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1.
《Insect Biochemistry》1989,19(8):775-779
Solubilization of the adenylate cyclase from neural membranes of the dipterous Ceratitis capitata, by using several detergents, and regulatory characteristics of the solubilized enzyme were examined. Triton X-100 is the most effective detergent in solubilizing this enzyme activity. The adenylate cyclase in Triton X-100-solubilized preparations (105,000 g supernatant) does not respond to either guanine nucleotides or fluoride and it apparently seems to be devoid of a functional regulatory component. When this preparation is centrifuged again at 300,000 g for 30 min no enzyme activity is detectable in the supernatant, however only 8% of total activity is recovered in the pellet. The activation pattern for the enzyme in the 300,000 g pellet is similar to that observed for the enzyme in the 105,000 g supernatant. Incorporation of solubilized enzyme into dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) or cholesterol-enriched DOPC liposomes increases the 300,000 g pellet adenylate cyclase activity in a similar extension; thus, this increase in enzyme activity appears to be independent not only on the phospholipid composition but also on the liposome fluidity. 相似文献
2.
《Phytochemistry》1987,26(2):353-357
Cell-free enzyme preparations from oat leaves effectively catalyse the conversion of both phytosterols and nuatigenin (a furostanol sapogenin) to the corresponding 3β-d-glucosides, with UDPG acting as a sugar donor. Subcellular fractionation has shown that UDPG: sterol glucosyltransferase activity is present almost exclusively in the membranous fraction (105 000 g pellet) while a large part (ca 70 %) of UDPG : nuatigenin glucosyltransferase activity occurs in the cytosol (105 000 g supernatant). The results obtained indicate clearly that oat leaves contain at least two UDPG-dependent glucosyltransferases catalysing glucosylation of 3β-hydroxysteroids which are localized in different cell compartments and exhibit different specifirity patterns. 相似文献
3.
Highly purified rough endoplasmic reticulum and three subfractions of golgi were prepared from 105,000g pellet of the homogenate by centrifugation in floatation and sedimentation discontinuous sucrose gradients. Highly purified plasma membranes were also prepared from 9,000g pellet of the same homogenates for assessment under the same experimental conditions. Although 5′-nucleotidase, a marker for plasma membranes, was markedly enriched in plasma membranes, very little or none of this enzyme activity was found in other fractions. Very little or no NADH cytochrome c reductase activity, a marker for rough endoplasmic reticulum, was found in fractions other than rough endoplasmic reticulum. Galactosyl transferase, a marker for golgi, was found and enriched in all the fractions; however, enrichment in golgi fractions was higher than in other fractions. Very little or no lysosomal marker activity, i.e., acid phosphatase, was found in rough endoplasmic reticulum or golgi fractions as compared to lysosomes. These marker enzyme data suggested that rough endoplasmic reticulum and golgi fractions were relatively pure with little or no cross contamination with other organelles. The [125I]human choriogonadotropin ([125I]hCG), [3H]prostaglandin (PG)E1, and [3H]PGF2a specifically bound to rough endoplasmic reticulum and golgi fractions in addition to plasma membranes. The enrichments of binding in the former two fractions, in some cases, were as high as plasma membranes itself. The specific binding of some of the ligands was found to be partially latent in rough endoplasmic reticulum and golgi fractions but not in plasma membranes. Marker enzyme data, ratio between bindings and marker enzyme activities (an index of organelle contamination), and partial latency of binding suggest that rough endoplasmic reticulum and golgi fractions intrinsically contain gonadotropin and PGs binding sites. 相似文献
4.
