Factors affecting the absorption maxima of acidic forms of bacteriorhodopsin. A study with artificial pigments.

The absorption maximum (568 nm) of light-adapted bacteriorhodopsin bR568 undergoes reversible changes after acidification. At pH 2.9, the absorption shifts to 605 nm (forming bR605) and it blue shifts to 565 nm, after further acidification to pH approximately 0.5 (forming bR565). Molecular models accounting for such acid-induced changes are relevant to the structure and function of bacteriorhodopsin. In the present study we approached the problem by applying artificial bR pigments based on selectively modified synthetic retinals. This may allow direct identification of the specific regions in the retinal binding site where the above changes in the protein-retinal interactions take place. We investigated the spectroscopic effects of acid in a variety of artificial pigments, including cyaninelike retinals, retinals bearing bulky groups at C4, short polyenes, and retinals in which the beta-ionone ring was substituted by aromatic rings. The results provide direct evidence for the hypothesis that the generation of bR605 is due to changes in polyene-opsin interactions in the vicinity of the Schiff base linkage. The second transition (to bR565) was not observed in artificial pigments bearing major changes in the ring structure of the retinal. Two approaches accounting for this observation are presented. One argues that the generation of bR565 is associated with acid-induced changes in retinal-protein interactions in the vicinity of the retinal ring. The second involves changes in polyene-opsin interactions in the vicinity of the Schiff base linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Raman microscope studies on the primary photochemistry of vertebrate visual pigments with absorption maxima from 430 to 502 nm

Raman microscope vibrational spectra have been recorded from single photoreceptor cells frozen at 77 K. Spectra of photostationary steady-state mixtures of visual pigments and their primary photoproducts were obtained from toad red rods (lambda max 502 nm), angelfish rods (lambda max 500 nm), gecko blue rods (lambda max 467 nm), and bullfrog green rods (lambda max 430 nm). All four photoproducts have enhanced low-wavenumber Raman lines at approximately 850, 875, and 915 cm-1 and show the anomalous decoupling of the 11- and 12-hydrogen out-of-plane (HOOP) wagging vibrations, as is observed in the bovine primary photoproduct. The low-wavenumber lines are enhanced in the resonance Raman spectrum by conformational distortion, and the uncoupling of the 11- and 12-hydrogen wags is caused by additional protein perturbations. The similarity of the HOOP modes in all four photoproducts indicates that the protein perturbations that uncouple the 11- and 12-hydrogen wags and that enhance the HOOP modes are very similar. Thus, these perturbations of the photoproduct Raman spectrum cannot be caused by the same protein-chromophore interactions that are responsible for wavelength regulation in these pigments.     

Macromolecular components of the vitelline membrane of hen's egg. I. Membrane structure and its deterioration with age.

The vitelline membrane of hen's egg has been successfully solubilized in sodium dodecyl sulfate (SDS), guanidine hydrochloride and urea solutions, and its macromolecular components examined. SDS-gel electrophoresis of the membrane solution revealed the presence of three major components designated I, II, and III, all containing carbohydrate and protein. The approximate molecular weights of components I and II were 32,000 and 260,000, respectively, and the sedimentation coefficients were 2.2S and 4.3S. Component III was in an aggregated form which disintegrated into smaller components upon reduction with 2-mercaptoethanol. It was found that component II (4.3S component) deteriorated during storage of the egg with the concomitant formation of degraded components. The loss of this component was accompanied by a gradual decrease of the neutral sugar content of the vitelline membrane. On the basis of these data, the membrane structure and its deterioration during storage are discussed.     

Changes in membrane dynamics associated with myogenic cell fusion

Changes in membrane dynamic properties associated with membrane fusion are studied employing in vitro myoblast fusion as a model system. We utilize a microscopic fluorescence relaxation approach which makes feasible the study of local variations in membrane dynamics within surface subdomains of single intact cells. Studies of the average rotational mobility of the fluorescent probe-1-anilino-naphthalene-8-sulfonate by this technique indicate that myoblast fusion activity is preceded by a generalized increased in membrane fluidity and that areas of cell contact between fusing cells exhibit higher fluidity and polarity, locally, than non-fusion regions.     

Light sensitivity of plastids and plastid pigments present in the albescent maize mutant

Dark grown albescent corn seedlings are deficient in colored carotenoids but accumulate phytoene, phytofluene and an unidentified substance in the carotenol fraction. They bleach upon exposure to bright light and appear albino. Seedlings grown under low level incandescent light are normal in appearance and contain almost as much colored carotenoid as control seedlings. The existing leaf tissue of seedlings grown under low level light does not bleach upon exposure to bright light. The enhanced carotenoid synthesis and stabilization of plastids is not affected by brief illumination with red light but requires several hours of low level incandescent light.     

Macromolecular Changes Associated with the Growth of Crustacean Tissues

Tissue mass, rate of protein synthesis, content of ribosomalRNA and rates of synthesis of ribosomal RNA have been studiedthroughout the molting cycle in the midgut gland, epithelium,and somatic muscle in the land crab, Gecarcinns lateralis. Inall tissues there is an increase in ribosomal RNA followed byan increase in the rate of synthesis of protein in the premoltperiod. Subsequently, the three tissues differed in that (a)in the midgut gland the level of ribosomal RNA and protein synthesisreturned to the intermolt rates before ecdysis whether or notthe mass of the tissue was increasing or decreasing; (b) ribosomalRNA and protein synthesis in epithelium reached a maximum ata time when epithelial cells reached a maximal size; subsequently,all three parameters decreased toward intermolt levels beforeecdysis; (c) ribosomal RNA and protein synthesis reached a maximumin the premolt period in somatic muscle while the muscle wasin fact decreasing in mass. Muscle ribosomes are very stableand appear to be conserved for weeks or months to be reusedafter ecdysis in a second burst of protein synthetic activityat the time when there is replacement and growth of new musculartissue. The relation of these events with hormonal control ofgrowth is discussed.     

