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Pattern of lipofuscin pigmentation in nitrergic and non-nitrergic, neurofilament immunoreactive myenteric neuron types of human small intestine.

Lipofuscin, an autofluorescent age pigment, occurs in enteric neurons. Due to its broad excitation and emission spectra, it overlaps with commonly used fluorophores in immunohistochemistry. We investigated the pattern of lipofuscin pigmentation in neurofilament (NF)-reactive nitrergic and non-nitrergic human myenteric neuron types. Subsequently, we tested two methods for reduction of lipofuscin-like autofluorescence. Myenteric plexus/longitudinal muscle wholemounts of small intestines of five patients undergoing surgery for carcinoma (aged between 18 and 69 years) were double stained for NF and neuronal nitric oxide synthase (nNOS). Lipofuscin pigmentation patterns were semiquantitatively evaluated by using confocal laser scanning microscopy with three different excitation wave lengths (one for undisturbed lipofuscin autofluorescence and two for specific labellings). Two pigmentation patterns could be detected in the five NF-reactive neuron types investigated. In nitrergic/spiny as well as in non-nitrergic/stubby neurons, coarse, intensely autofluorescent pigment granules were prominent. In non-nitrergic type II, III and V neurons, a fine granular, diffusely distributed and less intensely autofluorescent pigment was obvious. After incubation of wholemounts in either CuSO(4) or Sudan black B solutions, unspecific autofluorescence could be substantially reduced whereas specific NF and nNOS fluorescence remained largely unaffected. We conclude that NF immunohistochemistry is useful for morphological representation of subpopulations of human myenteric neurons. The lipofuscin pigmentation in human myenteric neurons reveals at least two different patterns which can be related to distinct neuron types. Incubations of multiply stained whole mounts in both CuSO(4) or Sudan black B are suitable methods for reducing autofluorescence thus facilitating discrimination between specific (immunohistochemical) and non-specific (lipofuscin) fluorescence.

Adolescent↗

Retinopretectal projections in albino and pigmented rabbits: an autoradiographic study.

The ipsilateral and contralateral retinal projection was studied in pigmented rabbits and in 3 strains of albino rabbits by anterograde transport of [3H]proline and [3H]fucose combined with autoradiographic techniques. Special attention was paid to the terminals in the pretectal area of both the pigmented and albino strains. On the contralateral side terminal labeling was found in both pigmented and albino rabbits in the nucleus of the optic tract (NOT), the anterior pretectal nucleus (PA), the posterior pretectal nucleus (PP) and the pretectal olivary nucleus (PO). Ipsilaterally labeling was found only in the pigmented strain in small patches in the PP. Ipsilateral projection was not found in the albinos in the pretectal area. The results are in agreement with the findings of Scalia in pigmented rabbits. The absence of ipsilateral labeling in the pretectal region in albinos is in contrast with earlier findings of Giolli and Takahashi et al., in pigmented rabbits but is in agreement with the observations of Takahashi and Oyster. Since no radioactively labeled fibers were found to project to the NOT in either pigmented or albino rabbits, these results do not support the hypothesis of Collewijn that the inverted optokinetic nystagmus in albinos is due to misrouting of the ipsilateral retinal fibers to the NOT.

Animals↗

Influence of soil substrate and ozone plus acid mist on the pigment content and composition of needles from young Norway spruce trees.

The influence of an ozone + acid mist treatment on photosynthetic pigments has been examined with the needles of the (Picea abies) clones 11, 14, 16, and 133 by spectrophotometric analysis of the total pigment extract and of single components upon HPLC separation (Part A), and in terms of a detailed pigment analysis of the 1987 and 1986 needles of clone 14 by TLC (Part B). Clone 14 had been already analysed prior to the onset of the experiment. At the end of the 14-month experiment, which incorporated frost events during a simulated winter period, neither symptoms corresponding to those of spruce Type I or IV decline, nor those of ozone damage could be observed. However, the 1986 needles of the trees on soil 1, which exhibit an adequate nutrient content, showed zonal chlorosis independently of the ozone + acid mist treatment. Analysis of variance of chlorophyll contents and needle ages showed a clear reduction to nearly 50% in the 1986 needles of clone 11, soil 1, and clone 16, soil 2. In contrast, clones 14 and 16 (soil 1) formed significantly more chlorophylls during the shorter exposure time in the 1987 flush. The detailed analysis of the individual pigment components of clone 14 needles provided no evidence for a destructive influence of the treatment on the chlorophylls, xanthophylls and beta-carotene in the two needle generations which had developed during the experiment, in spite of the distinct K deficiency of the 1986 needles of the trees on soil 2 and the common chlorosis of the needles of the trees on soil 1. The observed increase in violaxanthin content upon O(3)-treatment observed in clone 14 can be considered as an expression of the protective function of the xanthophylls against photooxidative processes. In conclusion, the observed differences in the chlorophyll and carotenoid contents are better correlated with the individual clone and soil character than with the ozone + acid mist treatment. Comparing the results of the pigment analyses of the needles the differences in the pigment concentrations reflect the N and K contents (Pfirrmann et al., 1990), which differ significantly between the clones. Thus it is not possible to pool the pigment data of all clones without considering the different nutrient levels.