In Locusta migratoria a cyclic AMP-specific phosphodiesterase (PDE) was found in the following tissues: flight muscles, leg muscles, gonads, fat body, Malpighian tubules, and midgut. In all tissues the enzyme is present in a soluble and a structure-bound form. The relative activities of these two forms are characteristic for each tissue. The intracellular localization of the enzyme in muscle was studied by differential centrifugation. It was found to be present only in the fraction which sedimented at 1500 g and in the 105,000 g supernatant. In the 1500 g pellet PDE seems to be strongly associated with the contractile proteins. No cAMP was hydrolysed by the mitochondrial and microsomal fraction. 相似文献
5.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1997,1344(1):47-58
An enzyme (Mr 240 000) with high fatty acid α-oxidation activity has been purified from the fruit of cucumber (Cucumis sativus). The specific α-oxidation activity in the purified fraction was 370 nmol/min per mg protein determined as liberation of 14CO2 from [1-14C]palmitic acid. α-Oxidation activity was observed both in the 12 000×g pellet and 150 000×g pellet by differential fractionation of cucumber homogenate. The enzyme was purified about 220-fold to near homogeneity from a 12 000×g fraction by solubilisation with Triton X-100R, ammonium sulphate precipitation, hydrophobic interaction and anion-exchange chromatographies and Superose 12 gel filtration. The molecular mass of the native enzyme was 240 000, and the major subunit molecular mass of 40 000 indicated an oligomeric structure. 相似文献
6.
Derek J. Baisted 《Phytochemistry》1979,18(10):1639-1641
Label appeared in several cell fractions isolated from the cotyledons of pea seeds germinated for 48 hr with mevalonate-[2-14C]. The major radioactive metabolite in each fraction was amyrin. In a similar experiment, a fraction sedimenting between 1000 and 25 000 g and a microsomal pellet were labeled with 3H from mevalonate-[2-3H]. Each of these tritiated fractions on incubation with UDP-glucose-[U-14C] yielded CHCl3-MeOH-soluble material bearing 14C and 3H. TLC of the extracts gave a compound chromatographically identical with a glucoside and bearing the two isotopes. Acid hydrolysis of this compound gave an ether-soluble material carrying 3H alone. On TLC it co-chromatographed with amyrin. Of the two tritiated cotyledon fractions, the microsomal pellet had the lower glucosyltransferase activity. The labeled amyrin residing in this fraction served as an acceptor for glucose from UDP-glucose in the presence of a glucosyltransferase from pea seedling axis tissue. In such a mixed preparation, the axis tissue transferase suffers a marked inhibition by the cotyledon preparation. 相似文献
7.
Abdullah B. Sipat 《Phytochemistry》1982,21(11):2613-2618
The activity of hydroxymethylglutaryl CoA reductase (NADPH) (EC 1.1.1.34) was studied in the latex of regularly tapped mature trees of Hevea brasiliensis. The reductase activity was found mainly (95% of the total activity) in the pellet fraction (40 000 g) of the centrifuged latex. The enzyme in this fraction had a specific requirement for NADPH as the cofactor and, while not obligatory for activity, was activated by dithiothreitol at the optimum concentration of 2 mM. The pH optimum was found to be 6.6–6.9 in 0.1 M phosphate buffer. Mevalonate and CoA (at 2 mM each) did not affect enzyme activity, while hydroxymethylglutarate (2 mM) was slightly inhibitory. p-Chloromercuribenzoate (1 mM) completely inhibited this enzyme. The reductase activity in the 40 000 g pellet was not easily solubilized either using Triton X-100 or by sonication. The apparent Km for the washed, membrane-bound enzyme (103 000 g pellet) was 56 μ M (RS-HMG-CoA). Magnesium-ATP (4 mM) inactivated the reductase but this effect was greatly diminished or was absent upon washing the 40 000 g pellet. 相似文献
8.
