Superoxide dismutase requires ascorbate for activation of cerebral soluble guanylate cyclase

Guanylate cyclase in 100,000 × g supernatant fraction prepared from the rat brain was markedly activated by superoxide dismutase. Although guanylate cyclase in pH 5.0 precipitated fraction prepared from the supernatant fraction failed to be stimulated by superoxide dismutase, the addition of ascorbate restored the responsiveness. Guanylate cyclase in the supernatant fraction which was preincubated with ascorbate oxidase also showed no response to superoxide dismutase. The superoxide dismutase-induced activation of guanylate cyclase in the supernatant fraction as well as in the ascorbate-added pH 5.0 precipitated fraction was completely eliminated by the addition of KCN, diethyldithiocarbamate, Tiron, retinol or hemoglobin.     

Aspects of the protein and the lipid composition of myelinoid marchi-positive bodies from mammalian spinal cord

The fraction floating on 0.32 M sucrose was isolated from normal mammalian spinal cord and analyzed with regard to protein and lipid composition. Comparisons were made with the myelin fraction isolated from the same spinal cord. A close relationship between the two fractions was indicated by a similar protein banding on SDS-polyacrylamide gel electrophoresis. The relative amounts of various proteins however were different and some high molecular weight proteins appeared unique to the floating fraction. The phospho- and galactolipid patterns, as revealed by thin-layer chromatography, were similar in the floating and the myelin fractions. The proportion of hydrophobic lipids, such as sterols and isoprenyl derivatives, was higher in the floating fraction. Bands co-migrating with cholesterol esters were detected only in the floating fraction from guinea pigs. Marchi-positive material of possible paranodal origin is enriched in the floating fraction. The present findings of a biochemical composition of the floating fraction closely resembling that of myelin is in line with the view that myelin turnover includes a step of degradation localized to the paranodal regions.     

Flower-inducing and -inhibiting activities of Phloem Exudate from Cotyledons of Pharbitis Seedlings

The flower-inducing and -inhibiting activities of phloem exudate (PE) prepared from cotyledons of Pharbitis seedlings were examined, using apex cultures in vitro from Pharbitis as a bioassay system.The PE was prepared from photoperiodically-induced cotyledons (SD-PE). The SD-PE was subjected to the following fractionations: When the SD-PE was extracted with CHCl₃ and then ethyl acetate, the inducing activity was located in the final aqueous fraction. The activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). The diffusate was fractionated by ion exchange chromatography, and flower-inducing activity was found in the fraction adsorbed onto anion exchange resin. When the fraction was applied to a Sep-Pak C18 cartridge, the activity eluted with 25% MeOH. As a result of the above fractionation, activity was increased about 30-fold.The nature of the flower-inhibiting activity of the PE taken from cotyledons exposed to continuous-light conditions was examined (CL-PE). The inhibiting activity was decreased as the cotyledons were exposed to longer dark periods; it appeared to be heat-stable. The CL-PE also inhibited flowering in Lemna. The CL-PE was subjected to the following fractionations: When the CL-PE was extracted with CHCl₃ and ethyl acetate, activity was located in the final aqueous fraction. Activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). When the diffusate was fractionated by ion exchange chromatography, the activity was found in the flow-through fraction. When the fraction was applied to a hydroxyapatite cartridge, the activity eluted with 25 mM sodium phosphate buffer. When the fraction was re-dialyzed (molecular weight cut off was 1,000), the diffusate contained the activity. As a result of the above fractionation, activity was increased about 10-fold.     

Affinity of a DNA with highly repetitive sequence for nuclear proteins from rat liver

A nuclear scaffold fraction (designated P fraction elsewhere) comparable to a nuclear matrix was prepared from rat liver. This fraction was composed mainly of 45-49 kDa proteins and high-molecular-weight proteins (more than 90 kDa). In addition, a 370-bp repetitive sequence DNA fragment was derived predominantly from the EcoRI digest of the deproteinized P fraction. By an immunoblot affinity assay with the P fraction, the fragment was shown to have affinity for each of the 107- and 115-kDa proteins. Moreover, by a filter binding assay with a mixture of these proteins, the affinity level was estimated to be about 6 times as high in the native (double-stranded) fragment as in the denatured (single-stranded) fragment.     

