Soluble Supernatant Fraction Research Articles

Detoxification of DNA hydroperoxide by glutathione transferases and the purification and characterization of glutathione transferases of the rat liver nucleus.

DNA peroxidized by exposure to ionizing radiation in the presence of oxygen is a substrate for the Se-independent GSH peroxidase activity of several GSH transferases, GSH transferases 5-5, 3-3 and 4-4 being the most active in the rat liver soluble supernatant fraction (500, 35 and 20 nmol/min per mg of protein respectively) and GSH transferases mu and pi the most active, so far found, in the human liver soluble supernatant fraction (80 and 10 nmol/min per mg respectively). Although the GSH transferase content of the rat nucleus was found to be much lower than that of the soluble supernatant, nuclear GSH transferases are likely to be more important in the detoxification of DNA hydroperoxide produced in vivo. Two nuclear fractions were studied, one extracted with 0.075 M-saline/0.025 M-EDTA, pH 8.0, and the other extracted from the residue with 8.5 M-urea. The saline/EDTA fraction contained subunits 1, 2, 3, 4 and a novel subunit, similar but not identical to 5, provisionally referred to as 5*, in the proportions 40:25:5:5:25 respectively. The 8.5 M-urea-extracted fraction contained principally subunit 5* together with a small amount of subunit 6 in the proportion 95:5 respectively. GSH transferase 5*-5* purified from the 8.5 M-urea extract has the highest activity towards DNA hydroperoxide of any GSH transferase so far studied (1.5 mumol/min per mg). A Se-dependent GSH peroxidase fraction from rat liver was also active towards DNA hydroperoxide; however, since this enzyme accounts for only 14% of the GSH peroxidase activity detectable in the nucleus, GSH transferases may be the more important source of this activity. The possible role of GSH transferases, in particular GSH transferase 5*-5*, in DNA repair is discussed.

Trypsin-like enzyme activity of the extracellular membrane vesicles of Bacteroides gingivalis W50.

Trypsin-like enzyme activity in spent culture media from 3-d-old batch cultures of Bacteroides gingivalis W50 was measured by using the hydrolysis of N alpha-benzoyl-L-arginine-p-nitroanilide. The cell-free culture medium was fractionated by differential centrifugation at 10,000 g and 75,000 g, yielding two particulate fractions and a soluble supernatant fraction. About 80% of the total recoverable activity was associated with the particulate fractions, the remainder being in the supernatant. Electron microscopy of ruthenium-red/osmium stained ultrathin sections of the pellet fractions showed them to be composed of vesicular particles (extracellular vesicles), between 50 and 250 nm in diameter. Enzyme activity in all three fractions was enhanced by dithiothreitol. Gel-permeation chromatography of the soluble fraction yielded one peak of activity which contained 64 kDa and 58 kDa polypeptides. Enzyme activity from the vesicular fractions could be solubilized by sonication, giving a similar chromatographic profile to the supernatant fraction. The main peak of activity was composed of 64 kDa and 58 kDa polypeptides. In addition, there was a higher molecular mass enzyme activity peak composed of the 64 kDa and 58 kDa components along with 111 kDa, 93 kDa and 70 kDa polypeptides. We conclude that the trypsin-like enzyme of B. gingivalis is released as a soluble protein and is also associated with extracellular vesicles, in which it may exist as a soluble component and also as a protein complex.

