Purification of membrane polypeptides of rat liver peroxisomes

Peroxisomes were obtained by sucrose density gradient centrifugation from the livers of di(2-ethylhexyl)phthalate-fed rats, and the membranes were prepared by carbonate extraction (Fujiki, Y., Fowler, S., Shio, H., Hubbard, A.L., & Lazarow, P.B. (1982) J. Cell Biol. 93, 103-110). The integrated membrane polypeptides were solubilized with sodium dodecyl sulfate, and purified by repeated polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Separation of 70 and 68 kDa polypeptides was not attempted in the present study because of their close migration in polyacrylamide gel electrophoresis. Other polypeptides with apparent molecular masses of 41, 27, 26, and 22 kDa were purified to near homogeneity. Antibodies were raised against these purified preparations. The 68 kDa polypeptide is suggested to be produced by the proteolytic modification of 70 kDa polypeptide, since the former increased concomitantly with decrease of the latter when the liver homogenate was incubated, and this change was prevented in the presence of leupeptin during the incubation. The 41 kDa polypeptide was a minor component. The 70 and 68 kDa polypeptides and 41 kDa polypeptide and their antibodies were cross-reactive, but the relation of these polypeptides was not clear. The 27 and 26 kDa polypeptides seemed to be another species of membrane polypeptides, although the relationship of these two polypeptides remains to be clarified. The 22 kDa polypeptide is not related to other membrane polypeptides. The results of immunoblot analysis of subcellular fractions of the liver and an electron microscopic immunocytochemical study to locate the antigenic sites with protein A-gold complex suggest that all of these polypeptides are localized on peroxisomal membranes. On proliferation of rat liver peroxisomes by administration of di(2-ethylhexyl)phthalate, a peroxisome proliferator, all of these polypeptides were markedly increased.     

Reaction of the hydrated electron with histone H1 and related compounds studied by e.s.r. and spin-trapping

The reactions of the hydrated electron with histone H1, protamine and related compounds (poly-L-lysine, poly-L-arginine and poly-D,L-alanine) were investigated by the spin-trapping technique. In order to identify the radical structure of the spin-adducts originating from macromolecules, the usual spin-trapping technique was developed as follows: N2-saturated aqueous solutions of proteins containing sodium formate were X-irradiated (4.5 kGy) in the presence of 2-methyl-2-nitrosopropane (MNP) as a spin-trap. The side-products due to the self trapping of MNP radicals were then removed from the spin-adducts of the proteins by a Sephadex G-25 column. Finally the spin-adducts were enzymatically digested to transform the broad e.s.r. signals due to slow tumbling of nitroxyl radicals to identifiable ones. The e.s.r. spectra obtained for all samples showed that the deaminated radical, R--CH--CO--NH--(R:amino acid side chain), was produced. Furthermore, polyacrylamide gel electrophoresis of the irradiated protamine and histone H1 indicated reduction of molecular size. These results confirm that hydrated electrons react with proteins and induce the deamination reaction which leads to main-chain scission.     

Separation and Characterization of Thylakoid and Cell Envelope of the Blue-green Alga (Cyanobacterium) Anacystis nidulans

The thylakoid and the cell envelope of the blue-green alga Anacystisnidulans were separated by mechanical disruption of lysozyme-treatedcells followed by differential and density gradient centrifugation.The prepared envelope was composed of an outer membrane, a peptidoglycanlayer and possibly a part of the cytoplasmic membrane. The preparedthylakoid retained the size and intricate structure typicalof the thylakoid membrane of this alga. Light absorption andfluorescence spectra revealed that the envelope contained carotenoids,a pigment with an absorption maximum at 748 nm (P750), and asmall amount of pheophytin-like pigment with an absorption maximumat 673 nm. The thylakoid contained chlorophyll a and carotenoidsbut no P750. The thylakoid contained five kinds of carotenoids,the major ones being rß-carotene and zeaxanthin, whereasthe cell envelope contained two kinds of carotenoids, zeaxanthinand nostoxanthin. Four kinds of lipids, abundant in the blue-greenalgae, were present in both the thylakoid and the cell envelope.However, the content of sulfolipid was very low in the cellenvelope. The polypeptide compositions differed between thethylakoid and the cell envelope. Similarities between blue-greenalgal cells and eukaryotic chloroplasts are discussed with respectto the spectrophotometric and biochemical characteristics ofthe thylakoid and the envelope. (Received March 7, 1981; Accepted May 22, 1981)     

The fine structure of the compound eye of a damsel-fly

Summary The compound eyes of two species of damsel-flies, Ishunura senegalensis and Cersion calamorum, were examined by electron microscopy. Each ommatidium is composed of eight retinula cells which are semistratified in the receptor layer. The retinula cells are divided into four types from the difference of levels in the rhabdom formation; one distal large cell having the rhabdomere only in the distal layer, four middle cells forming the rhabdom in the middle layer, two proximal cells making up the rhabdom in the proximal layer and one distal small cell having no rhabdomere in any layers. In addition, the lamina ganglionaris was partly observed. Some retinula axons terminate at an different level from the other axons. The functional differentiation among these different types of cells is discussed with relation to the analysis of the polarized light and the discrimination of the diffraction images.This work is supported by a grant from the U.S. Army Research and Development Group (Far East), Department of the Army (DA-CRD-AG-S29-544-67-G61).The authors wish to express their gratitude to Drs. H. Morita and H. Tateda for their helpful discussions throughout this study.     

