Synthesis of wheat leaf nitrite reductase de novo following induction with nitrate and light

Nitrite reductase has been purified almost 3000-fold, in 35% yield, to a specific activity of 77 units (mg protein)-1 from wheat leaves using a multi-step procedure with affinity chromatography on ferredoxin-Sepharose as the final step. The purified enzyme, although not homogeneous, exhibited absorption maxima at 278, 390, 568 and 687 nm. Minor contaminants were removed by gel filtration in the presence of sodium dodecyl sulphate to yield a single polypeptide of Mr 60 500 as judged by polyacrylamide gel electrophoresis. Antibodies raised against this polypeptide were shown to cross-react with native nitrite reductase and were used to study the synthesis of nitrite reductase in vivo and in vitro. The increase in nitrite reductase activity following exposure of dark-grown plants to nitrate and light was shown by immunodecoration of Western blots to be due to synthesis de novo. Poly(A)-rich RNA isolated from plants actively synthesising nitrite reductase was shown to direct the synthesis in a rabbit reticulocyte lysate of a polypeptide of Mr 64000 which was immunoprecipitated by antibodies to nitrite reductase.     

Interactions of cholesterol esters with phospholipids: cholesteryl myristate and dimyristoyl lecithin.

The ternary phase diagram of cholesteryl myristate--dimyristoyl lecithin--water has been determined by polarizing light microscopy, scanning calorimetry, and x-ray diffraction. Hydrated dimyristoyl lecithin forms a lamellar liquid--crystalline phase (L alpha) at temperatures greater than 23 degrees C into which limited amounts of cholesteryl myristate (less than 5 wt. %) can be incorporated. The amount of cholesterol ester incorporated is dependent upon the degree of hydration of the L alpha phase. Below 23 degrees C dimyristoyl lecithin forms ordered hydrocarbon chain structures (L beta' and P beta') which do not incorporate cholesterol ester. Comparison with other phospholipid--cholesterol ester--water phase diagrams suggests the following general principles: i) the incorporation of cholesterol ester occurs only into liquid crystalling phospholipid bilayers, ii) the extent of incorporation is temperature-dependent, with increasing amounts of cholesterol ester being incorporated at higher temperatures, and iii) unsaturated cholesterol esters induce increased disordering of the phospholipid bilayers.     

Body fat,rank, and nutritional status in a captive group of Rhesus Macaques

Using weight and skinfold thickness to calculate relative body fat, the fat content of 21 captive female rhesus macaques was estimated. Although age and. pregnancy had no effect on fat, rank was significant, especially during winter, with high-rank females having the highest fat scores. Rank therefore has a significant effect on the health of captive animals and may also affect the health of individuals in feral groups.     

Photoreactivation and dark repair of ultraviolet light-induced pyrimidine dimers in chloroplast DNA.

A UV-specific endonuclease was used to detect ultraviolet light-induced pyrimidine dimers in chloroplast DNA of Chlamydomonas reinhardi that was specifically labeled with tritiated thymidine. All of the dimers induced by 100 J/m2 of 254 nm light are removed by photoreaction. Wild-type cells exposed to 50 J/m2 of UF light removed over 80% of the dimers from chloroplast DNA after 24 h of incubation in growth medium in the dark. A UV- sensitive mutant, UVS1, defective in the excision of pyrimidine dimers from nuclear DNA is capable of removing pyrimidine dimers from chloroplast DNA nearly as well as wild-type, suggesting that nuclear and chloroplast DNA dark-repair systems are under separate genetic control.     

Calponin. Developmental isoforms and a low molecular weight variant

Two-dimensional gel analysis of basic proteins in developing human smooth muscle identifies calponin as a prominent marker of the differentiated phenotype. Adult tissue (human and mouse) typically expresses up to four calponin isoforms, three of which appear sequentially during fetal development: adult myometrial cells express the same three isoforms in primary culture in vitro and these are down-regulated, in reverse order, during the subsequent modulation of phenotype. Monospecific, polyclonal antibodies against calponin identify a lower molecular weight variant of calponin (L-calponin) that is strongly and specifically expressed in adult smooth muscles of the human urogenital tract. L-calponin is down-regulated in benign smooth muscle derived tumors (leiomyoma) and is not expressed in primary cultures of normal uterine tissue.     

