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1.
J Casas L G Harshman A Messeguer E Kuwano B D Hammock 《Archives of biochemistry and biophysics》1991,286(1):153-158
In vitro metabolism of juvenile hormone III (JH III) and juvenile hormone III bisepoxide was investigated using purified mouse liver cytosolic epoxide hydrolase (cEH) and cell fractions from Drosophila melanogaster. JH III was metabolized faster than JH III bisepoxide by epoxide hydrolase activity in D. melanogaster cell fractions and by cEH. After incubation with JH III bisepoxide, all cell fractions and cEH produced epoxy-diol, cis- and trans-tetrahydrofuran-diols, and tetraol as metabolites. An increase in the concentration of cEH resulted in an increase in the proportion of tetraol as a JH III bisepoxide metabolite but this trend was not observed in the D. melanogaster cell fractions. Differences between cell fractions in the metabolism of JH III and JH III bisepoxide suggests the presence of juvenile hormone epoxide hydrolase isozymes. 相似文献
2.
Sarjeet S. Gill 《Biochemical and biophysical research communications》1984,122(3):1434-1440
The light and heavy mitochondrial fractions of mouse liver have relatively high levels of epoxide hydrolase (EH) activity when monitored with trans-stilbene oxide as substrate. Using double diffusion analysis and immunoprecipitation experiments it was shown that EH activity in the mitochondrial fractions is immunologically similar to cytosolic EH, but immunologically dissimilar from microsomal EH. The EHs in the mitochondrial and cytosolic fractions also have a similar pI. 相似文献
3.
T Nakayama K Matsuura M Nakagawa A Hara H Sawada 《Archives of biochemistry and biophysics》1988,264(2):492-501
On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung. 相似文献
4.
Trypsin-like enzyme activity of the extracellular membrane vesicles of Bacteroides gingivalis W50 总被引:16,自引:0,他引:16
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. 相似文献
5.
Recombinant expression and purification of human androgen receptor in a baculovirus system 总被引:1,自引:0,他引:1
Zhu Z Bulgakov OV Scott SS Dalton JT 《Biochemical and biophysical research communications》2001,284(3):828-835
A full-length human androgen receptor (hAR) cDNA was used to produce recombinant baculovirus. Spodoptera frugiperda (Sf9) cells infected with this virus expressed protein with an N-terminal hexahistidine tag (His(6)-hAR) in soluble and insoluble forms. The soluble cytosolic His(6)-hAR demonstrated similar association and dissociation half-times for mibolerone, similar binding affinity for mibolerone, and similar steroid specificity as bona fide AR. Under native conditions, the soluble cytosolic His(6)-hAR was purified to apparent homogeneity in the presence of dihydrotestosterone, using metal ion affinity chromatography. The insoluble pellet fraction was solubilized with strong denaturant 6 M guanidine HCl, and His(6)-hAR was purified from it in the presence of 6 M guanidine HCl. Both the solubilized crude pellet fraction and the solubilized/purified His(6)-hAR could be renatured to bind mibolerone. The baculovirus system will therefore provide an efficient means for producing hAR for ligand-binding assays, as well as purifying hAR for detailed molecular analyses. 相似文献
6.