Autocrine factor participation in defence of cytotoxic CTLL-2 cells from oxidative stress

The role of autocrine factors (AF) secreted by cytotoxic IL-2-dependent CTLL-2 cells along with pyruvate in cell defense from oxidative stress was investigated. The addition of conditioned medium (CM) containing pyruvate and AF into CTLL-2 cell cultures increased significantly cell survival under oxidative stress condition. The kinetics of hydrogen peroxide removal from cell cultures under oxidative stress in the case of CM addition has been obtained. The removal of H2O2 mostly by means of its reaction with pyruvate that is contained in CM has been shown at the beginning of oxidative stress (up to 15 min). Pyruvate content in CM was determined as 138 +/- 7 microM. Cell filtration on column with Bio-Gel P-10 was used for removal pyruvate from CM. Three fractions of CM (A, B and C) were obtained as a result of gel filtration. Pyruvate was not detected in any fraction. The fraction A was eluted from column as the first one and contained the largest molecules. Cell survival test showed the fraction B to have the highest ability to protect CTLL-2 cells under oxidative stress. The fraction A supported cell survival to a less degree and fraction C was shown to have no protective ability. The addition of the fraction B to the cell cultures resulted in preservation of significantly higher intracellular ATP level in the cells under oxidative stress than in the control ones. Moreover, AF of the fraction B was shown to react directly with hydrogen peroxide and inactivate it in the absence of cells. AF of the fraction A did not have such properties.     

Identification of the membrane form of phenylalanine hydroxylase from the human liver and characteristics of its subunit composition

Phenylalanine hydroxylase was detected among human liver bioptats and autoptats extracted with 0.4% Triton X-100 from the 105,000 g homogenate fraction. In contrast to the membrane form of the rat liver enzyme, human liver phenylalanine hydroxylase is detected both by its enzymatic activity and immunochemically under non-denaturating conditions. The enzymatic activity of phenylalanine hydroxylase makes 5-15% of that of the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction as can be evidenced from rocket immunoelectrophoresis data. Immunoblotting of proteins performed after denaturating electrophoresis of the membrane and cytoplasmic fractions revealed an antigen band with a similar electrophoretic mobility. The subunit composition of the enzyme in both fractions was characterized by two-dimensional electrophoresis with subsequent immunoblotting. It was found that the membrane fraction of the enzyme is represented only by the L-subunit with Mr of 55 kD, whereas the cytoplasmic fraction, besides the predominant L-subunit, also contains 2H-subunits of the enzyme with Mr = 57 kD. These 2H-subunits differ between themselves as well as from the L-subunit by the pI value.     

The Presence of Phospholipase D In Rat Central Nervous System Axolemma

An axolemma-enriched fraction prepared from a purified myelinated axon fraction isolated from rat CNS was found to contain phospholipase D at a specific activity similar to that of a microsomal fraction isolated from whole brain. There was a concomitant threefold enrichment in the specific activity of phospholipase D and acetylcholinesterase in the axolemma-enriched fraction compared with the specific activities of these enzymes in the starting white matter whole homogenate. This axonal phospholipase D may be involved in remodeling of phospholipid, which in turn may affect axonal functions such as ion translocation.     

Partial purification of fatty-acid binding protein by ammonium sulphate fractionation

By fractionation of rat liver cytosol with 70% saturation ammonium sulphate, a soluble fraction showing high affinity for oleic acid was obtained. The binding of oleic acid to this fraction was inhibited by flavaspidic acid. The molecular weight of the main protein present in this fraction was 12 000 as determined by SDS-poly-acrylamide-gel electrophoresis. This soluble fraction stimulated the transfer of oleic acid from microsomes to phosphatidylcholine liposomes as demonstrated by a transfer assay in vitro. The behaviour of this fraction is similar to that described for fatty-acid binding protein.     

Inhibitory effect of the partially resolved coordination isomers of chromic desferricoprogen on coprogen uptake in Neurospora crassa.

Two partially resolved chromatographic fractions of geometrical and optical isomers of the chromic complexes of desferricoprogen, a siderophore from Neurospora crassa, were obtained from high-pressure liquid chromatography on a reverse-phase matrix. The first fraction was identified as a cis complex with a 20% diastereomeric excess of the lambda isomer. The second fraction was identified as a mixture of several of the possible trans isomers with a net 20% diastereomeric excess of the delta isomers. These fractions were used to evaluate the stereospecificity of the coprogen-mediated iron uptake system with respect to the metal coordination center. Fraction II competitively inhibited coprogen uptake, whereas fraction I showed only slight inhibition. N. crassa accumulated chromium from fraction II faster than the rate of chromium uptake from fraction I. Neither fraction had a significant effect on the uptake of ferricrocin, suggesting that coprogen and ferricrocin are taken up by different receptor systems.     

