . Whether this was formed by unfolded portions of the 28,000-dalton gap junction protein is not known, but its existence could explain the observed lattice invariance during the connexon structural transition.     

Lysine biosynthesis and the evolution-of pennate marine diatoms

The classification of lysine biosynthetic pathways in various organisms have been used to investigate their descent in evolution. We have attempted these determinations in the diatoms Amphora coffeaeformis var:perpusilla (Grunow Cleve.) and Phaeodactylum tricornutum (Bohlin). Additionally, we have verified earlier results of Vogel in a green alga, Chlorella pyrenoidosa strain Tx 71105 (Texas Culture Collection). Our research indicates that the diaminopimelic acid route is involved in all three organisms. While these studies do not exclude the possible co-existence of the α-aminoadipic acid route, the results imply a closer evolutionary relationship of pennate diatoms to bacteria and “classical” photosynthetic plants rather than to heterotrophic or mixotrophic fungi and atypical algal strains such as the Euglenophyta.     

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: I. Receptor Characterisation

Characterisation of receptor-mediated breakdown of inositol phospholipids in rat cortical slices has been performed using a direct assay which involves prelabelling with [3H]inositol. When slices were preincubated with [3H]inositol, lithium was found to greatly amplify the capacity of receptor agonists such as carbachol, noradrenaline, and 5-hydroxytryptamine to increase the amount of radioactivity appearing in the inositol phosphates. Using a large variety of agonists and antagonists it could be shown that cholinergic muscarinic, alpha 1-adrenoceptor, and histamine H1 receptors appear to be linked to inositol phospholipid breakdown in cortex. The large responses produced by receptor agonists allowed a clear discrimination between full and partial agonists as well as quantitative analysis of competitive antagonists for each receptor. Whereas carbachol and acetylcholine (in the presence of a cholinesterase inhibitor) were full agonists, oxotremorine and arecoline were only partial agonists. Very low concentrations of atropine shifted the carbachol dose-response curve to the right and allowed inhibition constants for the antagonist to be easily calculated. The nicotinic antagonist, mecamylamine, was ineffective. Noradrenaline adrenaline were full agonists at alpha 1-adrenoceptors, but phenylephrine and probably methoxamine were partial agonists. Prazosin, but not yohimbine, potently and competitively antagonised the noradrenaline inositol phospholipid response. Mepyramine but not cimetidine competitively antagonised the histamine response. These data provide strong confirmation for the potentiating effect of lithium on neurotransmitter inositol phospholipid breakdown and emphasise the ease with which functional responses at a number of cortical receptors can be characterised.     

Tetrahymena pyriformis as a protective antigen against Ichthyophthirius multifiliis infection: comparisons between isolates and ciliary preparations

Channel catfish, Ictalurus punctatus , were immunized with cilia from three isolates of Tetrahymena pyriformis and challenged with Ichthyophthirius multifiliis using a reproducible quantitative procedure. Two different methods of deciliation were used in antigen preparation. Results indicate that T. pyriformis cilia elicit an immune response in channel catfish against I. multifiliis , and that the protective ability of the cilia varies between T. pyriformis strains.     

Cellular requirements for induction of human primary proliferative responses to trinitrophenyl-modified cells

Cellular requirements for induction of primary proliferative responses by human T cells to trinitrophenylated autologous stimulators have been characterized. Substantial proliferative responses were observed with each of the Ia+ stimulator populations tested. Nevertheless, major differences in the hapten specificity of such responses were observed. Thus purified macrophages/monocytes (M phi) when TNP-modified induced responses that were relatively modest in absolute magnitude, but were highly hapten specific. This reflected the very limited capacity of purified M phi to induce proliferation when unmodified, i.e., an autologous mixed leukocyte response (AMLR). In contrast, unmodified M phi-depleted B plus null cells were potent stimulators of AMLR, but hapten modification did not significantly enhance the responses induced by these cells. Moreover, when M phi were added to B plus null cell stimulators AMLR responses were reduced and, with TNP-modified stimulators, hapten-specific responses were restored. The data thus suggest that M phi may have important roles in induction of primary T cell responses to conventional antigens but function largely as regulators rather than stimulators of AMLR. Finally, we have introduced a novel antigen-presenting cell population, the irradiated Ia+ TNP-specific cloned T cell. The possibility that such cells may utilize autostimulatory positive feedback circuits for activation of naive T cells and in interactions between subpopulations of hapten-reactive T cells is discussed.     

Selection for yield and yield components in the early generations of a potato breeding programme

Summary A number of unselected potato (Solanum tuberosum L.) clones were grown at two locations (a seed site and a ware site) in three consecutive years. The repeatability of total yield and yield components in the first two clonal years was compared with the same characters recorded in the third clonal year. Selection for yield in the first clonal year was only marginally more effective than a random reduction in number of genotypes, while selection in the second clonal year appeared to be somewhat more effective as judged by performance in the third clonal year. The inefficiency of selection in the first clonal year was ascribed, at least in part, to the inaccuracy of yield assessment as well as the carry-over effect of the mother tubers. Correlations of total yield were higher between different years in the same location than between different locations. Selection under growing conditions suitable for production of seed tubers tended to result in selection of early maturing clones which would not necessarily be optimal for ware growing conditions.