Light-induced absorbance changes in Fraction I particles prepared from spinach chloroplasts by French-press treatment

Light-induced changes of absorbance were measured in Fraction 1 particles prepared from spinach chloroplasts according to a method developed by

Full-size image

⁶ that employs breakage of chloroplasts in the French pressure cell and centrifugation of the resulting fragments on a sucrose density gradient. Absorbance changes were measured in the presence of ascorbate and 2,3,5,6-tetramethyl-1,4-phenylenediamine as an electron donating system. In addition to the oxidation of P₇₀₀ and cytochrome f, that was investigated in our previous study¹¹, three other light-induced absorbance changes were observed; reduction of cytochrome b with a peak at 562 nm, the so-called 515-nm change showing an absorbance increase at 515 nm and a decrease at 480 nm, and a broad band of unknown absorbance increase covering wave-lengths from 490 to 570 nm. Uncouplers such as carbonylcyanide-m-chlorophenylhydrazone and gramicidin D inhibited the 515-nm change under continuous light and accelerated the dark decay of flash-light-induced change. The other broad absorbance change was insensitive to these inhibitors.     

Demonstration of intracytoplasmic glycogen of megakaryocytes and blood platelets by means of the periodic acid-thiocarbohydrazide-silver proteinate method

Summary The glycogen of megakaryocytes and blood platelets has been investigated in glutaraldehyde and osmium tetroxide fixed tissues by the periodic acid-thiocarbohydrazide-silver proteinate method (PA-TCH-SP). The PA-TCH-SP method involves the staining of intracytoplasmic glycogens more densely than the routine lead citrate method. Glycogen having a mean particle diameter of 21.1 nm has been shown localizing in the matrix of mature megakaryocytes, while that of glycogen in the platelets was 26.2 nm. The staining pattern of the glycogen in blood platelets was classified into three groups according to staining intensity.It is found that the PA-TCH-SP method is a very suitable one for the demonstration of intracytoplasmic glycogen from the viewpoints of reaction specificity, reproducibility, fineness of reaction products, sufficiency of electron density, and experimental cost. This method is also a very useful one for differentiating intracytoplasmic glycogens and ribosomes.     

Molecular cloning of the Pseudomonas putida glyoxalase I gene in Escherichia coli

The glyoxalase I gene of Pseudomonas putida was cloned onto a vector plasmid pBR 322 as a 7.5 kilobase Sau 3AI fragment of chromosomal DNA and the hybrid plasmid was designated pGI 318. The gene responsible for the glyoxalase I activity in pGI 318 was recloned in pBR 322 as a 2.2 kilobase Hin dIII fragment and was designated pGI 423. The P. putida glyoxalase I gene on pGI 318 and pGI 423 was highly expressed in E. coli cells and the glyoxalase I activity level was increased more than 150 fold in the pGI 423 bearing strain compared with that of E. coli cells without pGI 423. The E. coli transformants harboring pGI 318 or pGI 423 could grow normally in the presence of methylglyoxal, although the E. coli cells without plasmid were inhibited to grow and showed the extremely elongated cell shape.     

The histochemistry of asparagine-linked oligosaccharides of glycoproteins in mammalian and avian tissues as studied by digestion with almond glycopeptidase

Summary In a series of mammalian and avian tissues, asparagine-linked oligosaccharides of glycoproteins have been localized histochemically by mearis of combined periodic acid-Schiff (PAS) and almond glycopeptidase digestion procedures. According to the present results, the particular saccharide residues were found to be localized primarily in glycoproteins of connective and muscular tissue elements but were hardly visualizable or absent in glycoproteins of epithelial tissue elements. In view of the substrate specificity of almond glycopeptidase and the known staining selectivity of the PAS reaction, the majority of connective and muscular tissue elements are thought to be the main sites where plasma types of glycoproteins are involved.     

Phenotype character of the methylglyoxal resistance gene in Saccharomyces cerevisiae: expression in Escherichia coli and application to breeding wild-type yeast strains.

The gene responsible for the methylglyoxal resistance of Saccharomyces cerevisiae was cloned, and its phenotypic characteristics were investigated. S. cerevisiae cells with the gene could accumulate large amounts of glutathione in the medium and should remarkably high resistance to various toxic compounds such as methylglyoxal, tetramethylthiuram disulfide, iodoacetamide, and heavy-metal ions. The gene was also expressed in Escherichia coli cells, and the resistance of E. coli cells to toxic compounds also increased as observed for S. cerevisiae cells. The phenotypic characteristics of the gene were applicable to the selection of the transformants of wild-type yeast strains having no genetic markers.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins, pigment system II particles and chlorophyll a in diethylether solution by the curve-fitting method.

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25 degrees C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol 49, 421--429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm. The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0--2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6--4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively. Absorption spectra at 25 degrees C and at --196 degrees C of the water-soluble chlorophyll proteins were compared by the curve-fitting methods. The component bands at --196 degrees C were blue-shifted by 0.8--4.1 nm and narrower in half widths as compared to those at 25 degrees C.     

Temperature dependence on the delayed fluorescence of chlorophyll a in blue-green algae.

1. The delayed fluorescence of chlorophyll a was measured with a phosphoroscope by changing the temperature in a range of room temperatures in intact cells of blue-green algae, Anacystis nidulans, two strains of Anabaena variabilis and Plectonema boryanum, and other kinds of algae, Cyanidium caldarium and Chlorella pyrenoidosa. The induction of delayed fluorescence remarkably depended on the temperature of measurment. Nevertheless, the induction pattern was characterized by three levels of intensity; the initial rise level at the onset of excitation light, the maximum level after a period of excitation and the steady-state level after 10 min of excitation. 2. In A. nidulans and a strain of A. variabilis grown at various temperatures, close relationship was found between the phase transition of membrane lipids and the initial rise and the steady-state levels of delayed fluorescence. The initial rise level showed the maximum at the temperature of phase transition between the liquid crystalline and the mixed solid-liquid crystalline states, The steady-state levels showed a remarkable change from a high in the liquid crystalline state to a low level in the mixed solid-liquid crystalline state. 3. The millisecond decay kinetics of the delayed fluorescence measured at the steady-state level in A. nidulans grown at 38 degrees C consisted of two components with different decay rates. The half-decay time of the fast component was about 0.17 ms and was constant throughout the temperature range of measurement. The half decay time of slow component ranged from 0.6 to 1.5 ms, depending on the temperature of measurment.     

Combined gas chromatography-chemical ionization mass spectrometry of sphingolipids. I. Glucosyl sphingosine,ceramides and cerebrosides of the spleen in Gaucher's disease

Trimethylsilylated glucosyl sphingosine, ceramides and glucocerebrosides were analysed by combined gas chromatography (GC)-chemical ionization (CI) mass spectrometry. Isobutane, methane and ammonia were used as reactant gases for chemical ionization. Essentially the same fragment ions were detected in the spectra with the different reactant gases.Purified glucocerebrosides isolated from the spleen of a patient with Gaucher's disease were clearly separated into their 8 molecular species by gas chromatography. Three other minor components were detected by spectrometry, and these 11 molecular species of glucocerebrosides from the spleen of the patient with Gaucher's disease have been analysed.The ceramides obtained by periodate oxidation and then alkaline reduction of the glucocerebrosides were also separated into 11 molecular species by GC-CI mass spectrometry.Molecular weight could be determined using the major fragment ion of m/e (M⁺?90) in the spectra of ceramides and cerebrosides. The chemical ionization method is useful for structural analyses and determination of the molecular species of sphingoglycolipids.