Vitamin E inhibits platelet phospholipase A 2

One of the most important functions of phospholipase A 2 is the release of arachidonic acid from membrane phospholipids for the synthesis of biologically active eicosanoids. We have demonstrated in our laboratory that vitamin E inhibits platelet phospholipase A 2 in a dose-dependent manner. Rats fed a 100 ppm or a 1000 ppm vitamin E diet exhibit diminished phospholipase A 2 activity compared to those fed a vitamin E-free diet. Addition of vitamin E to a sonicated platelet suspension resulted in further suppression of the phospholipase A 2 activity in all groups of rats. In order to gain insight into the mechanism of vitamin E inhibition of platelet phospholipase A 2, we partially purified this enzyme by gel filtration chromatography. Enzyme activity was localized in the soluble supernatant fraction of a high-speed spin. This partially purified rat platelet phospholipase A 2 had an absolute requirement for Ca 2+ and was inhibited by various forms of tocopherol. Tocol inhibited the enzyme to a greater extent than either d- or dl-α-tocopherol, while there was little or no effect from dl-α-tocopherol acetate. These results emphasize the importance of the hydroxyl moiety on the chromanol of the vitamin E molecule for its inhibitory action, compared to that of the methyl groups which are absent in tocol. This inhibitory action of vitamin E on platelet phospholipase A 2 suggests a crucial function for vitamin E in regulating arachidonate release from the membrane phospholipids and its subsequent metabolism.

Vanadium accumulation and subcellular distribution in relation to vanadate induced cytotoxicity in vitro.

A bovine kidney cell culture system was used to assess the relationship of vandium uptake and subcellular distribution to orthovanadate induced cytotoxicity. Twenty-four hr incubations with 20-1000 microM vanadium elicited 15-75% cytotoxicity. Concentration-related morphological changes from the control polygonal shape to the treated biopolar appearance were detected. Vanadium accumulated in cells via a multiphasic process; peak accumulation was achieved by 1 hr post-treatment and was followed by apparent decline, completed by 3 hr. A slower second phase of accumulation occurred during the remainder of the 24 hr incubation period. A concentration-dependent accumulation resulted in a 50-fold increase in cellular vanadium content. Near maximum toxicity was achieved at a cellular vanadium burden of approximately 5 nmoles/10(6) cells; further accumulation (up to 35 nmoles/10(6) cells) resulted in only a slight increase in the degree of toxicity. Subcellular distribution studies indicated 90% accumulation of vanadium in the soluble supernatant fraction (105,000xg supernatant) at varying stages of cytotoxicity. It was concluded that the multifaceted dependency of vanadium cytotoxicity on its cellular content may have resulted from a cellular balancing between proposed regulatory functions for vanadium and the interactions incurred with an excessive content.

Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon

A reconstituted system for examining directed organelle movements along purified microtubules has been developed. Axoplasm from the squid giant axon was separated into soluble supernatant and organelle-enriched fractions. Movement of axoplasmic organelles along MAP-free microtubules occurred consistently only after addition of axoplasmic supernatant and ATP. The velocity of such organelle movement (1.6μ/sec) was the same as in dissociated axoplasm. The axoplasmic supernatant also supported movement of microtubules along a glass surface and movement of carboxylated latex beads along microtubules at 0.5 μ/sec. The direction of microtubule movement on glass was opposite to that of organelle and bead movement on microtubules. The factors supporting movements of microtubules, beads, and organelles were sensitive to heat, trypsin, AMP-PNP and 100 μM vanadate. All of these movements may be driven by a single, soluble ATPase that binds reversibly to organelles, beads, or glass and generates a translocating force on a microtubule.

Glycosides of N-hydroxy-N-arylamine derivatives. Part 1. Synthesis and mutagenicity of O-glucosides of N-Hydroxy-N-arylamines and their acetohydroxamic acids

N-Acetyl-N-arylamino β-D-glucopyranosides (7a–d) were synthesized by the orthoester glycosylation method via N-arylamino β-D-glucopyranosides (6a–d), and the N-acetyl amides, having an N–O–C-1 linkage in their molecules, were characterized by chemical, enzymatic, and spectral analyses. In the mutation assay using Salmonella typhimurium TA100 strain with or without various intracellular fractions of guinea pig liver, these glucosides (7a–d) were non-mutagenic per se, but showed mutagenic activity in the presence of the post-mitochondrial supernatant (S9) or the microsomal fraction (Ms), except for the glucoside (7a). Of the glucosides (7b–d), the compounds having fewer chlorine atoms were more effective in inducing mutations than were those having multiple chlorines. No mutagenic activity was observed in the presence of the soluble supernatant fraction (S10.5). The mutagenicity of the glucosides (7b–d) seemed to be due to the corresponding N-deacetylated compounds (6b–d) formed through hydrolysis by a microsomal deacetylase(s). The pathway of the metabolic activation of the glucosides (7b–d) is discussed.