Journal Article↗

Retinal decussation patterns in pigmented and albino ferrets.

The decussation patterns of retinal ganglion cells in adult pigmented and albino ferrets were determined from the distribution of cells labelled after large unilateral injections of horseradish peroxidase into the visual pathway, involving the lateral geniculate nucleus and fibres of passage to the superior colliculus. About 6000 retinal ganglion cells project ipsilaterally in pigmented ferrets compared with only about 1500 in albino ferrets. In both strains, the vast majority of these cells (99 and 87% in pigmented and albino animals, respectively) are located in the temporal crescent, although we describe one albino ferret in which an aberrant uncrossed projection arises from nasal retina. In pigmented ferrets, there is a sharp nasotemporal division that runs through the area centralis; a small proportion of the ganglion cells in temporal crescent (less than 10%) does project contralaterally. In albinos, however, the majority of cells in temporal retina project contralaterally. There is no clear nasotemporal division in the albino retina; the density of uncrossed ganglion cells is reduced throughout temporal crescent and at no location exceeds the comparable density of the crossed projection. The peak density within the reduced uncrossed projection is also displaced away from the area centralis into temporal retina. Analysis of cell type on the basis of soma size indicates that whereas large horseradish peroxidase injections into the visual pathway of pigmented ferrets label all types of ganglion cell in the crossed projection, injections restricted to the superior colliculus label only those ganglion cells with large or small somata. The distribution of cell sizes in the crossed projection from temporal retina is biased towards small cells in the pigmented ferret but in albinos resembles that seen in the crossed projection from nasal retina. Thus the adult pigmented ferret has both a well developed nasotemporal division in which decussation lines are obvious in the crossed and uncrossed pathways and also, unlike rodents but like cats, a class of ganglion cell that does not project to the superior colliculus. The albino mutation both reduces the uncrossed projection throughout temporal retina, although the reduction is greatest close to the area centralis, and also commensurately increases the crossed projection from temporal retina.

Albinism↗

Differential susceptibility to noise-induced permanent threshold shift between albino and pigmented guinea pigs.

Evidence that reduced levels of cochlear melanin are associated with increased auditory sensitivity, increased levels of auditory fatigue and an increased susceptibility to noise-induced hearing loss led us to investigate the effects of noise exposure on the cochlear microphonic (CM) in albino and pigmented English shorthair guinea pigs. CMs were recorded from the round window prior to and at 90 min and 7 days after exposure to 45 min of 126 dB noise. Thresholds for the first detectable elicitation of the CM for four pure tones were determined and the output voltage of each cochlea was measured in 10 dB steps through intensity levels which produced a maximum voltage amplitude in the CM and voltage rollover. This analysis demonstrated that: albino guinea pigs displayed significantly lower auditory thresholds than did pigmented animals before exposure to noise; thresholds were elevated to comparable levels in both groups 90 min after noise exposure; pigmented guinea pigs showed a reliable recovery in CM thresholds 7 days after exposure to noise while thresholds in the albinos remained elevated to the same degree at both 90 min and 7 days after noise; 90 min after noise exposure, the maximum voltage output of albino cochleas was significantly less than that recorded from the cochleas of the pigmented guinea pigs. These results demonstrate that albino guinea pigs are more susceptible to the ototoxic effects of high intensity noise than pigmented guinea pigs. Converging evidence indicates that some aspects of cochlear function involve melanin pigment and that its absence may produce auditory abnormalities. Reduced melanin pigmentation may also contribute to such phenomena as noise-induced threshold shifts and individual differences in noise-induced hearing loss.

