Partial purification of an intercalated disc-containing cardiac plasma membrane fraction

The sarcolemma of cardiac muscle cells contains a specialised junctional region, the intercalated disc which includes three types of intercellular junction, the macula and fascia adherens and the nexus or gap junction. To facilitate the isolation of these junctions a procedure for the partial purification from mouse hearts of a subcellular fraction containing the intercalated disc region of the sarcolemma was developed. This involved investigating methods of tissue disruption that preserve the integrity of the intercalated disc and minimise myofibrillar entrapment of organelles. Examination of the distribution of marker enzymes showed that relative to the homogenate the intercalated disc fraction prepared by sucrose density centrifugation was only enriched 1.5- to 3-fold in 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas mitochondrial and sarcoplasmic reticulum marker enzymes were low. The properties of the intercalated disc-containing fraction were compared with the vesicular sarcolemmal fractions devoid of junctional complexes prepared by other methods.     

The membrane role in an anaerobic membrane bioreactor for purification of dairy wastewaters: a numerical simulation

A simplified modelling and a simulation of a membrane-coupled anaerobic bioreactor, AMBR were performed to assess the potential of controlled retention of solutes by the membrane, R, on biomass growth and of purified water quality. R was shown to be a major parameter, which enables to uncouple the hydraulic resistance time, HRT from the solute retention time, independent of biomass retention, and has a significant effect on purified water quality. Therefore, from a theoretical point of view, it facilitates reaching high biodegradation in a small volume membrane reactor. The simulation makes it possible: (i) to anticipate the effect and relative weight of model parameters in the mechanisms that rule the AMBR behaviour and (ii) to identify the AMBR parameters and operating modes in order to avoid reactor washout or overload, amplified by R. From the analysis, it appears that it is possible to use any type of membrane, which at least retain the biomass: (i) low R values using microfiltration or ultrafiltration membranes require long HRT or small influent concentration and larger reactor volume to achieve good water quality; (ii) high R values using nanofiltration or reverse osmosis membranes, which will retain the solutes as well as the small-degraded molecules within the anaerobic reactor volume, require short HRT for highly purified water, but necessitate a large investment.     

Solubilization and partial purification of ATPase from a rose cell plasma membrane fraction

Some isolates of the fungus Nectria haematococca Berk. and Br. can demethylate pisatin, a phytoalexin from pea (Pisum sativum L.). Pisatin demethylation appears to be necessary for tolerance to pisatin and virulence on pea, and is catalyzed by a microsomal cytochrome P-450. We now report solubilization of this enzyme from N. haematococca microsomes. Pisatin demethylase activity was obtained in the high speed supernatant of detergent treated microsomes, if detergent was removed before assay. The CO-binding spectrum of the soluble enzyme preparation indicated the presence of cytochrome P-450. Cholic acids were the most effective of the detergents tested for solubilizing enzyme activity. Loss of enzyme activity during solubilization was reduced by certain protease inhibitors, but not by substrate, reducing agents, antioxidants, or phospholipids. The most effective solubilization medium tested was 1% sodium cholate, 100 millimolar potassium phosphate, 500 millimolar sucrose, 1 millimolar phenylmethylsulfonyl fluoride, pH 7.5, which yielded approximately 30% of the pisatin demethylase and over 95% of the NADPH-cytochrome c reductase in the soluble fraction. Demethylase activity was lost when the reductase was removed by adsorption on 2′,5′-ADP-agarose. The demethylase activity of reductase-free fractions could be restored by adding a reductase preparation purified approximately 100-fold from microsomes of N. haematococca isolate 74-8-1, which does not demethylate pisatin. We conclude that pisatin demethylase requires NADPH-cytochrome c reductase for activity. The inability of some isolates to demethylate pisatin appears to be due to the absence of a suitable cytochrome P-450, rather than to a lack of functional reductase.     