Subcellular Localization of Sorbitol-6-phosphate Dehydrogenase in Protoplast from Apple Cotyledons 总被引:1,自引:0,他引:1
The subcellular localization of sorbitol-6-phosphate (S6P) dehydrogenasein protoplasts of apple cotyledons was examined by differentialcentrifugation and linear sucrose density gradient centrifugation(3060%, w/w). The distribution of S6P dehydrogenase activitywas 55% in the 500 x g pellet of the homogenate and 35% in thesupernatant of 105,000 x g. When the x g pellet was recentrifugedin a linear sucrose density gradient, one major peak of activitywas found at a density of 1.23. This peak coincided with themajor peak of chlorophyll and NADP+-triose-P dehydrogenase activity.When the 500 x g pellet was sonicated, the major peak of S6Pdehydrogenase activity shifted to a lighter density (d=1.18).The shifted peak also coincided with the peak of chlorophyll.The enzyme detected in the major peak of chlorophyll (d=1.23)was partially solubilized by sonic or detergent treatment, butnot by hypotonic solution. The results supported the localizationof S6P dehydrogenase in chloroplasts, and presumably their associationwith thylakoid membranes. Part of the enzyme was assumed tobe naturally present in the cytosol, too. (Received November 4, 1980; Accepted January 21, 1981) 相似文献
9.
Acetyl-CoA carboxylase was purified 300-fold from rat liver, in the absence of added citrate, by precipitation from an 18,000g supernatant in the presence of Triton X-100 at 105,000g and 20 °C, followed by chromatography on phosphocellulose. Acetyl-CoA carboxylase activity in this preparation was activated by preincubation with GTP (0.1–2.0 mm) and with citrate (20 mm). Colchicine (10?6–10?3m) inhibited enzyme activity and counteracted the effects of GTP and citrate. Sucrose density gradient centrifugation demonstrated that GTP and citrate preincubation promoted the formation of the polymeric, active enzyme, while colchicine engendered disassembly. Preincubation of the purified acetyl-CoA carboxylase at 4 °C caused inactivation and disassembly, which was countered by preincubation at 37 °C in the presence of GTP or citrate. These results suggest that GTP, like citrate, activates acetyl-CoA carboxylase by enhancing the conversion of the protomeric form of the enzyme to its more active, polymeric state. 相似文献
10.
RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : III. Electron Microscopy and Analysis of the Cytochromes
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The membranous nature of pellets obtained from broken Escherichia coli spheroplasts by successive centrifugation at 3500 g (P1), 20,000 g (P2), and 105,000 g (P3), has been established by electron microscopy. Spectrophotometric analysis has shown that about 90% of the cytochromes are concentrated in the particulate fractions. The crude ribosomal pellet (P3) contained as much of the total cytochromes as did the pellet obtained at 20,000 g (P2). The high cytochrome content of P3 is consistent with its high oxidative activity (1) and the presence of membrane vesicles in this fraction. Analysis at 77°K intensified the optical extinction of all the cytochrome absorption bands, but the degree of intensification was not uniform for each fraction nor for each band within a given fraction. Carbon monoxide had little or no inhibiting effect on NADH oxidation. Reduced plus carbon monoxide difference spectra yielded artifactual absorption bands in the wave length regions where reduced vs. oxidized absorption bands normally occur. Succinate and NADH, either together or separately, reduced nearly all of the cytochromes, indicating that the cytochrome portion of the electron-transport chain is shared by both substrates. A tentative formulation of the electron-transport chain is presented. 相似文献
11.
The content of spinach-leaf cells was compartmented by differential centrifugation. Three fractions were obtained,i.e. chloroplasts, pellet of remaining organelles sedimenting at 97 000g and cytosol. Enzyme activities of L-tryptophan aminotransferase (TAT) as well as L-tryptophan dehydrogenase (TDH) were demonstrated
in all cell fractions. The highest activities of both enzymes were found in the pellet of organelles followed by the enzyme
activities in the chloroplasts. The cytosol had the lowest enzyme activities. Chloroplasts are characterized by a relatively
higher TDH activity, organelles sedimenting at 97 000g were marked by a relatively higher TAT activity. In all fractions both pyridine nucleotide coenzymes catalyzed the TDH activity.
Ca2+ in a concentration of 0.8 minol l−1 increased markedly the TDH activity in both directions of its activity.
An erratum to this article is available at . 相似文献
12.
13.