Purification and partial characterization of a methylglyoxal reductase from goat liver

An enzyme which catalyzes the reduction of methylglyoxal to lactaldehyde has been isolated and purified from goat liver to apparent homogeneity. NADH was found to be a better substrate than NADPH for methylglyoxal reduction. Stoichiometrically equivalent amounts of lactaldehyde and NAD are formed from methylglyoxal and NADH. Enzyme activity was located only in the soluble supernatant fractions of liver cells. Of the various carbonyl compounds tested, methylglyoxal was found to be the best substrate. The pH optimum of the enzyme was found to be 6.5, and K mfor methylglyoxal was 0.4 mM. The molecular weight of the enzyme was found to be 89 000 by gel filtration on a Sephadex G-200 column. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel revealed that the enzyme is composed of two subunits. The enzyme is highly sensitive to sulfhydryl group reagents. The inactivation by p-chloromercuribenzoate could be substantially protected by methylglyoxal in combination with NADH, indicating a possible involvement of one or more sulfhydryl group(s) at the active site of the enzyme.

Biosynthesis in vitro of Ii core glycosphingolipids from neolactotetraosylceramide by beta 1-3- and beta 1-6-N-acetylglucosaminyltransferases from mouse T-lymphoma.

The N-acetylglucosaminyltransferases probably involved in the biosynthesis in vitro of Ii core glycosphingolipids have been solubilized from a membrane preparation of mouse lymphoma P-1798 and partially characterized. The detergent-extracted membrane supernatant contains both beta 1-3- and beta 1-6-N-acetylglucosaminyltransferase activities that transfer [3H]GlcNAc from UDP-[3H]GlcNAc to the terminal galactose of neolactotetraosylceramide (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; nLcOse4ceramide), to form the Ii core structures. The linkage of [3H]N-acetylglucosamine incorporated into the terminal galactose of nLcOse4Cer was determined from identification of 2,4,6-tri-O-methyl[3H]galactose and 2,3,4-tri-O-methyl[3H]galactose after hydrolysis of the permethylated enzymatic products, GlcNAc beta-[3H]Gal-GlcNAc-Gal-Glc-ceramide. In addition to the presence of beta-N-acetylglucosaminyltransferases, we have detected a galactosyltransferase activity in this soluble supernatant fraction that catalyzes the transfer of [14C]galactose from UDP-[14C]galactose to lactotriaosylceramide (GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; LcOse3ceramide) to form nLcOse4ceramide, the acceptor in the N-acetylglucosaminyltransferase-catalyzed reaction.

Inhibition of microsomal lipid peroxidation by glutathione and glutathione transferases B and AA. Role of endogenous phospholipase A2.

Lipid peroxidation in vitro in rat liver microsomes (microsomal fractions) initiated by ADP-Fe3+ and NADPH was inhibited by the rat liver soluble supernatant fraction. When this fraction was subjected to frontal-elution chromatography, most, if not all, of its inhibitory activity could be accounted for by the combined effects of two fractions, one containing Se-dependent glutathione (GSH) peroxidase activity and the other the GSH transferases. In the latter fraction, GSH transferases B and AA, but not GSH transferases A and C, possessed inhibitory activity. GSH transferase B replaced the soluble supernatant fraction as an effective inhibitor of lipid peroxidation in vitro. If the microsomes were pretreated with the phospholipase A2 inhibitor p-bromophenacyl bromide, neither the soluble supernatant fraction nor GSH transferase B inhibited lipid peroxidation in vitro. Similarly, if all microsomal enzymes were heat-inactivated and lipid peroxidation was initiated with FeCl3/sodium ascorbate neither the soluble supernatant fraction nor GSH transferase B caused inhibition, but in both cases inhibition could be restored by the addition of porcine pancreatic phospholipase A2 to the incubation. It is concluded that the inhibition of microsomal lipid peroxidation in vitro requires the consecutive action of phospholipase A2, which releases fatty acyl hydroperoxides from peroxidized phospholipids, and GSH peroxidases, which reduce them. The GSH peroxidases involved are the Se-dependent GSH peroxidase and the Se-independent GSH peroxidases GSH transferases B and AA.