Albinism↗

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment-protein complexes: peridinin-chlorophyll a protein (PCP) of Amphidinium carterae.

Peridinin-chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a "building block" approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving "optically dark" states of carotenoids in pigment-protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).

Amino Acids↗

Epithelial pigment slide in contact lens wearers: a possible marker for contact lens-associated stress on corneal epithelium.

PURPOSE: To compare the incidence of epithelial pigment slide among wearers of various types of contact lenses. METHODS: Prospectively, we studied 432 eyes of 432 patients. The patients were separated into 6 groups: hard contact lens (HCL) wearers (n = 166), conventional soft contact lens (CSCL) wearers (n = 30), extended disposable lens (EDCL) wearers (n = 46), frequent replacement SCL (FRCL) wearers (n = 60), daily disposable SCL (DDCL) wearers (n = 65), and normal controls (n = 65). The incidence of prominent pigment slide, defined as spike-like epithelial opacities in the corneal limbus and longer than 1 mm from the base to the apex of the wedges-shaped process detected by slit-lamp examination, was compared in these 6 groups. The relationship between the incidence of prominent pigment slide and the length of contact lens wear was examined. RESULTS: The overall incidence of prominent pigment slide in the CSCL, EDCL, HCL, FRCL, DDCL, and normal groups was 63.3, 23.9, 13.9, 8.3, 7.7, and 4.6%, respectively. The incidence of prominent pigment slide in the CSCL and EDCL groups was significantly higher than that in the control group. A higher incidence of prominent pigment slide was correlated with longer wearing period in each group. CONCLUSIONS: The presence of epithelial pigment slide may be a marker for contact lens-associated stress to the corneal epithelium.

Biomarkers↗

Organisation of xanthophyll pigments lutein and zeaxanthin in lipid membranes formed with dipalmitoylphosphatidylcholine.

Carotenoid pigments and in particular xanthophylls play several physiological functions in plant and animal membranes. Xanthophylls are present in biological membranes in the form of pigment-protein complexes but also as direct components of lipid phase. The biological activity of carotenoids in membranes depends on a molecular organisation of pigments in lipid bilayers, in particular the localisation, orientation and aggregational state. In the present work the organisation of lutein- and zeaxanthin-containing lipid membranes was analysed with the application of electronic absorption spectroscopy. Both xanthophyll pigments incorporated to the dipalmitoylphosphatidylcholine (DPPC) unilamellar liposomes form H-type molecular aggregates, manifested by the hypsochromic shift of the main absorption band of carotenoids. The aggregation of lutein and zeaxanthin in DPPC membranes was observed even at relatively low concentrations of a pigment in the lipid phase (1-5 mol%). Gaussian analysis of the absorption spectra of lutein and zeaxanthin in DPPC membranes in terms of the exciton splitting theory revealed the formation of different molecular structures of pigments interpreted as dimers, trimers, tetramers and large aggregates. The fraction of lutein and zeaxanthin in the monomeric form was found to depend on the physical state of the lipid phase. Pronounced monomerisation of lutein and zeaxanthin was observed as accompanying the transition from the P(beta)' phase to the L(alpha) phase of DPPC, mostly at the expense of the trimeric and tetrameric forms. The fraction of monomers of lutein is always lower by 10-30% than that of zeaxanthin under the same experimental conditions. Different organisational forms of lutein and zeaxanthin in the model system studied are discussed in terms of possible physiological functions of these pigments in the membranes of the retina: zeaxanthin in the protection of the lipid phase against oxidative damage and lutein in absorbing short wavelength radiation penetrating retina membranes.

1,2-Dipalmitoylphosphatidylcholine↗

Intraleucocytic malaria pigment and clinical severity of malaria in children.

Intraleucocytic malaria pigment has been suggested as a measure of disease severity in malaria. We have tested this hypothesis by studying 146 children aged 6 months to 14 years in 4 categories--cerebral malaria, mild malaria, asymptomatic malaria and 'no malaria'--in Ibadan, Nigeria, an area of intense malaria transmission in Africa. Children with cerebral malaria were studied at the university hospital, those with mild malaria at 2 primary health centres and the other 2 groups were studied in a primary school. The proportion of pigment-containing neutrophils showed a clear rise across the spectrum no malaria--asymptomatic malaria--mild malaria--cerebral malaria (median values 2.0%, 6.5%, 9.0% and 27.0%, respectively; P < 0.0001). The proportion of pigment-containing monocytes did not differ significantly between the mild malaria, asymptomatic malaria and no malaria groups but the cerebral malaria group had a higher median value than the other 3 groups. The ratio of pigment-containing neutrophils to pigment-containing monocytes showed the same trend across the groups of subjects as was observed with the number of pigment-containing neutrophils. It is concluded that the pigment-containing neutrophil count is a simple marker of disease severity in childhood malaria in addition to the parasite count.

