Michio Itoh

Shrinkage of whole chloroplasts upon illumination

Abstract The effect of light on the size and shape of whole spinach chloroplasts were studied by observing the changes of the volume distribution, the packed volume, the light-scattering cross-sectional area and the axial ratio measured by electron microscopy. Upon illumination of a chloroplast suspension, the volume at the distribution maximum decreased to 51–78% of the volume before illumination, and this volume change was reversed when the light was turned off. This phenomenon of shrinkage was confirmed also by the observations of the packed volume. On the other hand, the light-scattering cross-sectional area increased in the light and decreased in the dark. These changes of the area were interpreted as due to the changes of the axial ratios of chloroplasts, which were confirmed by electron-microscopic observations. These results are presented in this paper together with the effects of ATP and the inhibitors of photophosphorylation and the Hill reaction.

Photo-bleaching of carotenoids related to the electron transport in chloroplasts

Aerobic photo-bleaching of carotenoids in isolated spinach chloroplasts was investigated by measuring the absorption spectrum of chloroplasts and by analysing carotenoid components by means of thin-layer chromatography on microcrystalline cellulose. Carotenoids were irreversibly bleached when chloroplasts were illuminated by red light in the presence of ferricyanide or an inhibitor of the Hill reaction such as azide, hydroxylamine and carbonylcyanide m-chlorophenylhydrazone, while carotenoids in the chloroplasts suspended in a phosphate buffer in the absence of these agents underwent no bleaching on illumination. The photo-bleaching of carotenoids in the presence of these agents was suppressed by another group of inhibitors of the Hill reaction such as o-phenanthroline, substituted phenylureas and symmetrical triazines, or by reducing agents such as ascorbate and ascorbate plusN,N,N′,N′-tetramethyl-p-phenylenediamine. Chromatographic analysis of carotenoids extracted from the chloroplasts illuminated in the presence of carbonylcyanide m-chlorophenyl-hydrazone or ferricyanide showed that three major carotenoid components, carotenes, lutein and violaxanthin, were bleached to similar and considerable extents. Neoxanthin was much more resistant to the action of light. The photo-bleaching of lutein and violaxanthin proceeded more rapidly in air than in a nitrogen atmosphere, but the bleaching of carotenes proceeded at an equal rate under these conditions. These results are discussed in relation to the photochemical electron transport in chloroplasts.

The site of manganese function in photosynthetic electron transport system

Abstract The effects of Mn 2+ on aerobic photobleaching of carotenoids, on photoreduction of 2,6-dichlorophenolindophenol (DCIP) and on fluorescence above 600 mμ of spinach chloroplasts washed with 0.8 M Tris-HC1 buffer were investigated. Carotenoids (mostly carotenes, lutein and violaxanthin) in the Tris-washed chloroplasts were irreversibly bleached by illumination with red light, while carotenoids in normal chloroplasts prepared with a low concentration of Tris-HC1 underwent no bleaching upon illumination. The photobleaching of carotenoids observed with Tris-washed chloroplasts was inhibited by Mn 2+ (MnCl 2 or MnSO 4 ) as well as by some inhibitors of the Hill reaction such as dichlorophenyl-1,1-dimethylurea (DCMU), methylthio-4,6-bis-isopropylamino- s -triazine and o -phenanthroline or by reducing agents such as ascorbate plus tetramethyl- p -phenylene diamine (TMPD). DCIP photoreduction, which was deactivated by Tris, was reactivated to 50–80% of the rate for normal chloroplasts upon addition of Mn 2+ . The restored photoreduction of DCIP was inhibited by DCMU and carbonylcyanide m -chlorophenylhydrazone (CCCP). The steady-state fluorescence yield of normal chloroplasts measured at room temperature was lowered by Tris treatment, and the decreased yield was restored by adding Mn 2+ as well as ascorbate plus TMPD. CCCP also lowered the yield; the yield was recovered by adding ascorbate plus TMPD. Determination of manganese in normal and Tris-washed chloroplasts showed that 30% of the manganese in chloroplast was removed with Tris. It was postulated that Mn 2+ functions in the electron transport on the oxidizing side of Photosystem II at a site between water and an electron carrier ( Y ). CCCP as well as Tris inhibits the reduction of Y + by Mn 2+ , and carotenoids are oxidized by Y + which is reduced by ascorbate plus TMPD.

ACTION SPECTRUM FOR THE SHRINKAGE OF CHLOROPLASTS.