Cytosolic factors in bovine neutrophil oxidase activation. Partial purification and demonstration of translocation to a membrane fraction

The O2(.-)-generating oxidase of bovine neutrophils is activated in a cell-free system consisting of a particulate fraction enriched in plasma membrane and containing the dormant oxidase, a high-speed supernatant from neutrophil homogenate (cytosol), Mg ions, GTP gamma S, and arachidonic acid [Ligeti, E., Doussiere, J., & Vignais, P.V. (1988) Biochemistry 27, 193-200]. The cytosolic components participating in the activation of the membrane-bound oxidase have been investigated. These components were resolved into several active peaks by Q Sepharose chromatography. The oxidase-activating potency of these peaks was synergistically enhanced by combining samples from separate peaks, or by supplying them with a threshold amount of crude cytosol. Partial purification of two active fractions containing a limited number of proteins of 65, 56, 53, and 45 kDa was achieved by gel filtration of cytosol on Ultrogel AcA44, followed by chromatography on hydroxylapatite and Mono Q. The specific oxidase-activating potency of these partially purified fractions (activating potency per milligram of soluble protein) was 6-8-fold higher than that of crude cytosol; it was enhanced up to 75-fold by complementation with a minute amount of crude cytosol, which per se had a limited efficiency. These data indicate that oxidase activation requires more than one cytosolic component to be activated. To check whether translocation of cytosolic proteins to the membrane occurred concomitantly with oxidase activation, use was made of radiolabeled cytosolic proteins. Cytosol was treated with phenyl[14C]isothiocyanate ([14C]PITC), such that 60% of its activation potency was still present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification of a dicyclohexylcarbodi-imide-sensitive membrane adenosine triphosphatase complex from the obligately anaerobic bacterium Clostridium Pasteurianum.

The membrane adenosine triphosphatase complex of vegetatively growing Clostridium pasteurianum, solublized with Triton X-100, has been recovered as a significantly purified particulate preparation that is still sensitive to inhibition by dicyclohexylcarbodiimide and butyricin 7423.     

Partial purification of presynaptic plasma membrane by immunoadsorption

Chlamydomonas flagella exhibit force transduction in association with their surface. This flagellar surface motility is probably used both for whole cell gliding movements (flagella-substrate interaction) and for reorientation of flagella during mating (flagella-flagella interaction). The present study seeks to identify flagellar proteins that may function as exposed adhesive sites coupled to a motor responsible for their translocation in the plane of the plasma membrane. The principal components of the flagellar membrane are a pair of glycoproteins (approximately 350,000 mol wt), with similar mobility on SDS polyacrylamide gels. A rabbit IgG preparation has been obtained which is specific for these two glycoproteins; this antibody preparation binds to and agglutinates cells by their flagellar surfaces only. Treatment of cells with 0.1 mg/ml pronase results in a loss of motility-coupled flagellar membrane adhesiveness. This effect is totally reversible, but only in the presence of new protein synthesis. The major flagellar protein modified by this pronase treatment is the faster migrating of the two high molecular weight glycoproteins; the other glycoprotein does not appear to be accessible to external proteolytic digestion. Loss and recovery of flagella surface binding sites for the specific antibody parallels the loss and recovery of the motility-coupled flagellar surface adhesiveness, as measured by the binding and translocation of polystyrene microspheres. These observations suggest, but do not prove, that the faster migrating of the major high molecular weight flagellar membrane glycoproteins may be the component which provides sites for substrate interaction and couples these sites to the cytoskeletal components responsible for force transduction.     

Partial purification and biochemical characterization of a T cell suppressor factor produced by human glioblastoma cells

The human glioblastoma cell line 308 constitutively secretes a soluble factor with biologic and biochemical characteristics of human monocyte-derived interleukin 1 (IL 1). The 308 cells also produce a 97,000 m.w. factor that inhibits the effects of IL 1 and interleukin 2 (IL 2) on T lymphocytes. By using sequential chromatography on Blue Affigel, hydroxyapatite, and Ultrogel AcA54, the inhibitory factor, termed glioblastoma-derived T cell suppressor factor (G-TsF), was separated from IL 1 and purified 2000-fold with respect to the protein present in the crude 308 cell supernatant. This G-TsF preparation was sensitive to tryptic proteolysis, showed a peak of pI 4.6 on isoelectric focusing, and when labeled with 125I, revealed six protein bands in the range of 30 to 100 kdaltons on SDS gel.     