S. Matsuzaki R. Pochet E. Schell-Frederick 《Biochimica et Biophysica Acta (BBA)/General Subjects》1973,313(2):329-337
The validity of 5′-nucleotidase as a plasma membrane marker enzyme in beef thyroid has been tested by comparing the subcellular distribution of its activity to that of (Na+K+)-activated ATPase and adenyl cyclase. The specific activity and total activity of (Na+K+)-ATPase and adenyl cyclase were greatest in the 1000 × g (“nuclear”) and 33 000 × g (“mitochondrial and lysosomal”) fractions. In contrast, 5′-nucleotidase activity was concentrated in the 165 000 × g (“microsomal”) pellet and supernatant. Partially purified plasma membranes were separated from the 1000 (N2), 30 000 (M2) and 165 000 × g (P2) pellets by discontinuous sucrose gradient centrifugation. Again a discordant distribution of these enzyme activities was observed. (Na+K+)-ATPase specific activity was increased approximately 30-fold over the homogenate in Fractions N2 and M2. Basal, thyroid-stimulating hormone-and fluoride-stimulated adenyl cyclase activities were concentrated in the same fractions. 5′-Nucleotidase activity was preferentially located in M2 and P2. These differences in distribution pattern suggest that 5′-nucleotidase activity is not uniquely located in the plasma membrane in the thyroid. 相似文献
14.
John Kamsteeg Jan van Brederode Gerrit van Nigtevecht 《Biochemical genetics》1978,16(11-12):1059-1071
An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 5-hydroxyl group of cyanidin-3-rhamnosyl-(1→6)-glucoside has been demonstrated in petal extracts of Silene dioica plants. This glucosyltransferase activity was not detectable in green parts of these plants. The enzyme activity is controlled by a single dominant gene M; no glucosyltransferase activity could be demonstrated in petals of m/m plants. The enzyme was purified eightyfold by PVP and Sephadex G50 chromatography. The glucosyltransferase had a pH optimum of 7.4, had a molecular weight of about 55,000, was stimulated by divalent metal ions, and had a “true Km” value of 0.5×10?3 m for UDP-glucose and 3.6×10?3 m for cyanidin-3-rhamnosylglucoside. Pelargonidin-3-rhamnosylglucoside also could serve as acceptor. The enzyme did not catalyze the glucosylation of the 5-hydroxyl group of cyanidin-3-glucoside, although in petals of M/- n/n mutants cyanidin-3,5-diglucoside is present. ADP-glucose could not serve as a glucosyl donor. 相似文献
15.
Rapid Enrichment of CHAPS-Solubilized UDP-Glucose: (1,3)-beta-Glucan (Callose) Synthase from Beta vulgaris L. by Product Entrapment : Entrapment Mechanisms and Polypeptide Characterization
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Rapid enrichment of CHAPS-solubilized UDP-glucose:(1,3)-β-glucan (callose) synthase from storage tissue of red beet (Beta vulgaris L.) is obtained when the preparation is incubated with an enzyme assay mixture, then centrifuged and the enzyme released from the callose pellet with a buffer containing EDTA and CHAPS (20-fold purification relative to microsomes). When centrifuged at high speed (80,000g), the enzyme can also be pelleted in the absence of substrate (UDP-Glc) or synthesis of callose, due to nonspecific aggregation of proteins caused by excess cations and insufficient detergent in the assay buffer. True time-dependent and substrate-dependent product-entrapment of callose synthase is obtained by low-speed centrifugation (7,000-11,000g) of enzyme incubated in reaction mixtures containing low levels of cations (0.5 millimolar Mg2+, 1 millimolar Ca2+) and sufficient detergent (0.02% digitonin, 0.12% CHAPS), together with cellobiose, buffer, and UDP-Glc. Entrapment conditions, therefore, are a compromise between preventing nonspecific precipitation of proteins and permitting sufficient enzyme activity for callose synthesis. Further enrichment of the enzyme released from the callose pellet was not obtained by rate-zonal glycerol gradient centrifugation, although its sedimentation rate was greatly enhanced by inclusion of divalent cations in the gradient. Preparations were markedly cleaner when product-entrapment was conducted on enzyme solubilized from plasma membranes isolated by aqueous two-phase partitioning rather than by gradient centrifugation. Product-entrapped preparations consistently contained polypeptides or groups of closely-migrating polypeptides at molecular masses of 92, 83, 70, 57, 43, 35, 31/29, and 27 kilodaltons. This polypeptide profile is in accordance with the findings of other callose synthase enrichment studies using a variety of tissue sources, and is consistent with the existence of a multi-subunit enzyme complex. 相似文献
16.
