CDP-diglyceride hydrolase from pig liver mitochondria

1984 ◽  
Vol 62 (11) ◽  
pp. 1205-1216 ◽  
Author(s):  
D. W. Nicholson ◽  
W. C. McMurray
Keyword(s):  
Specific Activity ◽  
Divalent Cations ◽  
High Specific Activity ◽  
Liver Mitochondria ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Regulation Of Biosynthesis ◽  
Preparatory Method ◽  
Ph Range ◽  
Triton X

A CDP-diglyceride hydrolase activity, measured by the release of [3H]CMP from labeled CDP-diglyceride, has been identified in pig liver mitochondria. A modified preparatory method for the synthesis of [3H]CDP-diglyceride of high specific activity and purity is also reported. Activity of the hydrolase is enriched 2.5-fold in mitochondrial membranes (over whole mitochondria) and can be solubilized by nonionic detergents such as Triton X-100 with further enrichment of activity (i.e., 7.9-fold). The CDP-diglyceride hydrolase has a Km of 12.8 μM for CDP-diglyceride and a broad pH range with optimum activity at approximately pH 6.2. Of the CDP-diglycerides tested, the hydrolytic rate is highest for dioleoyl CDP-diglyceride. Activity is inhibited by all divalent cations in whole mitochondria, except in the presence of phosphatidylglycerol in which CMP release is stimulated by Co2+ and Mn2+. The increase in CMP release in the presence of Co2+ or Mn2+ can be accounted for entirely by diphosphatidylglycerol synthase activity which requires either cation. This effect is not seen in Triton X-100 solubilized mitochondrial membranes which contain no diphosphatidylglycerol synthase. All preparations are inhibited by mixed phospholipids (Asolectin) and by Trixon X-100 which abolishes activity completely at concentrations greater than 0.5% (w/v). CDP-diglyceride hydrolase is also inhibited by AMP (46%) and by cytidine nucleotides (CTP > CDP > cytidine) except CMP. A role for this activity in the regulation of biosynthesis of mitochondrial polyglycerophosphatides is proposed.

Purification and properties of phosphatidylgkycerophosphate synthetase from mammalian liver mitochondria

1978 ◽  
Vol 56 (6) ◽  
pp. 414-419 ◽  
Author(s):  
W. C. McMurray ◽  
E. C. Jarvis
Keyword(s):  
Divalent Cations ◽  
Maximal Activity ◽  
Liver Mitochondria ◽  
Mitochondrial Membranes ◽  
Purification And Properties ◽  
Standard Assay ◽  
Mammalian Liver ◽  
Triton X ◽  
Cytidine Diphosphate ◽  
Additional Stimulation

The enzyme which catalyzes the synthesis of phosphatidylglycerophosphate from sn-glycerol-3-phosphate and cytidine diphosphate diacylglycerol was released from rat or pig liver mitochondrial membranes by extraction with Triton X-100 or Nonidet P-40. The detergent-extracted enzyme, like the activity of intact mitochondria, did not require added cations or lipids.The Triton extracts were fractionated by column chromatography on Bio-Gel A-1.5. The fractions obtained from the columns exhibited little activity in the standard assay system unless divalent cations were included. Additional stimulation (about twofold) was observed in the presence of added phospholipids.The cation requirement of the purified enzyme was relatively nonspecific with Mg2+, Ba2+, or Ca2+ providing maximal activity in the 10 mM range. Either Mn2+ or Co2+ were stimulatory at somewhat lower concentrations but higher concentrations were inhibitory. Other cations such as Cd2+, Zn2+, Hg2+, or Cu2+ were ineffective as cofactors, and in the presence of Mg2+ inhibited the reaction at concentrations greater than 0.5 mM.The phospholipid stimulation was obtained specifically with phosphatidylethanolamines from natural or synthetic sources. Other diacylglycerophosphatides or lysophosphatides including lysophosphatidylethanolamine were ineffective.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

scholarly journals Regional differentiation of the sperm surface as studied with 125I-diiodofluorescein isothiocyanate, an impermeant reagent that allows isolation of the labeled components.

1979 ◽  
Vol 82 (3) ◽  
pp. 742-754 ◽  
Author(s):  
C A Gabel ◽  
E M Eddy ◽  
B M Shapiro
Keyword(s):  
Sea Urchin ◽  
Specific Activity ◽  
High Specific Activity ◽  
Fluorescein Isothiocyanate ◽  
Surface Proteins ◽  
Regional Differentiation ◽  
Specific Cell ◽  
Sperm Surface ◽  
Erythrocyte Surface ◽  
Triton X