Spin trapping of precursors of thymine damage in X-irradiated DNA

A spin-trapping method combined with ESR spectroscopy was utilized to obtain evidence for the presence of precursor radicals leading to damage in X-irradiated DNA. Two technical improvements were introduced to the conventional spin-trapping method to make possible its application to large molecules such as DNA: prior to X irradiation, sonolysis of aqueous DNA solution by 19.5-kHz ultrasound was made to get a highly concentrated DNA solution and to lower the viscosity of the solution; after precursor radicals in X-irradiated DNA were trapped by a spin-trapping reagent, the DNA was digested to oligonucleotides by DNase I to get an ESR spectrum with a well-resolved hyperfine structure. Thus, it was recognized that the ESR spectrum obtained after X irradiation of the aqueous solution containing DNA and the nitroso spin-trapping reagent 2-methyl-2-nitrosopropane consisted of at least three sets of signals in the DNA. Identification of free radicals was made by comparing the spectrum with that of thymidine, which was precisely examined by a spin-trapping method combining two kinds of spin traps (nitroso and nitrone compounds) with liquid chromatography. As a result, all the signals were identified as the spin adducts of radicals produced at the thymine base moiety of DNA. The 5-hydroxy-5,6-dihydrothymin-6-yl radical was identified as a precursor of 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), the 6-hydroxy-5,6-dihydrothymin-5-yl radical as a precursor of 6-hydroxy-5,6-dihydrothymine, and the 5-methyleneuracil radical as a precursor of 5-(hydroxymethyl) uracil.     

Coprophagy in female mice during pregnancy and lactation

Coprophagy in female mice was observed predominantly in the reproductive stage. Female mice exhibited coprophagy more frequently during pregnancy and ingested larger amounts of feces during pregnancy and lactation than when they were not pregnant. Feces were found to be rich in vitamin B12 and folic acid. However, there were no marked fluctuations in the levels of either vitamin in the feces during pregnancy or lactation as compared with levels when animals were not pregnant. Acceleration of coprophagy during pregnancy and lactation seemed to correlate with the increased nutritional requirements of females during the reproductive stage.     

Stoichiometry of components in the photosynthetic oxygen evolution system of Photosystem II particles prepared with Triton X-100 from spinach chloroplasts

The stoichiometry of the proteins of the photosynthetic oxygen evolution system and of the electron transport components in Photosystem II particles prepared with Triton X-100 from spinach chloroplasts were determined. Per about 220 chlorophyll molecules, there were one reaction center II, one molecule each of the 33, 24 and 18 kDa proteins, four Mn atoms, two cytochromes b-559 (one high-potential, the other low-potential), and 3.5 plastoquinone-9 molecules, but practically no cytochrome b-563, cytochrome f, phylloquinone, α-tocopherol or α-tocopherylquinone.     

The cellular eye lens and crystallins of cubomedusan jellyfish

Summary The ultrastructure and major soluble proteins of the transparent eye lens of two cubomedusan jellyfish,Tripedalia cystophora andCarybdea marsupialis, have been examined. Each species has two complex eyes (one large and one small) on four sensory structures called rhopalia. The lenses consist of closely spaced cells with few organelles. The lens is situated next to the retina, with only an acellular layer separating it from the photoreceptors. SDS-PAGE showed that the large lens ofC. marsupialis has only two crystallin polypeptide bands (with molecular masses of approximately 20000 and 35000 daltons), while that ofT. cystophora has three bands (two with a molecular mass near 20000 daltons and one with a molecular mass near 35000 daltons). Interestingly, the small lens ofT. cystophora appears to be markedly deficient in or lack the lower molecular weight proteins. The crystallins behaved as monomeric proteins by FPLC and showed no immunological reaction with antisera of the major squid crystallin, chicken-crystallin or mouse-crystallin in western immunoblots. Very weak reactions were found with antimouse- and-crystallin sera. The 35000 dalton crystallin ofT. cystophora was purified and called J1-crystallin. It contained relatively high leucine (13%) and tyrosine (9%) and low methionine (2%). Several tryptic peptides were sequenced. Weak sequence similarities were found with- and-crystallins, which may account for some of the apparent weak immunological crossreactivity with these vertebrate crystallins. A polyclonal antiserum made in rabbits from a synthetic peptide of J1-crystallin reacted strongly with J1-crystallin ofT. cystophora andC. marsupialis in immunoblots; by contrast, no reaction was obtained with the lower molecular weight crystallins from these jellyfish, with the squid crystallin, or with any crystallins from the frog or human lens. Thus, despite the structural similarities between the cubomedusan, squid and vertebrate lenses, their crystallins appear very different.Abbreviations SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - bp base pairs - PTC phenylisothiocyanate - FPLC fast phase liquid chromatography - NBRF National Biomedical Research Foundation A portion of this work was presented by Joram Piatigorsky at the First Hans Bloemendal Lecture in June 1988 in Nijmegen, The Netherlands