James M. Trevillyan Agneta Nordstrom T.Juhani Linna 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,845(1):1-9
Tyrosine-specific protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity was measured in normal human nonadherent peripheral blood lymphocytes using synthetic peptide substrates having sequence homologies with either pp60src or c-myc. A high level of tyrosine-specific protein kinase activity was found associated with the cell particulate fraction (100 000 × g pellet). High-pressure liquid chromatography and phosphoamino acid analysis of the synthetic peptide substrates substantiated the phosphorylation of tyrosine residues by the particulate fraction enzyme. The human enzyme was also capable of phosphorylating a synthetic random polymer of 80% glutamic acid and 20% tyrosine. Enzyme activity was half-maximal with 22 μM Mg·ATP and had apparent Km values for the synthetic peptides from 1.9 to 7.1 mM. The enzyme preferred Mg2+ to Mn2+ for optimal activity and was stimulated 2–5-fold by low levels (0.05%) of some ionic as well as non-ionic detergents including deoxycholate, Nonidet P-40 and Triton X-100. The enzyme activity was not stimulated by N6;O2′-dibutyryl cyclic AMP (100 μM), N6;O2′-dibutyryl cyclic GMP (100 μM), Ca2+ (200 μM), insulin (1 μg/ml) or homogeneous human T-cell growth factor (3 μg/ml) under the conditions used. Alkaline-resistant phosphorylation of particulate proteins in vitro revealed protein bands with Mr 59 000 and 54 000 suggesting that there are endogenous substrates for the human lymphocyte tyrosine protein kinase. 相似文献
17.
Guanylate cyclase activity was determined in a 1000g particulate fraction derived from rabbit heart homogenates using Mg2+ or Mn2+ as sole cation in the presence and absence of Triton X-100. With Mg2+, very little guanylate cyclase activity could be detected in the original particulate fraction assayed with or without Triton, or in the particulate fraction treated with varying concentrations of Triton (detergent-treated mixture) prior to enzyme assay. However, the detergent-solubilized supernatants as well as the detergent-insoluble residues (pellets) derived from detergent-treated mixtures possessed appreciable Mg2+-supported enzyme activity. With Mn2+, significant enzyme activity was detectable in the original particulate fraction assayed without Triton. Much higher activity was seen in particulate fraction assayed with Triton and in detergent-treated mixtures; the supernatants but not the pellets derived from detergent-treated mixtures possessed even greater activity. The sum of enzyme activity in pellet and supernatant fractions greatly exceeded that of the mixture. When the pellets and supernatants derived from detergenttreated mixtures were recombined, measured enzyme activities were similar to those of the original mixture. With Mg2+ or Mn2+, the specific activity of guanylate cyclase in pellet and supernatant fractions varied considerably depending on the concentration of Triton used for treatment of the particulate fraction; treatment with low concentrations of Triton (0.2–0.7 μmol/mg protein) gave supernatants showing high activity whereas treatment with relatively greater concentrations of the detergent (>0.7 μmol/mg protein) gave pellets showing high activity. The relative distribution of guanylate cyclase in pellet and supernatant fractions expressed as a function of Triton concentration during treatment (of the particulate fraction) showed that 50 to 80% of the recovered enzyme activity remained in supernatants at low detergent concentrations whereas 50 to 80% of the recovered activity resided in the pellets at higher detergent concentrations. Inclusion of excess Triton in the enzyme assay medium did not alter the specific activity profiles and the relative distribution patterns of the cyclase in pellet versus supernatant fractions. The results demonstrate the inherent potential of cardiac particulate guanylate cyclase to utilize Mg2+ in catalyzing the synthesis of cyclic GMP. However, it appears that some factor(s) endogenous to the cardiac particulate fraction severely impairs the expression of Mg2+-dependent activity; Mn2+-dependent activity is also affected by such factor(s) but apparently less severely. Further, the results suggest that previously reported activities of cardiac particulate guanylate cyclase, despite being assayed with Mn2+ and in the presence of Triton X-100, represent underestimation of what otherwise appears to be a highly active enzyme system capable of utilizing physiologically relevant divalent cation such as Mg2+. 相似文献
18.