Determination of nonspecific lipid transfer protein in rat tissues and morris hepatomas by enzyme immunoassay

Rat tissues contain a nonspecific transfer protein which in vitro mediates the transfer of diacylphospholipids as well as cholesterol between membranes. This protein appears identical to sterol carrier protein 2. A specific enzyme immunoassay for this protein was developed using antibodies raised in rabbits, against a homogeneous protein from rat liver. This assay was based on the very high affinity of the nonspecific lipid transfer protein for polyvinyl surfaces. A reproducible adsorption was achieved by presenting the protein to the surface in the presence of a large excess of bovine serum albumin. The adsorbed protein was detected with specific immunoglobulin (IgG) isolated by antigen-linked affinity chromatography and a goat anti-rabbit IgG-enzyme conjugate. Adsorption was proportional to the amount of protein present, giving rise to a linear standard curve. The enzyme immunoassay measured transfer protein levels in the range 0.2–2 ng. The highest concentrations of transfer protein were found in liver and intestinal mucosa. Levels in other tissues including brain, lung, kidney, spleen, heart, adrenals, ovary and testis were 5–10-fold lower than in liver. In the fast-growing Morris hepatoma 7777 the concentration of nonspecific lipid transfer protein was approximately one-tenth of that measured in the host liver, whereas a reduction of 65% was observed in the slow-growing Morris hepatomas 7787 and 9633. Subcellular distribution studies showed that approx. 70% of the transfer protein was present in the soluble supernatant fraction.

Kidney urokinase activity following acute exposure to lead

Urokinase activity was measured in kidney homogenate fractions obtained 2 days after injecting rats with 0.5 mg Pb 2+/100 g body weight. The activity was higher in the membrane-containing fractions than in the soluble supernatant fractions and was markedly higher in the preparations derived from the lead-treated rats. The kidney poly(A) + RNA was obtained form these animals and translated in a rabbit reticulocyte lysate system. In the preparations obtained from the lead-treated rats, there was an increased synthesis of a protein, believed to be urokinase, of pl 8.6 and molecular weight ( M r ) 45,000.

Location of RNase activity in nuclear residual structures.

We report here the partial isolation of an RNase activity which copurifies with the nuclear residual structure prepared from both rat liver and Herpes-infected BHK cells. After the successive treatment of purified nuclei with DNase, low salt and high salt, the RNase activity is found both in the high salt soluble supernatant fraction and in the residual nuclear structure. Triton X-100 treatment of this structure solubilizes the RNase activity. From this we conclude that some of the RNase activity associated with the nuclear residual structure may be located in either the phospholipidic or protein moieties that were extracted with Triton X-100. This RNase cuts rRNA non-randomly into characteristic degradation products. Its molecular weight, on a glycerol gradient, was determined to be 25,000.

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of michondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the ‘mitochondrial’ isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The ‘cytosolic’ isozyme accounted for only 1–2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.

Appearance of a cytosolic protein that stimulates glyceraldehyde-3-phosphate dehydrogenase activity during initiation of renal epithelial cell growth.

Rats fed a K-deficient diet exhibit accelerated kidney growth and enhanced activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase, EC 1.2.1.12). Cultures of renal epithelial cells (BSC-1 line) served as a model for this phenomenon because a reduction in the medium K concentration from 5.4 to 3.2 mM resulted in augmented growth and increased G3PD activity. Mixing the soluble supernatant fraction from cells grown in low-K medium (3.2 mM) with that from cells grown in control medium indicated that the cytosol of low-K cells contained a positive modifier of G3PD activity. Appearance of modifier activity that occurred within 1 hr after exposure of cells to low-K medium was blocked by cycloheximide but not by actinomycin D. Modifier activity was also observed in mouse fibroblasts stimulated by low-K medium and in proliferating renal medullary tissue from rats fed a K-deficient diet. A single protein that contained G3PD-stimulating activity was isolated from the soluble supernatant of BSC-1 cells exposed to low-K medium. This protein was not detectable in control cells. The material yielded a single band on NaDodSO4/polyacrylamide gel electrophoresis with an apparent Mr of 62,000. These results suggest that a new protein with the capacity to stimulate G3PD activity appears in the cytosol during the initiation of cell growth.