Mitochondrial and Peroxisomal β-Oxidation of Stearic and Lignoceric Acids by Rat Brain 总被引:1,自引:1,他引:0
Crude subcellular fractions were prepared from adult rat brains by differential centrifugation of brain homogenates. Greater than 98% of the cellular mitochondrial marker enzyme activity sedimented in the heavy and light mitochondrial pellets, and less than 1% of the activity sedimented in microsomal pellets. Lysosomal marker enzyme activities mainly (71-78% of cellular activity) sedimented in the heavy and light mitochondrial pellets. Significant amounts of the lysosomal marker enzyme activity also sedimented in the crude microsomal pellets (9-13% of total) and high-speed supernatants (14-16% of total). The specific activities of microsomal and peroxisomal marker enzyme activities were highest in the crude microsomal pellets. Fractionation of the crude microsomal pellets on Nycodenz gradients resulted in the separation of the bulk of the remaining mitochondrial, lysosomal, and microsomal enzyme activities from peroxisomes. Fatty acyl-CoA synthetase activities separated on Nycodenz gradients as two distinct peaks, and the minor peak of the activities was in the peroxisomal enriched fraction. Fatty acid beta-oxidation activities also separated as two distinct peaks, and the activities were highest in the peroxisomal enriched fractions. Mitochondria were purified from the heavy mitochondrial pellets by Percoll density gradients. Fatty acyl-CoA synthetase and fatty acid beta-oxidation activities were present in both the purified mitochondrial and peroxisomal enriched fractions. Stearoyl-CoA synthetase activities were severalfold greater compared to lignoceroyl-CoA synthetase, and stearic acid beta-oxidation was severalfold greater compared to lignoceric acid beta-oxidation in purified mitochondrial and peroxisomal enriched fractions.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
Peroxisomes were purified from livers of control mice and from mice treated with three agents which induce proliferation of hepatic peroxisomes — namely two structurally unrelated hypolipidemic drugs, clofibrate (ethyl--p-chlorophenoxyisobutyrate) and Wy-14,643 (4-chloro-6[2,3-xylidino)-2-pyrimidinylthio] acetic acid), and a plasticizer, DEHP (di-(2-ethylhexyl)phthalate).Membranes were isolated from these purified peroxisomes and analysed by SDS-polyacrylamide gel electrophoresis. All membranes which were tested, displayed two predominant integral membrane proteins of apparent molecular weights of 68 kDa and 70 kDa respectively, as well as a number of minor components. Treatment of animals with clofibrate, Wy-14,643 and DEHP was observed to result in each case in an increased proportion of the 70 kDa protein in the peroxisomal membranes. These treatments also resulted in increased peroxisomal fatty acid oxidation in livers and an increase in the proportion of catalase activity in the cytosolic fraction of liver cells.These results have been discussed in relation to alterations in the molecular composition of the membranes, the mechanisms of peroxisome proliferation and the inducibility of peroxisomal membrane proteins. 相似文献
8.
Purification of an active opioid-binding protein from bovine striatum 总被引:12,自引:0,他引:12
T L Gioannini A D Howard J M Hiller E J Simon 《The Journal of biological chemistry》1985,260(28):15117-15121
We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta-naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat-sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors. 相似文献
9.
Enzymes of carnitine acylation. Is overt carnitine palmitoyltransferase of liver peroxisomal carnitine octanoyltransferase?
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Liver mitochondria prepared by differential centrifugation are contaminated by significant quantities of peroxisomes and microsomal fractions. 'Easily solubilized carnitine palmitoyltransferase' prepared from liver mitochondria is thought to originate from the outer surface of the mitochondrial inner membrane. We have characterized the carnitine palmitoyltransferase activities of freeze-thaw extracts of rat liver mitochondrial preparations. Chromatography on Sephadex G-100 yields two broad peaks of carnitine decanoyltransferase activity: one eluted at the end of the void volume, which can be removed (precipitated) by ultracentrifugation; the second peak represents the soluble activity and is eluted at an Mr near 70,000. The activity in the soluble peak is precipitated by an antibody raised against carnitine octanoyltransferase purified from mouse liver peroxisomes. In contrast, antibody raised against carnitine palmitoyltransferase purified from liver mitochondrial membranes had no effect (P. Brady & L. Brady, personal communication). The carnitine acyltransferase activities of the Mr-70,000 peak in the presence or absence of Tween 20 showed maximum activity with decanoyl-CoA and about one-third of this activity with palmitoyl-CoA, similar to peroxisomal carnitine octanoyltransferase. These data show that 7500 g preparations of liver mitochondria isolated by differential centrifugation are enriched by peroxisomal carnitine octanoyltransferase (approx. 20% of the protein of the fraction is peroxisomal) and indicate that this enzyme may be the one reported as 'overt' or 'easily solubilized' mitochondrial carnitine palmitoyltransferase. 相似文献
10.
T Yoshihara T Hamamoto R Munakata R Tajiri M Ohsumi S Yokota 《The journal of histochemistry and cytochemistry》2001,49(9):1123-1131
Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001) 相似文献
11.