J Katz W Troll M Levy K Filkins J Russo M Levitz 《Archives of biochemistry and biophysics》1976,173(1):347-354
Direct evidence was obtained for the presence of hormone-stimulated trypsin-like protease activity in the rat uterus. Ovariectomized rats were either untreated (U), treated with estradiol (E), or estradiol plus progesterone (EP). The uteri were excised and subcellular fractions were prepared. Each fraction was assayed for protease activity using protamine as substrate, the cleavage products being quantitated fluorometrically following reaction with 4-phenylspiro[furan-2(3H),1′-phthalan]-3,3′dione (Fluram). Fractions from U rats yielded negative results, whereas the 12,000g pellets and nuclei from the uteri of E and EP rats exhibited appreciable activities. No significant increase in protease activity was observed in thymus and diaphragm following hormone treatment, indicating organ specificity. The enzyme (or enzymes) from the 12,000g pellet was solubilized and some characteristics were determined. The apparent Km is about 1.0 × 10?6m, the temperature optimum is about 44 °C and maximum velocity is achieved in the alkaline range (pH ~ 8.5). The protease is a plasminogen activator and is inhibited by diisopropyl fluorophosphate, Antipain, and Leupeptin. These properties resemble those of trypsin. 相似文献
19.
Adenylate cyclase activity in Phanerochaete chrysosporium was present in cell fractions sedimenting at 1,000xg, 15,000xg, and in the 150,000xg supernatant. A small amount of activity in the 1,000xg pellet could be solubilised by treatment with Triton X-100, and the enzyme in all fractions required an ATP-Mn2+ substrate. Adenylate cyclase activity in the 150,000xg pellet was low (0.003 nmol/mg protein·min) and may have resulted from contamination by other fractions. Highest adenylate cyclase specific activity (0.37 nmol/mg protein ·min) was recorded in the 150,000xg supernatant at the onset of idiophasic metabolism. During this growth phase, adenylate cyclase activity also increased in the 1,000xg pellet and was maximally 4.5-fold greater than that in primary phase cultures. No significant cAMP-phosphodiesterase activity could be detected during growht in any of the cell fractions or in the growth medium with either Mn2+, Mg2+, or Ca2+ as added cations. The extracellular cAMP concentration increased logarithmically during primary growth; however, in cultures in idiophasic metabolism cAMP levels remained constant and relatively low. We suggest that excretion into the medium is the principal means by which intracellular cAMP levels are decreased in P. chrysosporium.Abbreviation EB
extraction buffer 相似文献
20.
THE rapidity with which angiotensin I is converted to angiotensin II1–5 suggests that the subcellular localization of the converting enzyme is important. Bakhle6 and Cushman and Cheung7 have demonstrated the particulate nature of this enzyme. Subcellular distribution studies and marker enzyme analysis indicate that converting enzyme activity in rabbit lung is most concentrated in the pellet sedimenting at between 1,000 and 25,000g (P2), subsequently characterized as the light and heavy mitochondrial fraction. To identify this fraction more fully, we have resuspended P2 and centrifuged it through a discontinuous density gradient, a procedure which separates converting enzyme activity from the mitochondria. Marker enzyme analysis and electron microscopy suggest plasma membrane as the major constituent of that fraction displaying highest specific activity of converting enzyme. 相似文献