Hierarchy of ozone scavenging reactions in the plant cell wall

To estimate protection of the plasmalemma against ozone by the cell apoplast, the decomposition network of ozone in the mesophyll cell wall is analysed in consideration of data on published bimolecular reaction rate constants and concentrations of the reactants involved. The effect of dimerization of ascorbate free radicals (AFR) on the stoichiometric ratio of ozone reduction by ascorbate is quantified over the range of cell wall acidity, pH = 5.0‐6.5. As the disproportionation of AFR decreases sharply towards higher pH, the flow of AFR through dimerization is low over the pH range 5.5‐6.5, allowing abstraction of the second electron from AFR and formation of dehydroascorbate with a nearly 1:1 stoichiometric ratio in relation to ozone. The direct reaction between ozone and ascorbate (AA) in cell walls 0.3‐0.5 µm thick and at an AA concentration of 0.5 m M is able to detoxify 50‐70% of the O₃ that impinges on the wall surface. Generation of singlet oxygen and the hydroxyl radical, which are more reactive to AA than O₃, decreases markedly the O₃ flow to the plasmalemma. The question is raised whether cell wall alkalinization under ozone may hasten the decomposition of the pollutant due to the more rapid generation of hydroxyl radical by phenolic compounds.     

Proteins and protein complexes involved in the biochemical reactions of anaerobic ammonium-oxidizing bacteria

It has been less than two decades since anammox (anaerobic ammonium oxidation) coupled to nitrite reduction has been discovered. Already, this process has been recognized as an important sink for fixed nitrogen in the natural environment and has been implemented as a cost-effective ammonium removal technology. Still, little is known about the molecular mechanism of this remarkable reaction. In this mini review, we present an insight into how ammonium and nitrite are combined to form dinitrogen gas.     

Partial purification and some biochemical properties of neonatal rat cutaneous glutathione S-transferases

1. Previous studies have demonstrated the presence of glutathione S-transferases in the skin of rodents and humans. This study represents the first attempt to purify cytosolic glutathione S-transferases from skin of 3-day-old rats. 2. A partial purification of the enzyme was achieved by a two-step procedure: affinity chromatography followed by HPLC. Two peaks, one major (P-1) and one minor (P-2), were resolved by HPLC containing about 82% and 10% of the recovered activity, respectively. 3. The major form exhibited an overall purification of about 2270-fold with a specific activity of about 73 mumoles/min/mg protein towards 1-chloro-2,4-dinitrobenzene. 4. The kinetic data for P-1 yielded mean Km values of 2.39 mM for 1-chloro-2,4-dinitrobenzene and 0.72 mM for reduced glutathione, while the respective average Vmax values were found to be 212 and 101 mumoles/min/mg protein. 5. Significantly inhibition of enzyme activity was noted in the presence of 0.2 mM HgCl2, 0.63 microM 1.2-naphthoquinone, 1.0 microM triphenyltin chloride, and 12.5 microM 17 beta-estradiol-3-sulfate.     

Partial purification of an insulin-releasing activity in human serum

An activity that enhances insulin release from perifused rat pancreatic islets has recently been isolated from human serum fractions (molecular weight 1,000–5,000 daltons). To characterize this activity we have studied the insulin-releasing effect of serum subfractions from obese and non-obese children obtained by reversed-phase high-performance liquid chromatography (HPLC). The serum insulin-releasing activity eluted in the HPLC system at 12–13 minutes, which corresponded to the retention time of the tridecapeptide insulin-glucagon liberin isolated from bovine hypothalamus. Insulin-releasing activity was found in serum subfractions from both obese and normal-weight children. The relative insulin-releasing potency of the active subfractions was higher than that of the original total serum fractions, indicating the presence of some substance(s) which inhibit insulin secretion in the total serum fractions. Oral glucose loading increased the relative insulin-releasing activity in the HPLC subfractions from obese children. This study suggests that the insulin secretagogue in human serum might be identical to hypothalamic insulin-glucagon liberin as these substances behave similarly on reversed-phase HPLC and have parallel insulin-releasing properties.     