Neunaber R Achazi RK 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1999,124(1):89-100
The cytosolic supernatant of bream (Abramis brama L.) liver homogenates inhibits the 7-ethoxyresorufin-O-deethylase (EROD) activity of pike (Esox lucius) microsomal fractions. The inhibitor shows no activity against 7-ethoxycoumarin-O-deethylase and benzo(a)pyrene hydroxylase indicating a high isoenzyme specificity. The inhibiting component is a heat-sensitive substance (56 degrees C for 5') which is not self regenerating after subsequent cooling. It can be isolated from the cytosolic fraction using two combined steps of ion exchange chromatography. The purification factor is 500-fold with a recovery rate of 70%. SDS-PAGE of the purified fractions indicate that electrophoretic purity was not achieved. However, a prominent band at about 97 kDa was present in all fractions in a close intensity activity relationship. The molecular weight of the native form of the purified protein was determined to be 175 +/- 35 kDa using gel filtration on a Sephacryl S 300 HR column. So far the inhibitor can be characterized as a protein. It shows strong tendencies to aggregate due to lipophilic interactions. These interactions can be repressed by the addition of 1% sodium cholate. The inhibitor has an optimum activity at 25 degrees C and pH 8.0. The inhibitor does not correspond to any of the known cytosolic, endogenous inhibitors of EROD activities in fish, including proteases, cytosolic phosphatases, kinases and resorufin reductase (e.g. DT-diaphorase), although a non-dicoumarol (10 microM)-inhibited menadione oxidoreductase activity of up to 46.7 +/- 0.4 nmol/min per mg inhibitory protein was measured. Kinetic studies using Michaelis-Menten kinetics with purified inhibitor fractions prove a non-competitive mode of inhibition. As this kind of inhibitor is not described yet it is named CERODIP (cytosolic, EROD-inhibiting protein). 相似文献
12.
The steryl-sulfatase of normal human placental microsomes was solubilized and enriched about 350-fold. Chromatography on Sepharose 6B of the purified enzyme preparation revealed a single protein peak which eluted according to an apparent molecular mass of 270 +/- 30 kDa; when electrophorized on sodium dodecyl sulfate polyacrylamide gel the sulfatase migrated according to a molecular mass of 64 +/- 4 kDa. Estrogensulfatase activity was co-purified with the steryl-sulfatase activity; obviously, both activities belong to the same enzyme species. The purified sulfatase was injected into three rabbits. Antisera produced by the rabbits yielded a single sharp immunoprecipitation line in Ouchterlony double diffusion experiments when tested with the isolated sulfatase or with a solubilized microsomal fraction of normal placentas. The activity of sulfatase preparations incubated with antiserum was precipitated by addition of polyethylene glycol followed by centrifugation; none of the antibodies reacting with the sulfatase therefore appeared to interfere with its enzymatic activity. Using these antisera, steryl-sulfatase protein could be detected by immunoblotting analysis in solubilized microsomal fractions of normal placentas but not in solubilized microsomal fractions of three steryl-sulfatase activity-deficient placentas. This finding argues in favour of human placental steryl-sulfatase deficiency being due to extremely diminished or absent enzyme protein in the placenta. 相似文献
13.
Activation of neutrophil NADPH oxidase in a cell-free system. Partial purification of components and characterization of the activation process 总被引:26,自引:0,他引:26
The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is converted from an inactive to an active form upon stimulation of the neutrophil. This activation process was examined using a recently developed cell-free system in which dormant oxidase is activated by arachidonic acid in the presence of a soluble factor from the neutrophil (Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743). NADPH oxidase from unstimulated human neutrophils was detected only in the membrane fraction. The soluble activation factor was localized entirely to the cytosolic fraction and exhibited two peaks of activity when partially purified under nondenaturing conditions: a major peak with a molecular mass of approximately 250 kDa and a variable minor peak with a mass of approximately 40 kDa. Both forms activated NADPH oxidase in a similar manner and did not exhibit synergy when combined. The cytosolic factor is not protein kinase C (or another kinase) as both peaks of factor activity could be resolved from the protein kinase C peak and neither required calcium or ATP to activate the oxidase. Activation of NADPH oxidase did require the simultaneous presence of the membrane fraction, the cytosolic factor, arachidonic acid, and magnesium. Following activation, however, only the membrane fraction was then required for O2- production. Cytosolic factor levels were normal in five patients with either X-linked or autosomal recessive cytochrome b-negative chronic granulomatous disease. In contrast, the membrane fractions from each failed to generate O2-, indicating that the defects in these two genetic forms of chronic granulomatous disease reside either in the oxidase itself or in a membrane component required for activation. 相似文献
14.