Plasma membrane vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis

Absolute orientations (sidedness) of plasma membrane vesicles obtained in highly purified fractions by preparative free-flow electrophoresis and by aqueous two-phase partition were determined based on ATPase latency and morphological criteria. Free-flow electrophoresis yielded two plasma membrane fractions. One, the least electronegative and designated fraction `E,' was pure plasma membrane. The other, more electronegative and designated fraction `C,' was heavily contaminated by various other cellular membranes. Plasma membrane vesicles from both fraction C and fraction E partitioned into the upper phase with aqueous two-phase partitioning. Purified plasma membrane obtained from microsomes by two-phase partition (upper phase) when subjected to free-flow electrophoresis also yielded two fractions, one fraction co-migrated with fraction C and another fraction co-migrated with fraction E. Both fractions exhibited an ATPase activity sensitive to vanadate and insensitive to nitrate and azide. ATPase activity was used as a structure-linked latency marker for the inner membrane surface. Concanavalin A binding (linked to peroxidase) was used as an imposed electron microscope marker for the outer membrane surface. Fraction E vesicles showed low ATPase latency (two-fold or less) and weak reactivity with concanavalin A peroxidase. In contrast, fraction C vesicles were characterized by much greater latencies upon detergent treatment (sevenfold) and a strong reaction with concanavalin A peroxidase. Two-phase partition as the initial procedure for plasma membrane isolation, yielded mixtures of vesicles of both inside out and right-side out orientation. Free-flow electrophoresis resolved the plasma membrane isolates into vesicles from fraction C which were right-side out (cytoplasmic side in), and vesicles from fraction E which were wrong-side out (cytoplasmic side out). Therefore, the two methods used in series, provided highly purified membrane preparations of apparently homogenous vesicles of opposite known absolute orientations.     

Cationic proteins of polymorphonuclear leukocyte lysosomes. II. Composition, properties, and mechanism of antibacterial action

Zeya, H. I. (University of North Carolina, Chapel Hill), and J. K. Spitznagel. Cationic proteins of polymorphonuclear leukocyte lysosomes. II. Composition, properties, and mechanism of antibacterial action. J. Bacteriol. 91:755-762. 1966.-A basic proteins fraction from guinea pig polymorphonuclear (PMN) granules was obtained by acid extraction and precipitation with 20% (v/v) ethyl alcohol. The fraction accounted for most of the antibacterial activity of the PMN granules and corresponded to the antibacterial cationic components of intact granules (bands I, II, and III) resolved by zone electrophoresis. Absence from the fraction of components identical to the enzymatic components of intact lysosomes showed that the fraction was essentially free from lysosomal enzymes. The amino acid analysis of proteins in the fraction gave a preponderance of basic amino acids (25%), especially of arginine (16%). The comparative amino acid analysis showed that the lysosomal cationic proteins (LCP) fraction was markedly different from nuclear histones. The LCP fraction manifested antibacterial activity against certain gram-positive and gram-positive microorganisms, including Candida albicans, and exhibited stoichiometric relationship in its activity. Microorganisms treated with LCP fraction were agglutinated. Anionic substances such as nucleic acids, heparin, and endotoxin effectively blocked the antibacterial activity of the fraction. The LCP fraction caused suppression of oxygen uptake by bacterial cells and damaged the permeability barriers of cells as manifested by rapid release of P(32) as well as ultraviolet-absorbing material at 260 mmu, in the supernatant fluid.     

Characterization of tonoplast-enriched vesicles isolated by gradient density fractionation of suspension-cultured cells

A tonoplast-enriched microsomal fraction was isolated from Catharanthus roseus cells. It was characterized by structural and functional criteria. This fraction presented a homogeneous size distribution as shown by quasi-elastic light scattering and a homogeneous density on self-generated gradients of Percoll. The mean diameter of the vesicles was estimated to be 0.30 micron and the buoyant density around 1.04 g/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of its polypeptide pattern was in good agreement with the one obtained for tonoplast purified from isolated vacuoles. According to enzymatic assays and inhibition tests, this fraction possessed pyrophosphate and ATP-dependent proton pumps and very low contamination by submitochondrial particles, endoplasmic reticula and Golgi membranes. In light of our previous published results on tonoplast purified from isolated vacuoles, the very low extent of AMP hydrolysis by the microsomal fraction is interpreted as supplementary proof in favor of a tonoplast-enriched fraction.     