The regional differentiation of the sperm surface has been studied with the aid of a novel covalent labeling technique that permits concurrent cytological, biochemical, and immunological analyses. For these studies isothiocyanate derivatives of fluorescein (FITC) and diiodofluorescein (IFC) were employed: the latter can be prepared with radioiodine to high specific activity (125IFC) and is an impermeant reagent for the erythrocyte surface. Sperm of sea urchin (Strongylocentrotus purpuratus), medaka )Oryzias latipes), and golden hamster bind the fluorescent chromophores with a nonuniform distribution, most of the fluorescence being associated with the midpiece. The radioactive derivative 125IFC permits an analysis of the proteins that are responsible for most of the binding. Additionally, 125 IFC-labeled sperm are capable of fertilizing eggs, as assessed by autoradiography. That IFC labels the surface of the sperm was inferred from the following: (a) the labeling of the surfaces of other cells by fluorescein isothiocyanate and its derivatives; (b) the agglutination of labeled sperm by antibodies directed against IFC; (c) the use of peroxidase-dependent immunocytochemical reaction using anti-IFC antibodies, with analysis by electron microscopy; and (d) extraction of labeled sea urchin sperm with Triton X-100 under conditions that preferentially solubilize the plasma membrane. The antiserum directed against IFC was used to isolate the labeled surface components from Triton X-100 extracts of whole sperm, by immunoprecipitation, with Staphylococcus-A protein serving as a coprecipitant. The results support previous data showing that the sperm surface is a heterogeneous mosaic of restricted domains, one notable zone being the midpiece, where common molecular properties may be shared by sperm with distinctly different morphologies. In addition, IFC-mediated covalent alteration of specific cell surface proteins may be used to label, to identify, and, with the use of anti-IFC antibodies, to isolate such proteins from other cellular constituents.

Expression of Aspergillus aculeatus β-mannanase in Pichia pastoris Yeast and Analysis of Industrially Important Properties of Enzyme

2019 ◽  
Vol 35 (1) ◽  
pp. 38-44
Author(s):  
M.N. Lazareva ◽  
E.I. Semenko ◽  
S.P. Sineoky
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
High Specific Activity ◽  
Yeast Cells ◽  
Aspergillus Aculeatus ◽  
Gene Encoding ◽  
Pichia Pastoris Yeast ◽  
Efficient Expression ◽  
Ph Range ◽  
First Time

β-Mannanases are enzymes for the industrial application and they can be used, in particular, in the feed industry. The most important requirements for feed enzymes are broad pH range, thermal stability and high specific activity. The efficient expression of the man1 gene encoding Aspergillus aculeatus β-1,4-mannanases in Pichia pastoris yeast cells has been obtained for the first time. The industrially valuable properties of the enzyme were confirmed. The obtained data indicate that the man1 gene from A. aculeatus is potentially useful for the construction of industrial mannanase producers on the basis of the Pichia pastoris yeast. recombinant β-mannanase, Pichia pastoris, Aspergillus aculeatus, overexpression. The work was financially supported by State project №595-00004-18 PR and used the help of the National Bioresource Center - Russian National Collection of Industrial Microorganisms NRC «Kurchatov Institute» - GosNIIgenetika (Moscow, Russia).

scholarly journals Molecular characterization of Plasmodium falciparum DNA-3-methyladenine glycosylase

2020 ◽  
Author(s):  
Nattapon Pinthong ◽  
Paviga Limudomporn ◽  
Jitlada Vasuvat ◽  
Poom Adisakwattana ◽  
Pongruj Rattaprasert ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Specific Activity ◽  
Divalent Cations ◽  
Biochemical Properties ◽  
Parasite Development ◽  
Dependent Manner ◽  
Human Enzyme ◽  
Wide Range ◽  
Nacl Concentration ◽  
Ph Range

Abstract Background The emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and if unresolved would eventually result in cell death. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum (Pf)MAG was characterized for its potential for development as an anti-malarial candidate.Methods Native PfMAG from crude extract of chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was partially purified using three chromatographic procedures. From bio-informatics analysis, primers were designed for amplification, insertion into pBAD202/D-TOPO and heterologous expression in Escherichia coli of recombinant PfMAG. Functional and biochemical properties of the recombinant enzyme were characterized. Results PfMAG activity was most prominent in parasite schizont stages, with a specific activity of 147 U/mg (partially purified) protein. K1 PfMAG contained an insertion of AAT (coding for asparagine) compared to 3D7 strain and 16% similarity to the human enzyme. Recombinant PfMAG (74 kDa) was twice as large as the human enzyme, preferred double-stranded DNA substrate, and demonstrated glycosylase activity over a pH range of 4-9, optimal salt concentration of 100-200 mM NaCl but reduced activity at 250 mM NaCl, no requirement for divalent cations, which were inhibitory in a dose-dependent manner.Conclusio PfMAG activity increased with parasite development being highest in the schizont stages. K1 PfMAG contained an indel AAT (asparagine) not present in 3D7 strain and the native enzyme was twice as large as the human enzyme. Recombinant PfMAG had a wide range of optimal pH activity, and was inhibited at high (250 mM) NaCl concentration as well as by divalent cations. The properties of PfMAG provide basic data that should be of assistance in developing anti-malarials against this potential parasite target.