Intracellular phospholipid movement and the role of phospholipid transfer proteins in animal cells.

The mechanism of the intracellular movement of phospholipids from their site of synthesis in the endoplasmic reticulum to mitochondria and other cell membranes is a major unsolved problem of cell biology. Phospholipid transfer proteins of varying specificity found in the soluble supernatant fractions of many tissues catalyze the transfer of phospholipids from microsomes to mitochondria in vitro. They are postulated to play a similar role in vivo, but evidence for their function in living cells is lacking. We have now used an analogue of choline, N-propyl-N,N-dimethylethanolamine [PDME, (2-hydroxyethyl)dimethylpropylammonium hydroxide], to devise a test for the function of the transfer proteins in living cells. The rates of translocation of newly synthesized phosphatidylcholine and the analogue phosphatidyl-PDME in living cells were compared with the rates of transfer in vitro catalyzed by soluble transfer proteins extracted from the same cells. Labeled PDME, choline, and ethanolamine were found to be rapidly incorporated into the lipids of isolated rat hepatocytes and of baby hamster kidney (BHK-21) cells in culture. The translocation of newly synthesized phosphatidylcholine and phosphatidyl-PDME was very rapid in both types of cells with a half-time for equilibration of a few minutes, while the translocation of phosphatidylethanolamine was much slower, with a half-time 20-80 fold longer than those of the other two phospholipids. We then compared these relative rates of movement with the activities of the phospholipid transfer proteins of the respective cells. Partially purified phosphatidylcholine transfer protein from rat liver transfers phosphatidylcholine and phosphatidyl-PDME at identical rates but transfers phosphatidylethanolamine at a rate too low to be detected. This result is consistent with an essential function of this transfer protein in vivo. In contrast, partially purified phosphatidylcholine phospholipid transfer protein from BHK cells transfers phosphatidylcholine rapidly, while no transfer of phosphatidyl-PDME and phosphatidylethanolamine was detected. We further found that the specific phosphatidylcholine transfer protein of BHK cells accounts for nearly all of the transfer activity detected in the crude soluble fraction. The rapid translocation of phosphatidyl-PDME in vivo in BHK cells is therefore inconsistent with the postulate that soluble phospholipid transfer proteins are responsible for the rapid movement of phospholipids from microsomes to mitochondria in living cells.

Effect of Vanadate, Molybdate, and Azide on Membrane-Associated ATPase and Soluble Phosphatase Activities of Corn Roots

The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 x MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K(+). K(+)-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K(+) transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.

Immunogenicity of potassium thiocyanate extracted and electrofocused Pasteurella multocida X-73I antigens in chickens and mice.

The immunogenicity of antigenic fractions obtained by the extraction of Pasteurella multocida strain X-73 (serotype 1) with potassium thiocyanate (KSCN) was determined in chickens and mice. The initial KSCN extract was centrifuged at 105 000 X g, and the antigens were separated into a particulate fraction (40p) and a soluble supernatant fraction (40s). The ultracentrifuged fractions were further resolved by preparative electrofocusing. The 40p fraction was resolved into two subgroups having isoelectric points of 3.5-3.9 and 5.5-6.0; the 40s fraction was resolved into five subgroups ranging in isoelectric points from 4.4 to 9.0. The 40p fractions were antigenically similar and contained lipopolysaccharide (LPS) and protein. The 40s fractions were antigenically distinct from the 40p fractions and from each other; they contained proteins and polysaccharides but no LPS. The 40p antigens were strongly immunogenic in mice and chickens, whereas the 40s antigens were weakly immunogenic in chickens and not immunogenic in mice. The incorporation of Freund's complete adjuvant increased the immunogenicity of the 40s antigens in chickens. The 40p antigens induced greater frequencies of serological responses in chickens than the 40s antigens as detected by counterimmunoelectrophoresis and immunodiffusion. This suggested that the increased protection associated with the 40p antigens may have been the result of better antibody response. The toxicity of all the fractions was evaluated by determination of lethality for 10-day-old chicken embryos because of the sensitivity and reliability of the test. The 40p fraction had an LD50 = 0.38 micrograms, and the 40s fraction had an LD50 = 2.5 micrograms. Since the 40s fraction contained no detectable LPS, it is likely that two toxins are present, one which contains LPS and one which does not.