The development of polyploidy in two classes of rat liver nuclei

Two classes of nuclei from livers of Sprague-Dawley rats were isolated, one pelleting in 2.3 M sucrose (H nuclei) and the second class sedimenting through 1.6 and 1.8 M sucrose and banding at the 1.8/2.3 M sucrose interface (L nuclei) of a three-step discontinuous gradient. In younger animals, the L nuclear fraction was the major fraction, but the percentage of nuclei found in the L fraction decreased as the animals grew. Nuclear ploidy was determined by flow microfluorometry using propidium iodide as a DNA stain. Both the H and L nuclear fractions contained diploid, tetraploid and octaploid nuclei; but the degree of polyploidy was greater in the H fraction. Concomitant with the change in distribution of nuclei between the H and L fractions with increasing age was a progressive increase in the degree of polyploidy in the H fraction. Polyploidy did not increase linearly with age in the H nuclear fraction but increased in cycles marked by large changes in the numbers of nuclei found in H and L nuclear fractions. By 12 weeks of age, 4n-H nuclei were the largest single population of nuclei in rat liver. These observations suggested that the shift of liver nuclei from the L fraction to the H fraction was associated with the development of polyploidy and with the differentiation of hepatocytes.     

Isolation of plasma membrane and tonoplast fractions from spinach leaves by preparative free-flow electrophoresis and effect of photoinduction

A membrane fraction enriched in plasma membrane and tonoplast vesicles was isolated from green leaves of Spinacia oleracea L. and subjected to subfractionation by free-flow electrophoresis. The most electronegative membrane vesicle fraction collected after the free-flow electrophoretic separation was identified as derived from tonoplast, while the least electronegative fraction was identified as derived from plasma membrane. The identification of the fractions was based on membrane morphology, and on the presence or absence of biochemical markers. The plasma membrane fraction was enriched in thick (9–11 nm) membranes which bound N-1-naphthylphthalamic acid (NPA), and reacted with phosphotungstic acid at low pH on thin sections for electron microscopy. The tonoplast fraction was enriched in vesicles with 7–9 nm thick membranes that neither bound NPA nor reacted with phosphotungstic acid at low pH. Both the plasma membrane and the tonoplast fraction were about 90% pure, with a cross-contamination of not more than 2%. Membrane vesicles originating from dictyosomes, endoplasmic reticulum, mitochondria, plastids, or peroxisomes contaminated the plasma membrane and the tonoplast fractions by a few % only. In leaves of photoinduced plants (24 h light period), the plasma membranes were thicker than in control leaves (8 h light, 16 h dark). The plasma membrane fraction obtained from photo-induced leaves by free-flow electrophoresis retained this increase in thickness, showing not only that photoinduction alters plasma membrane structure, but also that this change is stable to isolation.     

Nuclear Fraction of Bacillus subtilis as a Template for Ribonucleic Acid Synthesis

A "nuclear fraction" prepared from Bacillus subtilis was a more efficient template than purified deoxyribonucleic acid for the synthesis of ribonucleic acid by exogenously added ribonucleic acid polymerase isolated from B. subtilis. The initial rate of synthesis with the nuclear fraction was higher and synthesis continued for several hours, yielding an amount of ribonucleic acid greater than the amount of deoxyribonucleic acid used as the template. The product was heterogenous in size, with a large portion exceeding 23S. When purified deoxyribonucleic acid was the template, a more limited synthesis was observed with a predominantly 7S product. However, the ribonucleic acids produced in vitro from these templates were very similar to each other and to in vivo synthesized ribonucleic acid as determined by the competition of ribonucleic acid from whole cells in the annealing of in vitro synthesized ribonucleic acids to deoxyribonucleic acid. Treatment of the nuclear fraction with heat (60 C for 15 min) or trypsin reduced the capacity of the nuclear fraction to synthesize ribonucleic acid to the level observed with purified deoxyribonucleic acid.     

Involvement of a Cell Envelope Component of Escherichia coli in the Early Stages of Infection with Bacteriophage ϕX174

Envelope fraction I prepared from a ?X174 sensitive host, KD4301, showed a strong eclipsing activity, while the lipopolysaccharide (LPS) fraction showed a weak activity. The eclipsing activity in envelope fraction I was sensitive to heat treatment, while that in the LPS fraction was insensitive. When the complete phage particles (114S) were treated with envelope fraction I, the eclipsed particles (70S) and a rapidly sedimenting component were obtained, but when they were treated with LPS, only 70S eclipsed particles were obtained. Electron microscopic observation showed that there were two types of eclipsed particles formed on treatment with fraction I; in one of them phage DNA was extruded from the phage particles as a thick bundle, and in the other more than 95% of the phage DNA was extruded from the phage particles. The rapidly sedimenting component was the membrane-eclipsed particle complex. LPS gave only one type of eclipsed particles in which DNA was extruded as a thick bundle. These results indicate that a heat labile component in the cell envelopes other than LPS is involved in the extrusion of ?X174 DNA.     

