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Biomedical subjects

W L Butler

Publications and source records attributed to W L Butler.

At least 73 records · Page 4Linked to original sources
PubMed1972

A spectroscopic analysis of a high fluorescent mutant of Chlamydomonas reinhardi.

Chloroplast fragments of a high fluorescent mutant of Chlamydomonas reinhardi, hfd 91, were compared against those of Acl(+), a low chlorophyll variant of the wild type. The chloroplast fragments of the mutant which have a high invariant fluorescence yield lacked photochemical activities associated with photosystem II (PSII) but retained normal photosystem I (PSI) activities. The mutant fragments also lacked the low temperature (-196 degrees C) light-induced absorbance changes due to the photoreduction of C-550 and the photooxidation of cytochrome (cyt) b-559 which are PSII-mediated reactions. A fourth-derivative analysis of the absolute spectra of the chloroplast fragments at different stages of reduction (obtained with ferricyanide, ascorbate, and dithionite) showed both the oxidized and reduced forms of C-550 and the reduced forms of cyt c-553, b-559, and b-564 in wild-type fragments. The mutant fragments lacked C-550 and an ascorbate-reducible cyt b-559 but contained cyt c-553, a dithionite-reducible cyt b-559, and cyt b-564.

Ascorbic Acid↗
PubMed1972

On the primary nature of fluorescence yield changes associated with photosynthesis.

Absorbance changes of C-550 and cytochrome b(559), and fluorescence-yield changes were measured during irradiation of chloroplasts at -196 degrees . The photo-reduction of C-550 proceeded more rapidly than the photo-oxidation of cytochrome b(559), and the fluorescence-yield change had similar kinetics to the cytochrome b(559) change. The fluorescence yield of chloroplasts exposed to a 16-musec flash at -196 degrees did not increase during the flash, but increased in the dark after the flash. Both of these experiments indicate that the fluorescence yield follows the dark reduction of the primary electron donor of Photosystem II, not the photoreduction of the acceptor. This explanation would also account for the recent results of Mauzerall [Proc. Nat. Acad. Sci. USA (1972) 69, 1358-1362] showing that the fluorescence yield of chloroplasts at room temperature requires about 20 musec to reach a maximum after a very brief flash.

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PubMed1972

Extraction and Reconstitution of Photosystem II.

Hill activity (oxygen evolution with ferricyanide as the electron acceptor), light-induced absorbance changes at liquid nitrogen temperature associated with the primary activity of photosystem II, and fluorescence yield changes at both low temperature and room temperature were measured with lyophilized spinach chloroplasts before and after extraction with hexane and reconstitution with beta-carotene and plastoquinone A. Extraction eliminated the Hill activity, and both beta-carotene and plastoquinone A were required for maximal restoration of activity to the reconstituted chloroplasts.Extraction also eliminated the light-induced absorbance changes at -196 C due to the photoreduction of C-550 and photooxidation of cytochrome b(559), and reconstitution with beta-carotene and plastoquinone A restored the low temperature photoreactions. However, only beta-carotene was essential for the restoration of the photoreactions. Cytochrome b(559) was modified, as a result of the extraction, to a lower redox potential, autooxidizable form and remained as such after reconstitution with beta-carotene. The beta-carotene-restored chloroplasts showed the photoreduction of C-550 but not the photooxidation of cytochrome b(559) because the cytochrome was already oxidized. When beta-carotene-reconstituted chloroplasts were suspended in buffer containing ascorbate prior to freezing, the cytochrome b(559) was reduced and could be photooxidized by irradiation at low temperature. After reconstitution with beta-carotene plus plastoquinone A the cytochrome b(559) was partially restored to its original high potential form and was in the reduced state so that both the photoreduction of C-550 and the photooxidation of cytochrome b(559) occurred on irradiation of the beta-carotene plus plastoquinone A-reconstituted chloroplasts. Reconstitution with plastoquinone A alone had essentially no effect on restoring the photoreactions.The fluorescence yield of dark-adapted lyophilized chloroplasts at -196 C showed an irreversible increase of about 2.5-fold during irradiation. After extraction the fluorescence yield of the chloroplasts was high (at the maximal light-induced level of the lyophilized control chloroplasts) and showed very little change in the light. Reconstitution with beta-carotene alone restored some fluorescence quenching which was relieved by irradiation at low temperature. Reconstitution with plastoquinone A alone restored a high degree of quenching, but this quenching was not relieved by light at low temperature. Fluorescence emission spectra at -196 C showed that the fluorescence of variable yield in the lyophilized and beta-carotene-reconstituted chloroplasts involved only the 680 and 695 nm emission bands but not the larger 730 nm emission band, whereas the irreversible quenching in plastoquinone A-reconstituted chloroplasts involved all wavelengths of emission. Extraction of the chloroplasts also eliminated the sharp 695 nm emission band at low temperature, and reconstitution with beta-carotene partially restored it.The fluorescence yield changes at room temperature differed from the low temperature measurements in that the strong fluorescence quenching restored to the plastoquinone A-reconstituted chloroplasts was relieved by light and reappeared in the dark. Thus plastoquinone A appeared to be much more effective than beta-carotene in restoring the fluorescence of variable yield in room temperature measurements. However, it is argued from the results at low temperature that the quenching in plastoquinone A-reconstituted chloroplasts, which is probably due to the oxidized form of the quinone, is nonspecific and a different quenching mechanism from that which obtains in normal chloroplasts.The results suggest that extraction with hexane removes plastoquinone A, which interrupts electron transport, and beta-carotene, which disrupts the primary photochemical activity of photosystem II. Reconstitution of the extracted chloroplasts with beta-carotene alone restores C-550 and the primary photochemical activity of photosystem II, and when the photosystem II reaction centers are restored the additional requirement of plastoquinone A for the Hill reaction can be demonstrated.