Occurrence of phosphodiesterase IV in the developing human brain, liver and placenta.

The presence of phosphodiesterase IV has been demonstrated in the human fetal brain, liver and placenta as early as in the 6th week of intrauterine development. The enzyme activity in each tissue increases with gestation, being maximum at 18-21 wk and then decreases. The Km values of this enzyme for bis-(p-nitrophenyl)-phosphate hydrolysis in the brain, liver and placenta are 2.94 mM, 1.47 mM and 1.66 mM, respectively. Presence of sulfhydryl group in the active center of the placental enzyme has been demonstrated with the help of cationic study. EDTA inhibits the enzyme in all three tissues. Effect of concanavalin A reveals the absence or unexposition of glucose, mannose and N-acetylglucosamine moieties in the active site of the enzyme in each of the three tissues. Maximum enzyme activity has been found to be localized in the soluble supernatant fraction obtained on centrifuging the brain and liver homogenate at 105,000 x g and in 20,000 x g pellet of the placenta.

Characteristics of membrane-bound and soluble forms of xanthine oxidase from milk and endothelial cells of capillaries

Xanthine oxidase (xanthine:O 2 oxidoreductase, EC 1.2.3.2) was purified from bovine milk lipid globules to electrophoretic homogeneity ( M r 155000) and antibodies were raised against it in rabbits. By immunolocalization techniques, the xanthine oxidase antigen was detected in milk lipid globules and mammary gland epithelium, but also in capillary endothelium from various tissues, including liver, lung and intestine. These findings were paralleled by measurements of xanthine oxidase activities in the tissues, both in a membraneassociated and a soluble form. Addition of hypoxanthine to fractions containing native xanthine oxidase did not promote lipid peroxidation, in contrast to the widely used in vitro system for lipid peroxidation which involves addition of xanthine oxidase preparations. Extraction with buffers of high ionic strength and with nonionic detergents removed only part of the enzyme from the membranes. Immunoprecipitates from the soluble supernatant fractions, using anti-xanthine oxidase IgG, were enriched in the M r 155000 polypeptide. Patterns of proteolytic cleavage products of the xanthine oxidase monomer from capillaries and milk lipid globules were similar but not identical. Immunoprecipitates from soluble fractions of miik lipid globules and tissues were enriched in both xanthine oxidase and NADH-cytochrome c reductase activities. E!ectrophoretic separation of proteins from milk lipid globule membranes under non-denaturing conditions revealed a close correlation of xanthine oxidase and part of the NADH-cytochrome c reductase activity, but showed different activity profiles of NADH-ferricyanide reductase and xanthine oxidase.

Purification and characterization of measles virus haemagglutinin protein G.

The 79 000 mol. wt. measles virion membrane glycoprotein G has been isolated from purified measles virus. Ultracentrifugation of 2% Triton X-100-treated measles virus produced a soluble supernatant fraction containing both G and F, the other external viral membrane protein. Lentil lectin-Sepharose and Sephacryl S-300 column chromatography of this fraction gave a pure preparation of G protein. Sucrose density-gradient centrifugation and SDS-polyacrylamide gel electrophoresis revealed that G was isolated from the virion membrane in the form of a disulphide-linked dimer. Antiserum prepared against purified G reacted only with the G polypeptide of measles virus in a slab gel antibody overlay technique. The antiserum also exhibited haemagglutination inhibition, virus neutralization and haemolysis inhibition activities.