scholarly journals Application Potential of Cyanide Hydratase from Exidia glandulosa: Free Cyanide Removal from Simulated Industrial Effluents

Catalysts ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1410
Author(s):  
Anastasia Sedova ◽  
Lenka Rucká ◽  
Pavla Bojarová ◽  
Michaela Glozlová ◽  
Petr Novotný ◽  
...  
Keyword(s):  
Specific Activity ◽  
Copper Ions ◽  
High Specific Activity ◽  
Industrial Effluents ◽  
Free Cyanide ◽  
Highly Active ◽  
Physico Chemical ◽  
Application Potential ◽  
Ph Range ◽  
Few Data

Industries such as mining, cokemaking, (petro)chemical and electroplating produce effluents that contain free cyanide (fCN = HCN + CN−). Currently, fCN is mainly removed by (physico)chemical methods or by biotreatment with activated sludge. Cyanide hydratases (CynHs) (EC 4.2.1.66), which convert fCN to the much less toxic formamide, have been considered for a mild approach to wastewater decyanation. However, few data are available to evaluate the application potential of CynHs. In this study, we used a new CynH from Exidia glandulosa (protein KZV92691.1 designated NitEg by us), which was overproduced in Escherichia coli. The purified NitEg was highly active for fCN with 784 U/mg protein, kcat 927/s and kcat/KM 42/s/mM. It exhibited optimal activities at pH approximately 6–9 and 40–45 °C. It was quite stable in this pH range, and retained approximately 40% activity at 37 °C after 1 day. Silver and copper ions (1 mM) decreased its activity by 30–40%. The removal of 98–100% fCN was achieved for 0.6–100 mM fCN. Moreover, thiocyanate, sulfide, ammonia or phenol added in amounts typical of industrial effluents did not significantly reduce the fCN conversion, while electroplating effluents may need to be diluted due to high fCN and metal content. The ease of preparation of NitEg, its high specific activity, robustness and long shelf life make it a promising biocatalyst for the detoxification of fCN.

Mechanism and localization of cardiolipin biosynthesis revisited: evidence for the identical mechanism and different localization in mitochondrial and submitochrondrial membranes isolated from guinea pig and rat liver

1990 ◽  
Vol 68 (6) ◽  
pp. 922-935 ◽  
Author(s):  
Lidija Stuhne-Sekalec ◽  
Nikola Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Rat Liver ◽  
Divalent Cations ◽  
Experimental Results ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Membrane Bound ◽  
Rat Livers ◽  
Guinea Pig Liver

The mechanism of cardiolipin (diphosphatidylglycerol) biosynthesis was examined in mitochondria and outer and inner mitochondrial membranes prepared from guinea pig and rat livers to determine whether this formation from phosphatidylglycerol was absolutely dependent on cytidinediphosphodiglyceride, as previously reported for intact mitochondria. Experimental results confirmed that the biosynthesis of cardiolipin, from the membrane-bound radioactive phosphatidylglycerol in intact mitochondria isolated from guinea pig and rat liver, was absolutely dependent on CDP-diglycerides and required the addition of divalent cations. Furthermore, the same mechanism for the biosynthesis of cardiolipin was operational in the outer and inner mitochondrial membranes. This biosynthesis was associated with both the outer and inner mitochondrial membranes prepared from guinea pig liver, but only with the inner mitochondrial membranes prepared from rat liver. The release of radioactive glycerol was also measured, but the amount obtained did not satisfy the stoichiometric requirement for CDP-diglyceride-independent biosynthesis of cardiolipin from 2 mol of phosphatidylglycerol with the liberation of 1 mol of glycerol. Therefore, it was concluded that this mechanism is not involved in the biosynthesis of cardiolipin in mitochondrial and submitochondrial membranes prepared from guinea pig and rat liver.Key words: mitochondria, outer mitochondrial membranes, inner mitochondrial membranes, cardiolipin, biosynthesis.