E V Nupenko M P Panchenko M G Starikova A V Grishin V A Tkachuk 《Biochimica et biophysica acta》1991,1091(2):213-221
Lung cytosolic fraction (23500 x g supernatant) activates cAMP synthesis by lung membrane adenylate cyclase (AC). 23 kDa and 29 kDa proteins were isolated from rabbit lung cytosolic fraction in a homogeneous state, as 'activators' of lung membrane AC. Both of these proteins possess high adenylate kinase (AK) activity and are able to mimic the 'activating' effect of lung cytosol on the lung membrane AC in the standard incubation mixture devoid of adenylate kinase. The activating effect is abolished in the presence of adenylate kinase inhibitor DAPP and after heat- or trypsin-treatment of the cytosolic fraction. Commercial adenylate kinase or nonionic detergent Lubrol PX activate cAMP synthesis by lung membrane AC in a similar manner to that of cytosolic fraction. In the presence of commercial adenylate kinase or Lubrol PX no activating effect of the cytosolic fraction on lung membrane AC is revealed. The ability of cytosolic fraction, commercial adenylate kinase, Lubrol PX or purified 23 kDa and 29 kDa proteins to activate cAMP synthesis by lung membrane AC correlates with their ability to support the constant ATP (AC substrate) concentration in the AC assay mixture. Our data indicate that 'activation' of lung membrane AC in the presence of cytosolic fraction may be produced by cytosolic adenylate kinase activity which regenerates ATP from AMP in the presence of creatine kinase and creatine phosphate providing the substrate for cAMP synthesis by AC. 相似文献
15.
An impaired peroxisomal targeting sequence leading to an unusual bicompartmental distribution of cytosolic epoxide hydrolase. 总被引:3,自引:0,他引:3
To gain an understanding of the mechanism by which the subcellular distribution of cytosolic epoxide hydrolase (cEH) is directed, we have analyzed the carboxy terminal region of rat liver cEH by means of cDNA cloning to define the structure of its possible peroxisomal targeting sequence (PTS). Purified cEH was subjected to peptide analysis following endoproteinase Glu-C digestion and HPLC-separation of the fragments. The obtained sequence information was used to perform PCR experiments resulting in the isolation of a 680 bp cDNA clone encoding the carboxy terminus of cEH. The deduced amino acid sequence displays a terminal tripeptide Ser-Lys-Ile which is highly homologous to the PTS (Ser-Lys-Leu) found in other peroxisomal enzymes. This slight difference appears to be sufficient to convert the signal sequence into an impaired and therefore ambivalent PTS, directing the enzyme partly to the peroxisomes and allowing part to reside in the cytosol. 相似文献
16.
S Chiba K Shibuya K Miyazono A Tojo Y Oka K Miyagawa F Takaku 《The Journal of biological chemistry》1990,265(32):19777-19781
The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of 125I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-125I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein. 相似文献
17.
Two homodimeric isozymes, glutathione S-transferase (GST) 25 kDa and GST 27 kDa, in equal proportion comprise the majority (greater than 75%) of the pulmonary cytosolic GST of untreated rabbits. The subunits of GST 25 kDa and GST 27 kDa are distinguishable by electrophoretic mobility (25 and 27 kDa, respectively), apparent isoelectric points (pI 7.4 and pI 9.1, respectively), and immunoreactivity. Immunoblots indicated that these subunits may be minor components in hepatic cytosol. The pulmonary isozymes could not be distinguished by their activities toward chloro-2,4-dinitrobenzene (CDNB) or activity and stereoselectivity toward pyrene 4,5-oxide (PyO). The purified GST fractions represented less than or equal to 16% of the PyO activity for pulmonary cytosol. The stereoselectivity of the cytosolic GST for the pro-S-configured oxirane carbon of PyO was not maintained in the purified preparations which were virtually nonstereoselective. Immunoprecipitation of pulmonary cytosolic GST with anti-GST 27 kDa and anti-GST 25 kDa indicated that at least 84 and 60% of the activity toward CDNB and PyO, respectively, is mediated by the two isozymes. The specific PyO activities of GST 27 kDa, GST 25 kDa, and the rabbit hepatic preparations (approximately 0.2 unit/mg) were similar to that of hepatic GST purified from horse, cow, and pig, and to human placental GST pi (0.02-0.5 unit/mg) but one-tenth that of rat hepatic GST or human hepatic GST mu. However, the activity of the hepatic cytosol from rat and human was similar to that of rabbit. Thus, some GST isozymes may be particularly susceptible to modulation of activity/stereoselectivity that can be discerned with arene oxide substrates such as PyO. 相似文献
18.