Adolescent↗

Functional characterization of visual and nonvisual pigments of American chameleon (Anolis carolinensis).

Using only 11-cis 3, 4-dehydroretinal as a chromophore in the pure-cone retina, American chameleon (Anolis carolinensis) detects a wide range of color from ultraviolet (UV) to infrared. We previously characterized its visual opsin genes sws1Ac, sws2Ac, rh1Ac, rh2Ac, and LwsAc that encode SWS1Ac, SWS2Ac, RH1Ac, RH2Ac, and LWSAc opsins, respectively, and the pineal gland-specific opsin (PAc) gene. Here we present the light absorption profiles of the visual pigments obtained by expressing these opsins and reconstituting them with 11-cis retinal using the COS1 cell cDNA expression system. The purified SWS1Ac, SWS2Ac, RH1Ac, RH2Ac, LWSAc, and PAc pigments have the wavelengths of maximal absorption at 358, 437, 491, 495, 560, and 482 nm, respectively. SWS1Ac is the first vertebrate UV opsin whose spectral sensitivity has been directly evaluated. RH1 pigments, orthologous to the rod pigments of other vertebrates, are sensitive to hydroxylamine in the dark, exhibiting a cone pigment-like characteristic, probably reflecting their adaptation to the pure cone retina. Interestingly, the blue-sensitive SWS2Ac pigment shows an exceptionally low level of sensitivity to hydroxylamine, possessing a rod pigment-like characteristic.

Animals↗

A novel method for eliminating the melanin pigments from melanoma cells undergoing cytogenetic analysis in cases of uveal melanoma.

A method has been developed to eliminate melanin pigments from chromosomal plates of uveal melanoma tumors. On preparing the chromosomal plates of tumor masses of posterior uveal melanoma for cytogenetic analysis in intermediate pigmented population, it was found that the melanin pigments obscured the cells and thereby the chromosomes. Thus, cytogenetic analysis could not be carried out. Hence, a method was developed in our laboratory to eliminate these obscuring pigments. Several different attempts were made to eliminate the melanin pigments. Finally, washing the cells with phosphate buffered saline before the harvesting stage served the purpose. After harvesting and preparing the chromosomal plates, the cells were found to be devoid of melanin pigments. The protocol would help researchers trying to carry out cytogenetic analysis on melanoma tumor masses in populations with intermediate to dark pigmentation.

Cytogenetics↗

Comparative analyses of the pigment-aggregating and -dispersing actions of MCH on fish chromatophores.

In melanophores of the peppered catfish and the Nile tilapia, melanin-concentrating hormone (MCH) at low doses (<1 microM) induced pigment aggregation, and the aggregated state was maintained in the presence of MCH. However, at higher MCH concentrations (such as 1 and 10 microM), pigment aggregation was immediately followed by some re-dispersion, even in the continued presence of MCH, which led to an apparent decrease in aggregation. This pigment-dispersing activity at higher concentrations of MCH required extracellular Ca(2+) ions. By contrast, medaka melanophores responded to MCH only by pigment aggregation, even at the highest concentration employed (10 microM). Since it is known that medaka melanophores possess specific receptors for alpha-melanophore-stimulating hormone (alpha-MSH), the possibility that interaction between MSH receptors and MCH at high doses in the presence of Ca(2+) might cause pigment dispersion is ruled out. Cyclic MCH analogs, MCH (1-14) and MCH (5-17), failed to induce pigment dispersion, whereas they induced aggregation of melanin granules. These results suggest that another type of MCH receptor that mediates pigment dispersion is present in catfish and tilapia melanophores, and that intact MCH may be the only molecule that can bind to these receptors. Determinations of cAMP content in melanophores, which were isolated from the skin of three fish species and treated with 10 nM or 10 microM MCH, indicate that MCH receptors mediating aggregation may be coupled with Gi protein, whereas MCH receptors that mediate dispersion may be linked to Gs. The response of erythrophores, xanthophores and leucophores to MCH at various concentrations was also examined, and the results suggest that the distribution patterns of the two types of MCH receptors may differ among fish species and among types of chromatophore in the same fish.