Proteins released from liver after ischaemia induced an elevation of heart resistance against ischaemia-reperfusion injury: 1. Beneficial effect of protein fraction isolated from perfusate after ischaemia and reperfusion of liver

OBJECTIVES: Numerous mechanisms have been proposed to participate in adaptation of heart to ischaemia by ischaemic preconditioning. We have described previously a release of cardio-protective protein fraction during ischaemic preconditioning of dog heart. In the current study the effect of high soluble protein fraction (HS fraction) released from isolated perfused rat liver after ischaemia and reperfusion was examined on isolated perfused rat heart during ischaemia-reperfusion injury. METHODS: Livers were subjected to 30 or 60 min ischaemia followed with 120 min reperfusion. HS fraction was isolated using ammonium sulphate precipitation and dissolved in perfusion solution before Langendorf perfusion of isolated rat hearts. The protein pattern of HS fraction was detected with SDS-PAGE and western blot with ConA and anti ConA antibody. Hearts were then subjected to 20 min ischaemia followed by 20 min reperfusion. During reperfusion, the haemodynamic parameters of hearts were measured. Heart levels of adenine nucleotide were measured in HClO4 extracts using HPLC on C18 column. RESULTS: Liver ischaemia induced changes in protein pattern of HS fraction released from the liver during reperfusion period. Particularly, we registered an increase in amount of several low-molecular weight proteins and decreased amount of high-molecular weight proteins. Proteins in this fraction isolated from perfusate after liver ischaemia interact with ConA with lower intensity as proteins isolated from perfusate after control non-ischaemic condition. HS fraction isolated from perfusate after ischaemia and reperfusion of liver had beneficial effect on heart function during 20 min ischaemia and subsequent 20 min reperfusion, documented by: i) decrease of arrhythmia score from 2 to 1 in 5 min of reperfusion and from 2 to 0 in 10 min of reperfusion; ii) improved heart contractility monitored as stabilised [dP/dt]max and increased Q parameter; iii) increased coronary flow. Proteins isolated from liver perfused under control non-ischaemic condition did not induce similar effects. The stabilisation of heart haemodynamics, observed after administration of HS proteins isolated from perfusate after ischaemia and reperfusion was associated with slight increase in ATP and ADP levels as well as decrease in AMP level.     

THE PREPARATION AND PROPERTIES OF BACTERIAL CHROMATOPHORE FRACTIONS

Chromatophore material from the bacterium Rhodopseudomonas spheroides was freed of ribosomes by centrifugation in 27 per cent RbCl and then separated into "heavy" and "light" fractions by centrifugation through a sucrose gradient. The fractions differed from one another in the following ways. (a) The isopycnic density of the heavy fraction was between 1.15 and 1.18 gm/ml and that of the light fraction was 1.14 gm/ml. (b) The heavy fraction was able to bind ribosomes; the light fraction was not. (c) The light fraction was homogeneous in the ultracentrifuge and had a sedimentation constant, extrapolated to infinite dilution, of 153 s_20,w. The heavy fraction was grossly heterogeneous. (d) Both the amount of bacteriochlorophyll relative to protein and the ratio of bacteriochlorophyll to carotenoids were greater in the light fraction. (e) The spectra of the two fractions in the near infra-red were different. Comparisons of the chromatophore fractions from cells with different amounts of bacteriochlorophyll showed that the specific bacteriochlorophyll contents of the two fractions did not change to the same extent as did that of the whole cells. The amount of heavy fraction from pigmented cells was roughly independent of the cellular pigment content and was about equal to that from pigment-free cells. The amount of light fraction depended on the pigment content of the cells; no light fraction was obtained from cells devoid of bacteriochlorophyll. The cytochrome complements of both fractions underwent quantitative as well as qualitative changes with varying growth conditions. The size of the photosynthetic unit in R. spheroides appeared to increase as the total cellular bacteriochlorophyll content increased; however, the number of units per light fraction particle remained constant.