Altered G-protein expression and adenylate cyclase activity in platelets of non-insulin-dependent diabetic (NIDDM) male subjects

Adenylate cyclase activity and levels of guanine nucleotide regulatory proteins (G-proteins) were compared in platelets from normal and non-insulin-dependent diabetic (NIDDM) male subjects. Whilst no differences were noted in basal and NaF-stimulated adenylate cyclase activities the degree of stimulation achieved by both forskolin and prostaglandin, E1 was lower by some 34 and 52% respectively, in platelet membranes from diabetic subjects compared with those from normal control subjects. Altered alpha 1-adrenoceptor-mediated inhibition of prostaglandin E1-stimulated adenylate cyclase activity was evident; it being some 34% lower in platelet membranes from diabetic subjects compared to controls. Analysis of G-protein alpha-subunits, using specific anti-peptide antisera, showed that platelets from all subjects exhibited the Gi-2 and Gi-3, but not the Gi-1 forms of the inhibitory G-protein 'Gi' and all expressed the 42 kDa species of alpha-subunit of the stimulatory G-protein Gs. Whilst platelets of diabetic subjects had levels of Gs which were comparable to those found in control subjects their levels of Gi-2 and Gi-3 were some 49 and 75%, respectively, of those found in platelets from control subjects. It is suggested that changes in adenylate cyclase functioning and G-protein expression may contribute to altered platelet functioning in non-insulin-dependent diabetic subjects.     

Structure and polymorphism of saturated monoacid 1,2-diacyl-sn-glycerols

The 1,2-diacyl-sn-glycerols (1,2-DGs) are the predominant naturally occurring isomer found in cell membranes, lipid droplets, and lipoproteins. They are involved in the metabolism of monoacylglycerols, triacylglycerols, and phospholipids. The 1,2-DGs participate in the activation of protein kinase C, in phosphorylation of target proteins, and in transduction of extracellular signals into the cell. We have undertaken a study of the physical properties of a homologous series of synthetic optically active diacylglycerols. Stereospecific 1,2-diacyl-sn-glycerols were synthesized with saturated fatty acyl chains of 12, 16, 18, 22, and 24 carbons in length. Their polymorphic behavior was examined by differential scanning calorimetry and X-ray powder diffraction. The solvent-crystallized form for all the 1,2-DGs packs in the orthorhombic perpendicular subcell (beta') and melts with a single sharp endotherm to an isotropic liquid. On quenching, the C12, C16 and C18 compounds pack in a hexagonal subcell (alpha), whereas the C22 and C24 pack in a pseudohexagonal subcell (sub-alpha). The sub-alpha phase reversibly converts to the alpha phase. The long spacings of these compounds in both the alpha and beta' phases increase with chain length. In the alpha and beta' phases, the acyl chain tilts were found to be 90 degrees and 62 degrees from the basal methyl plane. The polymorphic behavior of 1,2-diacyl-sn-glycerol is quite different from that of the corresponding monoacid saturated 1,3-diacylglycerols which form two beta phases with triclinic parallel subcells.     

Salt induced transitions of chromatin core particles studied by tyrosine fluorescence anisotropy.

Chromatin core particles containing 146 base pairs of DNA have been found to undergo a single defined transition below 10 mM ionic strength as studied by both sedimentation velocity and tyrosine fluorescence anisotropy. A method is described for the preparation of such core particles from chicken erythrocytes with greater than 50% yield.     

Purification and properties of a light-inducible nuclease from Euglena gracilis.

The nuclease described by Carell, E.F., Egan, J.M. and Pratt, E.A. [Arch. Biochem. Biophys. (1970) 138, 26-31] has been purified 1000-fold from Euglena gracilis strain Z. The enzyme catalyzes the hydrolysis of both polyribonucleotides and polydeoxyribonucleotides. The relative rates of hydrolysis of synthetic and natural polynucleotides was found to be: poly (U) 100, poly (dT) 33, denatured calf-thymus DNA 33, yeast tRNA 9, E. coli total RNA 6, poly (dA dT) 5, poly (A) less than 1, poly (C) less than .05, and poly (G) less than .05. The enzyme attacks polynucleotides in an endonucleolytic fashion, yielding products terminated with a 3'-phosphate. Poly (U) appears to be hydrolyzed completely to 3'-UMP; both RNA and DNA appear to have some phosphodiester bonds resistant to enzyme catalyzed hydrolysis. Because of its mode of action and its inducibility by light, we propose the name endonuclease L for this enzyme.