In the absence of detergent, approximately 80-85% of the total cGMP-stimulated phosphodiesterase (PDE) activity in bovine brain was associated with washed particulate fractions; approximately 85-90% of the calmodulin-sensitive PDE was soluble. Particulate cGMP-stimulated PDE was higher in cerebral cortical gray matter than in other regions. Homogenization of the brain particulate fraction in 1% Lubrol increased cGMP-stimulated activity approximately 100% and calmodulin-stimulated approximately 400-500%. Although 1% Lubrol readily solubilized these PDE activities, approximately 75% of the cAMP PDE activity (0.5 microM [3H]cAMP) that was not affected by cGMP was not solubilized. This cAMP PDE activity was very sensitive to inhibition by Rolipram but not cilostamide. Thus, three different PDE types, i.e., cGMP stimulated, calmodulin sensitive, and Rolipram inhibited, are associated in different ways with crude bovine brain particulate fractions. After solubilization and purification by chromatography on cGMP-agarose, heparin-agarose, and Superose 6, the brain particulate cGMP-stimulated PDE cross-reacted with antibody raised against a cGMP-stimulated PDE purified from calf liver supernatant. The brain enzyme exhibited a slightly greater subunit Mr than did soluble forms from calf liver or bovine brain, as evidenced by protein staining or immunoblotting after polyacrylamide gel electrophoresis under denaturing conditions. Incubation of brain particulate and liver soluble cGMP-stimulated PDEs with V8 protease produced several peptides of similar size, as well as at least two distinct fragments of approximately 27 kDa from the brain and approximately 23 kDa from the liver enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes in a cell-free system by anionic amphiphiles requires the participation of both membrane and cytosolic components. We reported that ammonium sulfate fractionation (Pick, E., Kroizman, T., and Abo, A. (1989) J. Immunol. 143, 4180-4187) and affinity chromatography on 2',5'-ADP-agarose (Shaag, D., and Pick, E. (1990) Biochim. Biophys. Acta 1037, 405-412) permit separation of cytosol in two fractions (sigma 1 and sigma 2) that support O2- production by solubilized membrane synergistically. We now describe the purification of sigma 1 to near homogeneity and demonstrate that it represents a cytosolic component distinct from p47-phox and p67-phox, that are both found in fraction sigma 2. Sigma 1 was absolutely required for the full expression of amphiphile-activated NADPH-oxidase activity. This requirement was evident whether sigma 1 was added to cell-free systems composed of: (a) solubilized membrane and a sigma 2-enriched cytosolic fraction, or (b) purified cytochrome b559, incorporated in liposomes, and purified sigma 2. Sigma 1 was purified by a sequence comprising ammonium sulfate fractionation, hydrophobic chromatography on phenyl-Superose, absorption with CM-Sepharose, anion exchange chromatography on DEAE-Sepharose, and gel filtration on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sigma 1 of maximal purity, under both reducing and nonreducing conditions, demonstrated the presence of two proteins, of 24 and 22 kDa. On gel filtration, sigma 1 was eluted as a symmetrical peak of 46 kDa that by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed the presence of both 24- and 22-kDa bands. We suggest that, in its native form, sigma 1 might represent a complex of the 24- and 22-kDa proteins. The specific roles of each molecule in NADPH oxidase function remain to be determined. 相似文献
20.
Separation and distribution of thiosulfate-oxidizing enzyme, tetrathionate reductase, and thiosulfate reductase in extracts of marine heterotroph strain 16B. 总被引:1,自引:0,他引:1
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Thiosulfate-oxidizing enzyme (TSO), tetrathionate reductase (TTR), and thiosulfate reductase (TSR) were demonstrated in cell-free extracts of the marine heterotrophic thiosulfate-oxidizing bacterium strain 16B. Extracts prepared from cells cultured aerobically in the absence of thiosulfate or tetrathionate exhibited constitutive TSO and TTR activity which resided in the soluble fraction of ultracentrifuged crude extracts. Constitutive TSO and TTR cochromatographed on DEAE-Sephadex A-50, Cellex D, Sephadex G-150, and orange A dye-ligand affinity gels. Extracts prepared from cells cultured anaerobically with tetrathionate or aerobically with thiosulfate followed by oxygen deprivation showed an 11- to 30-fold increase in TTR activity, with no increase in TSO activity. The inducible TTR resided in both the ultracentrifuge pellet and supernatant fractions and was readily separated from constitutive TSO and TTR in the latter by DEAE-Sephadex chromatography. Inducible TTR exhibited TSR activity, which was also located in both membrane and soluble extract fractions and which cochromatographed with inducible TTR. The results indicate that constitutive TSO and TTR in marine heterotroph 16B represent reverse activities of the same enzyme whose major physiological function is thiosulfate oxidation. Evidence is also presented which suggests a possible association of inducible TTR and TSR in strain 16B. 相似文献