Partial purification of the apolipoprotein E receptor of rat liver membrane

The liver has two distinct lipoprotein receptors; one is the apolipoprotein (apo) B, E receptor and the other the apo-E receptor. In this study, the protein to which apo-E HDLc (cholesterol-induced high density lipoprotein containing apo-E as the predominant protein species) bound specifically was partially purified from the rat liver membrane by ion exchange chromatography and preparative gel electrophoresis. The molecular weight of the protein was estimated to be about 36K daltons and the protein bound to 125I-apo-E HDLc in a specific and saturable manner, suggesting that the protein is the apo-E receptor.     

Partial purification and characterization of an endothelial cell growth regulator from the bovine ovary

An inhibitor of endothelial cell thymidine incorporation in vitro was partly purified from cow ovaries using ammonium sulphate (AS) precipitation. Supernatant fluid from the 100,000 g pellet of freshly homogenized ovaries was subjected to stepwise AS precipitation. Precipitates were collected sequentially at 40%, 60%, 80% and 95% saturation, and then each was dissolved, dialysed (Mr 8000 cutoff) and examined in tissue culture for effects on cellular thymidine incorporation by cow pulmonary artery endothelial cells (CPAE) and mouse fibroblasts (L929 and 3T3). The 80% AS precipitate (ppt.) inhibited the in-vitro uptake of [3H]thymidine by CPAE and L929 cells, but not 3T3 cells. Heparin-Sepharose (HS) chromatography of the 80% AS ppt. revealed that the inhibitory activity on CPAE and L929 cells did not bind to HS; the inhibitory fraction was found in the HS column breakthrough (80% BT). The 80% BT fraction reduced CPAE[3H]thymidine uptake as determined by autoradiography and increased cellular uptake of trypan blue. Serial fractions from Sephacryl S-200 exclusion chromatography of the 80% BT contained CPAE inhibitory activity in the Mr range 30,000-50,000. The inhibitory activity on endothelial cells and L929 fibroblasts and the non-reduced molecular weight range of that fraction are similar to those of tumour necrosis factor alpha (TNF alpha). The results indicate that the cow ovary contains a fraction that inhibits endothelial cell growth in vitro and may have important roles in follicular atresia and luteal regression.     

Partial purification and characterization of the messenger RNA for cell fibronectin.

Fibronectin mRNA has been partially purified by guanidine extraction, oligo-(dT)-cellulose chromatography and sucrose density gradient centrifugation. We obtain a fraction which programs a wheat germ in vitro translation system to synthesize a polypeptide species which co-electrophoreses with fibronectin in SDS-polyacrylamide gels and which is immunoprecipitated with affinity purified fibronectin-specific IgG. Analysis of this RNA fraction by methyl mercury hydroxide-agarose gel electrophoresis reveals the presence of a band accounting for 30 percent to 50 percent of the ethidium bromide-staining material in the fraction. The RNA of this band has an estimated molecular weight of about 3 million daltons and is greatly reduced in the corresponding RNA fraction from RSV transformed CEF. This RNA has been tentatively identified as fibronectin mRNA.     

Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts

An alkaline 5-phosphodiesterase (5-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5-PDE was purified 40-fold to a specific activity of 30 U mg^–1 protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 °C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg²⁺ up to 168% of the original activity, while Zn²⁺, Mn²⁺ and Cu²⁺ ions, chelating agent (EDTA) and NaN₃ (1–10 mM), and 5-ribonucleotides (1–5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 °C), at 70 °C for up to 120 min and without loss of activity over 90 d at –18 °C.