Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.     

Localization of glyoxylic acid-induced histofluorescence in surgically isolated medial basal hypothalamus of the rat

Summary Examination of glyoxylic acid-induced catecholamine histofluorescence in the hypothalamic median eminence of adult male rats revealed a linear pattern of fine varicosities coursing through the ependymal and fibrous zones, suggestive of juxtaposition to tanycytes. In order to determine the origin of these terminals, adult rats were subjected to complete isolation of the medial basal hypothalamus, using a small Halasz-Pupp knife. As rapidly as 24h after this deafferentation degenerative axon profiles were observed dorsal, as well as anterior and lateral, to the knife track. Occasionally at three days postoperatively, and routinely by seven days after surgery, fine-sized new fibres were seen passing through the knife wound. The linear profiles of varicosities observed in the normal median eminence remained traceable in the experimental preparations; the site of origin for these terminals therefore appears to be neurons of the arcuate (A12) and rostral periventricular (A14) regions. The results also indicate that fibres innervating the isolated area are capable of morphologically demonstrable new growth. The observations bear functional implications in assessing endocrine regulation following MBH isolation of the type used in this study.This study was supported by USPHS Postdoctoral Fellowship 5-F₂2-HD00630 (CT) and USPHS Grant NS-11642 (JRS). The authors wish to express their appreciation to Yvonne Cheung and Patricia Walker for technical assistance     

Distribution of intrinsic nerve cell bodies and axons which take up aromatic amines and their precursors in the small intestine of the guinea-pig

Summary The sites of uptake, decarboxylation and retention of 1-dopa and the uptake and retention of dopamine and 6-hydroxytryptamine in the small intestine of the guinea-pig have been localised histochemically with a fluorescence technique for arylethylamines. In segments of ileum from untreated guinea-pigs only noradrenergic axons are fluorescent; these axons were eliminated by surgical denervation (crushing nerves running to the intestine through the mesentery) or by chemical denervation with 6-hydroxydopamine. In denervated segments of ileum, cell bodies and processes of intrinsic neurons become fluorescent after the injection of 1-dopa, dopamine or 6-hydroxytryptamine and the inhibition of monoamine oxidase, as do cells of Brunner's glands and Paneth cells. About 11% of the nerve cell bodies in the submucous plexus and 0.4% of those in the myenteric plexus become fluorescent. Varicose intrinsic axons which take up amines are found amongst the nerve cell bodies of the myenteric and submucous plexuses. They also ramify in the principal connections of the plexuses, in the tertiary strands of the myenteric plexus, in the deep muscular plexus and contribute sparse supplies of axons to arterioles in the submucosa and to the lamina propria of the mucosa. The axons are resistant to the degenerative actions of 6-hydroxydopamine.It is suggested that the intrinsic amine handling axons are more likely to utilise an indolamine related to 5-hydroxytryptamine than they are to utilise a catecholamine as a neurotransmitter.     

Effects of desiccation on the excitation energy distribution from phycoerythrin to the two photosystems in the red alga Porphyra perforata

Low temperature (77°K) fluorescence emission and excitation spectra were recorded for wet and desiccated thalli of Porphyra perforata . The photosystem I (F730) and photosystem II (F695) fluorescence emission kinetics during photosystem II trap closure were also recorded at 77°K. Desiccation induced a lowering of the fluorescence yield over the whole emission spectrum but the decrease was most pronounced for the photosystem II fluorescence bands, F688 and F695. It was shown that the desiccation-induced changes of the phycoerythrin sensitized emission spectrum were due to 1) a decrease in the fluorescence yield of the photosystem I antenna, 2) an even stronger decrease in the fluorescence of photosystem II, which was mediated by an increased spillover (k_T(II→I)) of excitation to photosystem I and an increase in the absorption cross section, α, for photosystem I. We hypothesize that the increase of both k_T(II→I) and α are part of a mechanism by which the desiccation-tolerant, high light exposed, Porphyra can avoid photodynamic damage to photosystem II, when photosynthesis becomes inhibited as a result of desiccation during periods of low tide.     

Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanidesensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 μM and 20 μM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured.Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity.The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.     

The fluorescence decay kinetics of in vivo chlorophyll measured using low intensity excitation

We report fluorescence lifetimes for in vivo chlorophyll a using a time-correlated single-photon counting technique with tunable dye laser excitation. The fluorescence decay of dark-adapted chlorella is almost exponential with a lifetime of 490 ps, which is independent of excitation from 570 nm to 640 nm.Chloroplasts show a two-component decay of 410 ps and approximately 1.4 ns, the proportion of long component depending upon the fluorescence state of the chloroplasts. The fluorescence lifetime of Photosystem I was determined to be 110 ps from measurements on fragments enriched in Photosystem I prepared from chloroplasts with digitonin.     

Correlation between temperature range of growth and structural transitions in membranes and lipids of Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild-type Escherichia coli grown at different temperatures were labelled with 1,6-diphenyl-1,3,5-hexatriene and anlyzed using fluorescence polarization techniques. Lipids extracted from the membranes were similarly analyzed using fluorescence polarization. The thermotropic structural transition in outer membranes changed as a function of growth temperature. The structural transition in cytoplasmic membranes and lipids extracted from either cytoplasmic or outer membranes did not change with growth temperature. These data suggest that adaptive changes which occur in the outer membrane determine the temperature range of growth of E. coli. These changes apparently require alterations in outer membrane components other than phospholipids.     

The composition and fluidity of adipocyte membranes prepared from young and adult rats

Membranes from adipocytes of adult and young rats have been compared. Phospholipid fatty acids from adult rats were more saturated than those from young rats. This difference was associated with a decreased fluidity in the membranes of the adult rats, which was inferred from measurements of fluorescence polarisation of the fluorescent probe, 1,6-diphenylhexa-1,3,5-triene.     

Quenching of the fluorescence emitted by P695-682 at room temperature in etiolated illuminated leaves

Chlorophyll(ide) fluorescence emission decreases at room temperature during completion of protochlorophyll(ide) reduction. The process responsible for this quenching is parallel to the P_688-676 P_695-682 transition. It proceeds equally well in darkness and in the light. It consists in a decrease of the fluorescence yield of chlorophyll(ide) in P_695-682. Apparently, room temperature P_695-682 fluorescence is regulated by a conjunction of factors such as energy transfers and photobiochemical activities.Abbreviations NADP nicotinamide-adenine dinucleotide phosphate - CPI chlorophyll-protein-complex I - CPII chlorophyll-protein-complex II Aspirant du Fond National de la Recherche Scientifique, Belgium