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PubMed1971

Greening of etiolated bean leaves in far red light.

Eight-day-old dark-grown bean leaves were greened by prolonged irradiation with far red light. Growth, chlorophyll content, oxygen-evolving capacity, photophosphorylation capacity, chloroplast structure (by electron microscopy), and in vivo forms of chlorophyll (by low temperature absorption and derivative spectroscopy on intact leaves) were followed during the greening process. Chlorophyll a accumulated slowly but continuously during the 7 days of the experiment (each day consisted of 12 hours of far red light and 12 hours of darkness). Chlorophyll b was not detected until the 5th day. The capacity for oxygen evolution and photophosphorylation began at about the 2nd day. Electron microscopy showed little formation of grana during the 7 days but rather unfused stacks of primary thylakoids. The thylakoids would fuse to give grana if the leaves were placed subsequently in white light. The low temperature spectroscopy of intact leaves showed that the chlorophyll a was differentiated into three forms with absorption maxima near 670, 677, and 683 nanometers at -196 C during the first few hours and that these forms accumulated throughout the greening process. Small amounts of two longer wavelength forms with maxima near 690 and 698 nanometers appeared at about the same time as photosynthetic activity.

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PubMed1971

Control of senescence in marchantia by phytochrome.

Mature green tissue of Marchantia polymorpha L. bleaches markedly when placed in continuous darkness for 4 days but remains green when given daily 1-hour photoperiods of white light. The tissue, however, is induced to bleach when each daily 1-hour photoperiod is terminated with a brief irradiation with far red light. The bleaching does not occur when each irradiation with far red light is followed by a brief irradiation with red light. The bleaching is taken as an index of senescence since the loss of chlorophyll in the bleached tissue is accompanied by a breakdown of cell organelles and cytoplasm. Phytochrome is clearly implicated in the control of senescence by light. It was also found that 5 minutes of red light given once a day was as effective as the 1-hour photoperiods with white light in preventing the bleaching and that bleaching was induced when each daily 5-minute irradiation with red light was followed by a 10-minute irradiation with far red light.

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PubMed1971

The development of photophosphorylation and photosynthesis in greening bean leaves.

Photophosphorylation and oxygen evolution were measured in 8-day-old dark-grown bean leaves (Phaseolus vulgaris) after various times of greening in far red light and in white light. The sequence of development was the same for both greening regimes, but the processes were much more rapid in white light. The capacity for photophosphorylation, as assayed by the firefly luciferase assay, appeared after 12 hours in far red light. At this stage and for times up to 24 hours, photophosphorylation was not inhibited by 10(-5)m 3-(3,4-dichlorophenyl)-1,1-dimethylurea. At 24 hours, the capacity for oxygen evolution appeared and photophosphorylation became partially inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea at concentrations which inhibited oxygen evolution. In white light photophosphorylation appeared after 15 minutes, and oxygen evolution at one hour. Photophosphorylation became partially sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea when oxygen evolution appeared. Carbonylcyanide m-chlorophenyl-hydrazone inhibited photophosphorylation and photosynthesis at low concentrations, 10(-5)m, with immature leaves, but the leaves developed resistance to carbonylcyanide m-chlorophenyl-hydrazone as they greened.

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PubMed1970

Immunochemical and spectroscopic evidence for protein conformational changes in phytochrome transformations.

Phytochrome was examined by immunochemical and spectroscopic techniques to detect differences between the protein moieties of red- and far red-absorbing phytochrome (P(r) and P(fr)). No differences in the reaction of P(r) and P(fr) with phytochrome antibody were discernible on Ouchterlony double diffusion plates. However, the microcomplement fixation assay showed a greater degree of antibody reaction with P(fr) than with P(r), indicating some difference in the surface characteristics of the two forms. Circular dichroism spectroscopy between 300 and 200 nanometers revealed differences between P(r) and P(fr) which may reflect differences in the protein conformation. The circular dichroism spectrum of P(r) showed a negative band at 285 nanometers which was not present in the spectrum of P(fr), and the large negative circular dichroism band at 222 nanometers with P(fr), associated with the alpha-helical content, was shifted 2 nanometers to shorter wave length with P(r) although there was no change of magnitude of this band. The absorbancy of P(r) and P(fr) is very nearly the same in the 280 nanometer spectral region, but sensitive difference spectra between P(r) and P(fr) did reveal spectra which were similar to solvent perturbation spectra obtained by others with different proteins. In total, the experiments indicate that there are conformational differences between the protein moieties of P(r) and P(fr) but that these differences are rather slight from a standpoint of gross structure.

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PubMed1970

The Cytochromes of Prototheca zopfii.

The respiratory pigments of Prototheca zopfii include seven cytochromes: two c-type cytochromes, a soluble c(549) and a membrane bound c(551); three b-type cytochromes, b(555), b(559) and b(564); and cytochromes a and a(3). Cytochromes a and a(3) could be resolved spectrally in the alpha-band region by reducing the cells in the presence of methanol and cyanide. Methanol shifted the absorption maximum of cytochrome a from 598 to 603 nanometers and permitted dithionite (or substrate) to reduce the cyanide-cytochrome a(3) complex to give a well defined 595-nanometer absorption band. Methanol did not interfere with CO binding by cytochrome a(3), and CO did not alter the methanol effect on cytochrome a. Azide and cyanide, which partially inhibited exogenous respiration, stimulated endogenous respiration. Frozen steady states of the electron transport chain in the presence of cyanide and azide indicated that the stimulation by these inhibitors was due to an increased autooxidation of one of the b-type cytochromes, possibly b(564).

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PubMed1970

Inhibition of Respiration in Prototheca zopfii by Light.

Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a(3). Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The alpha-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a(3) was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated.

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