Animals↗

In search of the visual pigment template.

Absorbance spectra were recorded by microspectrophotometry from 39 different rod and cone types representing amphibians. reptiles, and fishes, with A1- or A2-based visual pigments and lambdamax ranging from 357 to 620 nm. The purpose was to investigate accuracy limits of putative universal templates for visual pigment absorbance spectra, and if possible to amend the templates to overcome the limitations. It was found that (1) the absorbance spectrum of frog rhodopsin extract very precisely parallels that of rod outer segments from the same individual, with only a slight hypsochromic shift in lambdamax, hence templates based on extracts are valid for absorbance in situ: (2) a template based on the bovine rhodopsin extract data of Partridge and De Grip (1991) describes the absorbance of amphibian rod outer segments excellently, contrary to recent electrophysiological results; (3) the lambdamax/lambda invariance of spectral shape fails for A1 pigments with small lambdamax and for A2 pigments with large lambdamax, but the deviations are systematic and can be readily incorporated into, for example, the Lamb (1995) template. We thus propose modified templates for the main "alpha-band" of A1 and A2 pigments and show that these describe both absorbance and spectral sensitivities of photoreceptors over the whole range of lambdamax. Subtraction of the alpha-band from the full absorbance spectrum leaves a "beta-band" described by a lambdamax-dependent Gaussian. We conclude that the idea of universal templates (one for A1- and one for A2-based visual pigments) remains valid and useful at the present level of accuracy of data on photoreceptor absorbance and sensitivity. The sum of our expressions for the alpha- and beta-band gives a good description for visual pigment spectra with lambdamax > 350 nm.

Animals↗

A visual pigment from chicken that resembles rhodopsin: amino acid sequence, gene structure, and functional expression.

The amino acid sequence of a rhodopsin-like visual pigment from chickens has been determined by isolating and sequencing its gene. The predicted sequence is between 70% and 80% identical to bovine, human, and chicken rhodopsins and between 40% and 50% identical to human blue, green, and red cone pigments, the chicken red cone pigment, and cavefish long-wave cone pigments. The encoded pigment, produced by transfection of cDNA into cultured cells, absorbs maximally at 495 nm as determined from photobleaching difference spectra and reacts at 20 degrees C with 50 mM hydroxylamine with a half-time of 16 min. These properties, together with a high pI predicted from the amino acid sequence, suggest that this cloned gene encodes the chicken green pigment previously identified by biochemical and spectroscopic studies. This sequence defines a new branch of the visual pigment gene family.

Amino Acid Sequence↗

Interactions of the beta-ionone ring with the protein in the visual pigment rhodopsin control the activation mechanism. An FTIR and fluorescence study on artificial vertebrate rhodopsins.

The photoreactions of rhodopsin regenerated with three 9-cis retinal analogs, modified at or in the vicinity of the beta-ionone ring (namely 5,6-epoxy, 7,8-diH, diethyl-acyclic) have been investigated by UV-vis and FTIR difference spectroscopy. In parallel, the ability to catalyze the GDP-->GTP exchange of G-protein (transducin) has been monitored by time-dependent fluorescence spectroscopy. The first photoproduct obtained with all three pigments at liquid nitrogen temperature is a blue-shifted intermediate (BSI), followed by a lumi-like intermediate at 170 K. For the 5,6-epoxy-ISO and 7,8-diH-ISO pigment we obtain two further intermediates similar to the META-I and META-II states of native RHO. For the diethyl-acyclic-ISO pigment only one further intermediate can be stabilized at 280 K. As compared to META-II the respective photoproduct exhibits striking differences. The latter two pigments have also been investigated in the solubilized lipid-free state (detergent: dodecyl maltoside) at 280 K. For the 5,6-epoxy-ISO pigment, the UV-vis, FTIR, and activation data agree with the formation of a META-II-like photoproduct (81% activation). Less META-II formation is observed for the 7,8-dihydro-ISO pigment in membranes (65% activation), but full formation in detergent (100% activation). Neither the membrane-bound nor the solubilized diethyl-acyclic-ISO pigment forms a META-II-like intermediate (18% and 0% activation, respectively). Therefore, we conclude that the substitution of the beta-ionone ring by two ethyl groups abolishes steric interactions with the protein, which are essential for META-II formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Effect of variation of retinal polyene side-chain length on formation and function of bacteriorhodopsin analogue pigments.