Abstract The effect of monochromatic light on the rate of shrinkage of whole chloroplasts—a new response of the plastids to light, recently discovered by the present authors—was investigated. The relative rate of shrinkage of isolated spinach chloroplasts on illumination was determined using the Coulter counter. The action spectrum obtained showed two major peaks at 435 and 680 mμ with a minor peak and a shoulder at 720–740 and 490 mμ, respectively. The significance of this spectrum is discussed referring to the absorption spectrum of chloroplasts as well as the action spectra so far reported for other photosynthetic reactions.

The subunit structure of porcine ceruloplasmin

Abstract Treatment of porcine ceruloplasmin with 8 M urea results in the formation of heterogeneous components. However, when p -chloromercuribenzoate (PCMB) or 2-mercaptoethanol is added together with urea ceruloplasmin is dissociated into two subunits of identical molecular weight. Ceruloplasmin is also dissociated into half-molecules upon exposure of the native protein to pH 2.1 buffer or upon exposure of the succinylated protein to pH 12.5 buffer. Starch gel electrophoresis reveals that the subunits thus obtained are homogeneous. The N- and C-terminal amino acids were found to be two tyrosyl and two serine residues, respectively. These results may indicate that porcine ceruloplasmin is composed of two subunits. The frictional ratio values of the subunits are appreciably greater than the corresponding values for the native protein, suggesting a significant increase of asymmetry for the subunits on dissociation. On dissociation into subunits, the oxidase activity and blue color of native protein are lost, and the prosthetic copper becomes removable by EDTA.

Chromatographic separation of different species of cells with ion exchange resin

Abstract A technique to separate different species of cells chromatographically with anion exchange resins as the adsorbent was developed and applied to mixtures of several species of cells to examine the degree of separation. Resin granules adsorbing cells on the surface were placed on the top of a resin column without cells, and the elution of cells was made with salts (NaCl, sodium acetate, KI, or phosphates); the resin granules were stirred with a square rod inserted into the column in a cylindrical glass tube. The concentration gradient elution of mixed cells gave a separation pattern with peaks, each of a single species of cells. The separation was complete between bakers yeast and Escherichia coli or Chlorella cells, less satisfactory between bakers and wine yeast cells, and partial between bakers yeast and Trigonopsis cells. Rapidity in practicing the separation is one of the characteristics of this newtechnique, since the whole process of separation could be completed within an hour. The applicabilities of the technique for analysis or separation of mixed cells in suspension were discussed.

Effect of menadione on the electron transport pathway of yeast mitochondria

Abstract The respirations of yeast mitochondria with NADH and citrate as substrates were accelerated by the addition of menadione. The accelerated respirations were sensitive to cyanide, but insensitive to antimycin A. When the respirations of mitochondria with these substrates were inhibited by antimycin A, the addition of menadione resulted in a restoration of respiration which was sensitive to cyanide. When the respirations were inhibited by cyanide, the addition of menadione resulted in only a slight restoration of respiration. The menadione-accelerated or -restored respirations were both insensitive to dicumarol. When the respiration of mitochondria with NADH as substrate was inhibited by antimycin A, only the bands of cytochrome b were reduced. The addition of menadione to the antimycin A-blocked system resulted in a rapid and complete reduction of cytochromes c + c 1 and a . It may be suggested from these results that menadione bridged the portion of the electron transport pathway of NADH-oxidation of yeast mitochondria involving an antimycin A-sensitive site. Reduced menadione appeared to enter the pathway at the cytochrome c + c 1 region. Other quinones, such as 1,4-naphthoquinone, p -benzoquinone, coenzyme Q 6 and vitamin K 1 , could not replace menadione.

Absorbance changes of the 503 mμ pigment as related to the respiration of yeast cells

Abstract The 503 mμ band, which is detectable in the absorption spectrum of aerobically growing cells of bakers yeast, was further intensified in cells collected after prolonged cultivation. The cells showed another new band at 475 mμ, besides the 503 and 550 mμ bands in the visible region. Upon addition of various carbonyl reagents, the 475 and 503 mμ bands disappeared. These two bands, as well as the 550 mμ band, were flattened upon oxygenation and reappeared upon deoxygenation of cell suspensions. The reduction of the 503 mμ pigment and the c -type cytochrome (550 mμ pigment) started at the same oxygen tension of the suspension. In the presence of respiratory substrates or inhibitors other than cyanide and azide, onset of the reduction of both pigments was accelerated or delayed. In almost all of these cases, reduction of both pigments also started simultaneously. With the inhibitors, the reduction rate of the 503 mμ pigment was lowered while the oxidation rate was not appreciably affected. With cyanide or azide, oxidation of both pigments by aeration was completely inhibited.