Changes in the permeability of the inner mitochondrial membrane associated with plastid development

Summary Mitochondria isolated from greening etiolated laminae of Avena sativa L. show changes in the permeability of their inner membranes during chloroplast development similar to those described earlier for plastids. Oxalo-acetate, succinate and -keto-glutarate permeate most readily inner membranes of mitochondria isolated from laminae given 2 h illumination whilst glutamate and glycine show later and more general penetration into the matrix spaces of mitochondria from greening tissue. Aminolevulinic acid (ALA) by contrast does not readily enter Avena mitochondria especially those isolated from laminae illuminated for longer than 2 h.Abbreviations ALA amino-levulinic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane-sulphonic acid     

低温胁迫期间水稻光合膜色素与蛋白水平的变化

对４℃和１１℃两种低温胁迫过程中水稻类囊体膜色素与蛋白组成的变化进行了比较研究。结果表明：４℃低温不仅使类囊体膜中的光合色素（叶绿素、类胡萝卜素）含量降低,而且还引起膜蛋白组成的深刻变化,表现在大部分原有膜蛋白组分的含量在低温下明显降低,同时在低温处理的第３天诱导出一条３２．５ＫＤ的新蛋白带。与４℃处理相比,１１℃低温处理只引起了光合色素含量的降低,而对类囊体膜蛋白组成的影响不大,另外发现,两种低     

Changes in thylakoid structure associated with the differentiation of heterocysts in the cyanobacterium, Anabaena cylindrica.

The thylakoids of vegetative cells of the filamentous cyanobacterium, Anabaena cylindrica, are capable of oxygen-evolving photosynthesis and contain both Photosystems I and II (PSI and PSII). The heterocysts, cells specialized for nitrogen fixation, do not produce oxygen and lack Photosystem II activity, the major accessory pigments, and perhaps the chlorophyll a associated with PSII. Freeze-fracture replicas of vegetative cells and of heterocysts reveal differences in the structure of the thylakoids. A histogram of particle sizes on the exoplasmic fracture face (E-face, EF) of vegetative cell thylakoids has two major peaks, at 75 and 100 A. The corresponding histogram for heterocyst thylakoids lacks the 100 A size class, but has a very large peak at about 55 A with a shoulder at 75 A. Histograms of protoplasmic fracture face (P-face, PF) particle diameters show single broad peaks, the mean diameter being 71 A for vegetative cells and 64 A for heterocysts. The thylakoids of both cell types have about 5600 particles/micrometers2 on the P-face. On the E-face, the density drops from 939 particles/micrometers2 on vegetative cell thylakoids to 715 particles/micrometers2 on heterocyst thylakoids. The data suggest that the 100 A E-face particle of vegetative cell thylakoids is a PSII complex. The 55 A EF particle of heterocysts may be part of the nitrogenase complex or a remnant of the PSII complex. The role of the 75 A EF particle is unknown. Other functions localized on cyanobacterial thylakoids, such as respiration and hydrogenase activity, must be considered when interpreting the structure of these complex thylakoids.     

Accumulation of chlorophyllous pigments esterified with the geranylgeranyl group and photosynthetic competence in the CT2256-deleted mutant of the green sulfur bacterium Chlorobium tepidum

Green sulfur bacteria contain chlorophyllous pigments, chlorophyll (Chl) aPD and bacteriochlorophyll (BChl) aP, esterified with Delta2,6-phytadienol and phytol, respectively, which would be produced by reduction of the geranylgeranyl group at the C-17 propionate residue. In the genome of Chlorobium tepidum, two paralogous genes presumably encoding geranylgeranyl reductase, CT1232 and CT2256, are found. The deletion mutants of the CT1232 and CT2256 genes were constructed using an insertional inactivation method in order to clarify the biosynthetic process of the Delta2,6-phytadienyl and phytyl groups in green sulfur bacteria. The compositions of chlorophyllous pigments in the two mutants were determined by LC-MS analysis. The CT2256-deleted mutant accumulated Chl aGG and BChl aGG esterified with geranylgeraniol, indicating that CT2256 was involved in the production of both Delta2,6-phytadienyl and phytyl groups. The relatively high fluorescence emission from chlorosomes in the mutant also suggested some hindrance of the energy transfer from chlorosomes to the reaction center complex. However, the CT1232-deleted mutant almost showed no apparent phenotype compared to the wild type. Furthermore, the purple bacterium Rhodobacter capsulatus mutant defective in the bchP gene was partially complemented with the CT2256 gene; BChl aP was synthesized in the mutant in addition to accumulating other intermediates.     

Changes in membrane structure and function during chick embryonic erythropoiesis.

We found considerable differences in the pattern of membrane proteins as well as in the relative amounts of individual components in isolated chick red blood cell membranes during the course of embryonic development. Of special interest in the increase in the relative amounts of two major polypeptides, band 3 and 3.1 (MW 100,000 daltons) with increasing age of the cells. With respect to functional studies, we found that the magnitude of sulfate influx decreases with increasing age of the embryo. Furthermore, the activity of ouabain-sensitive ATPase increases with increasing age (2.5-day embryo to adult). In addition, both the basal and the fluoride-stimulated adenylate cyclase activities decrease as the embryo age increases, whereas the enzyme sensitivity to epinephrine increases with increasing age of the embryo.