The effect of the length of the retinal polyene side chain on bacterioopsin pigment formation and function has been investigated with two series of synthetic retinal analogues. Cyclohexyl derivatives with polyene chains one carbon longer and one or more carbons shorter than retinal and linear polyenes with no ring have been synthesized and characterized. Compounds of six carbons or less in the polyene chain form pigments very poorly or not at all with bacterioopsin. Compounds containing at least seven carbons in the chain are found to form reasonably stable bacterioopsin pigments that show a small shift in absorbance on irradiation. However, photocycling and proton photorelease are not detected. The analogue with nine carbons in the polyene chain (one less than retinal) forms a stable pigment with an M-type intermediate but demonstrates reduced amounts of photocycling and light-activated proton release. The analogue with a polyene chain identical with that of retinal, but containing no ring, forms a pigment that shows both an efficient light-activated proton photocycle and release. The pigment containing the chromophore with the polyene chain one carbon longer than retinal is likewise fully active. We thus conclude that the length of the polyene chain must be at least 9 carbons for the formation of a stable pigment that photocycles and must be 10 carbons for both the photocycle and light-activated proton release to have a high quantum efficiency.

Bacteriorhodopsins↗

Spectral tuning in the mammalian short-wavelength sensitive cone pigments.

The wild-type mouse ultraviolet (UV) and bovine blue cone visual pigments have absorption maxima of 358 and 438 nm, respectively, while sharing 87% amino acid identity. To determine the molecular basis underlying the 80 nm spectral shift between these pigments, we selected several amino acids in helices II and III for site-directed mutagenesis. These amino acids included: (1) those that differ between mouse UV and bovine blue; (2) the conserved counterion, Glu113; and (3) Ser90, which is involved in wavelength modulation in avian short-wavelength sensitive cone pigments. These studies resulted in the identification of a single amino acid substitution at position 86 responsible for the majority of the spectral shift between the mouse UV and bovine blue cone pigments. This is the first time that this amino acid by itself has been shown to play a major role in the spectral tuning of the SWS1 cone pigments. A single amino acid substitution appears to be the dominant factor by which the majority of mammalian short-wavelength sensitive cone pigments have shifted their absorption maxima from the UV to the visible regions of the spectrum. Studies investigating the role of the conserved counterion Glu113 suggest that the bovine and mouse SWS1 pigments result from a protonated and unprotonated Schiff base chromophore, respectively.

Amino Acid Substitution↗

Pigment quantitation and analysis by HPLC reverse phase chromatography: a characterization of antenna size in oxygen-evolving photosystem II preparations from cyanobacteria and plants.

Photosystem II, the photosynthetic water-oxidizing complex, can be isolated from both plants and cyanobacteria. A variety of methods have been developed for purification of this enzyme, which can be isolated in several functional and structural forms. Knowledge of the pigment content of photosystem II preparations is important for precise spectroscopic, biochemical, and functional analysis. We have determined pigment stoichiometries in oxygen-evolving photosystem II preparations from plants and cyanobacteria. We have employed a solvent system for the isocratic elution of a reverse phase HPLC column in which we have determined the extinction coefficients of the relevant pigments. Pigments were extracted from four photosystem II preparations. These preparations included spinach photosystem II membranes [Berthold, D. A., Babcock, G. T., & Yocum, C. F. (1981) FEBS Lett. 134, 231-234], spinach photosystem II reaction center complexes [Ghanotakis, D. F., & Yocum, C. F. (1986) FEBS Lett. 197, 244-248], spinach photosystem II complexes [MacDonald, G. M., & Barry, B. A. (1992) Biochemistry 31, 9848-9856], and photosystem II particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803 [Noren, G. H., Boerner, R. J., & Barry, B. A. (1991) Biochemistry 30, 3943-3950]. Pigment stoichiometries were determined using two different methods of data analysis and were based on the assumption that there are two pheophytin a molecules per photosystem II reaction center. The pigment stoichiometries obtained were comparable for the two methods of data analysis and agreed with previous biophysical and biochemical characterizations of the preparations. The average pigment stoichiometries (chlorophyll:plastoquinone-9 per 2 pheophytin a) determined using the two data analysis methods were as follows: photosystem II membranes, 274:3.2; photosystem II reaction center complexes, 78:2.5; Synechocystis PS II particles, 55:2.4; photosystem II complexes, 121:2.0.

Chlorophyll↗