Translocation of isomeric spin-labelled radioactive cytidinediphosphodiglycerides from isolated guinea pig liver microsomal to mitochondrial membranes

1979 ◽  
Vol 57 (6) ◽  
pp. 618-624 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Liver Mitochondria ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Guinea Pig Liver

Translocation of membrane-bound isomeric (5-, 12-, and 16-doxyl) spin-labelled radioactive cytidinediphosphodiglycerides (CDP-diglycerides) from guinea pig liver microsomal membranes to mitochondrial membranes was studied. When microsomal membranes containing known amounts of isomeric spin-labelled radioactive CDP-diglycerides were incubated with unlabelled mitochondrial membranes, reisolated mitochondria contained labelled lipids in an amount which could not be accounted for by microsomal contamination, indicating that translocation of labelled CDP-diglycerides from microsomal to mitochondrial membranes had occurred. The rate of loss of paramagnetism in microsomal and in reisolated mitochondrial membranes was found to be different, supporting the conclusion that the translocation of labelled lipids between membranes took place. When reisolated mitochondria containing translocated isomeric spin-labelled radioactive CDP-diglycerides were further incubated with sn-3-glycerophosphate, the formation of labelled phosphatidylglycerol was detected. Data from these studies established that the translocation of labelled CDP-diglycerides from microsomal membranes to both outer and inner mitochondrial membranes had occurred.This study established that the isolated guinea pig liver mitochondria are capable of biosynthesis of polyglycerophosphatides (phosphatidylglycerolphosphate, phosphatidylglycerol, and diphosphatidylglycerol or cardiolipin) but depend on the microsomal supply of CDP-diglyceride, an obligatory precursor in the formation of polyglycerophosphatides. This liponucleotide can be translocated, as shown here, to outer and inner mitochondrial membranes for further biosynthetic utilization.

Characterization of a lysosomal proteinase purified from haploid cells of Physarum flavicomum undergoing encystment

1983 ◽  
Vol 61 (5) ◽  
pp. 1357-1366 ◽  
Author(s):  
Henry R. Henney Jr. ◽  
Hiltrud U. White
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Acid Proteinase ◽  
Thiol Groups ◽  
Ph Optimum ◽  
Golgi Bodies ◽  
Cell Extracts ◽  
Thiol Reagent ◽  
Lysosomal Proteinase ◽  
Ph Range

Haploid cells of Physarum flavicomum differentiating to dormant microcysts exhibited prominent Golgi bodies, lysosomes, and autophagic vacuoles digesting intracellular materials. Total intracellular acid proteinase activities, as well as enzyme specific activity, increased during the encystment process. Lysosomes, isolated by fractionation of cell extracts on sucrose density gradients, were identified by their ultrastructural characteristics as well as their content of the following enzymes of high specific activity: acid proteinase, acid β-galactosidase, acid phosphatase, and β-glucuronidase. The lysosomal acid proteinase was chromatographically purified, and its molecular weight was estimated to be 32 000. The proteinase was most stable in the pH range of 2–3 which similarly corresponds to its pH optimum using hemoglobin and Azocasein, respectively, as substrates. Its isoelectric point was about 2. The enzyme exhibited little activity and was unstable at pH 7 and above. The rate of activity of the proteinase was maximal at 55 °C, and good stability of the enzyme was noted up to 45 °C. The proteinase required a thiol reagent for stability. Pepstatin, which specifically affects acid proteinases, inhibited the enzyme. Also, compounds reactive with enzyme thiol groups were highly effective inhibitors of the proteinase. The lysosomal enzyme is an acid (carboxyl) proteinase with essential thiol groups.

THE RESPIRATORY DEPENDENT SWELLING OF LIVER MITOCHONDRIA

1966 ◽  
Vol 44 (9) ◽  
pp. 1259-1270
Author(s):  
Roberto Cereijo-Santaló
Keyword(s):  
Reduced Glutathione ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Endogenous Respiration ◽  
Mitochondrial Membranes ◽  
Ph Values ◽  
Sucrose Medium ◽  
Lag Period ◽  
Ph Range ◽  
Reduced Nicotinamide Adenine Dinucleotide

The respiratory dependent swelling of isolated rat liver mitochondria was studied in sucrose and in potassium chloride media.In a sucrose medium it was found that mitochondria did not swell in the absence of respiration over the pH range 7.0–8.0. However, swelling of mitochondria readily occurred during the oxidation of endogenous or added respiratory substrates. Maximum rates of swelling at pH 7.0 were observed with ascorbate, reduced glutathione, and reduced nicotinamide–adenine dinucleotide (NADH). The swelling induced by substrate oxidation in a sucrose medium was markedly inhibited by the addition of KCl.In a KCl medium, mitochondria swelled in the absence of respiration at acid (6.0) and at alkaline (8.0) pH values, but were stable at pH 7.0–7.4. Endogenous respiration did not modify these results. The addition of β-hydroxybutyrate induced maximum swelling at pH 7.0. The extent of swelling produced by this substrate diminished when either the pH or the molarity of the buffer was increased. Ascorbate and reduced glutathione failed to induce swelling in a KCl medium. NADH in a KCl medium produced swelling to about the same extent as that in a sucrose medium. However, the onset of the swelling was delayed in the KCl medium. This lag period was shortened by the addition of rotenone and it was lengthened when either the molarity or the pH of the buffer was increased.An attempt has been made to relate these results to proton pressure produced in the mitochondrial membranes during electron transport.

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