Purification and Partial Characterization of the Soluble NADH Dehydrogenase from the Phototrophic Bacterium Rhodopseudomonas capsulata

1984 ◽  
Vol 39 (1-2) ◽  
pp. 68-72 ◽  
Author(s):  
Toshihisa Ohshima ◽  
Matsumi Ohshima ◽  
Gerhart Drews
Keyword(s):  
Cytochrome C ◽  
Nadh Dehydrogenase ◽  
Rhodopseudomonas Capsulata ◽  
Subunit Structure ◽  
Ph Optimum ◽  
Single Polypeptide Chain ◽  
Membrane Bound ◽  
Single Polypeptide ◽  
Sepharose 4B

Abstract Soluble NADH dehydrogenase was purified to homogeneity from chemotrophically grown cells of Rhodopseudomonas capsulata by ammonium sulfate fractionation, AH -Sepharose 4B chromatography and FMN-Sepharose 6B affinity chromatography. The enzyme contains a single polypeptide chain of an apparent M, of 37000, suggesting that the subunit structure is different from that of the membrane-bound enzyme. The purified soluble NADH dehydrogenase requires flavin compounds, e.g., FMN, FAD and riboflavin, for activity. Addition of FMN and FAD. but not riboflavin, to the enzyme solution stabilized the enzyme. The pH optimum for activity was at 7.5. The enzyme was specific for NADH as an electron donor while NADPH was inert. Menadione, ferricyanide, cytochrome c and DCIP served as an electron acceptor. The M ichaelis constants for NADH. DCIP, FM N. and cytochrome c were 45, 2.9. 7.9 and 15 μM, respectively. Many properties of soluble NADH dehydrogenase were substantially different from those of the membrane-bound enzyme, suggesting different functions.

Soluble neutral maltase–glucoamylase from the small intestine: separation and characterization of components with differing affinity for concanavalin A

1982 ◽  
Vol 60 (11) ◽  
pp. 1007-1013 ◽  
Author(s):  
G. Forstner ◽  
A. Salvatore ◽  
L. Lee ◽  
J. Forstner
Keyword(s):  
Concanavalin A ◽  
Gradient Elution ◽  
Soluble Form ◽  
Rat Intestine ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Neutral Ph ◽  
Membrane Bound ◽  
Sepharose 4B

Intestinal maltase with a neutral pH optimum exists in both a brush border membrane-bound form and a soluble form in suckling rat intestine. Previous experiments in our laboratory have shown that the soluble enzyme contains a component which binds much more tightly to concanavalin A (ConA) than solubilized forms of the membrane enzyme. We studied the origin of this component by subjecting neutral, soluble maltase activity to chromatography on Sepharose 4B at age 13, 18 (preweaning), and 25 (postweaning) days. At 13 days, two maltase peaks were obtained with approximate molecular weights of 400 000 (peak I) and 150 000 (peak II). Peak II was less prominent at 18 days and was absent at 25 days. At 13 days, the majority of peak I consisted of material which was bound between 0.025 and 0.05 M α-methyl mannoside on gradient elution chromatography of ConA-Sepharose. Peak II contained material which eluted between 0.075 and 0.3 M α-methyl mannoside. At 25 days, all of the soluble maltase eluted between 0.025 and 0.04 M α-methyl mannoside. Peak I and peak II maltases had similar pH optima and Km's for maltase. Peak II maltase had a fourfold greater activity toward glycogen than peak I maltase with approximately the same activity for palatinose, turanose, and trehalose. Both maltases were precipitated by an antibody raised against adult membrane-bound maltase. Soluble maltase with neutral pH activity in the suckling rat intestine, therefore, consists of two immunologically related isozymes which differ in their molecular weight, their binding by ConA, and their specificity for glycogen. The small isozyme disappears at or about the time of weaning.

Isolation and Partial Characterization of the Membrane-Bound NADH Dehydrogenase from the Phototrophic Bacterium Rhodopseudomonas capsulata

1981 ◽  
Vol 36 (5-6) ◽  
pp. 400-406 ◽  
Author(s):  
Toshihisa Ohshima ◽  
Gerhart Drews
Keyword(s):  
Nadh Dehydrogenase ◽  
Deae Cellulose ◽  
Sodium Deoxycholate ◽  
Pyridine Nucleotide ◽  
Sodium Cholate ◽  
Rhodopseudomonas Capsulata ◽  
Michaelis Constant ◽  
Membrane Bound ◽  
Ph Dependent

Abstract Chemotrophically grown cells of Rhodopseudomonas capsulata contain at least three different pyridine nucleotide dehydrogenases, i) a soluble, found in the supernatant (144000 × g) of cell free extracts, NADH-dependent, ii) a mem brane-bound, NADH-dependent, and iii) a soluble, found in the supernatant N AD PH dependent. The membrane-bound NADH dehydrogenase (E.C. 1.6.99.3) has been solubilized by sodium deoxycholate treatm ent of m em branes and purified 75 fold by column chrom atography on Sephadex G-150 and DEAE cellulose in the presence of sodium cholate. The native enzyme has an apparent molecular mass (Mr) of 97 000, containing polypeptides of Mr of about 15 000. The pH optim um was at 7.5. The enzyme was specific for NADH. The Michaelis constant for NADH and DCIP were 4.0 and 63 μm, respectively. The enzyme was inactivated by FMN, riboflavin and NADH. In contrast, the soluble NADH-dehydrogenase (i) was activated by FMN.

Isolation of a b-Type Cytochrome Oxidase from Membranes of the Phototrophic Bacterium Rhodopseudomonas capsulata

1982 ◽  
Vol 37 (3-4) ◽  
pp. 193-198 ◽  
Author(s):  
Hendrik Hüdig ◽  
Gerhart Drews
Keyword(s):  
Cytochrome C ◽  
Affinity Chromatography ◽  
Cytochrome Oxidase ◽  
Membrane Fraction ◽  
Rhodopseudomonas Capsulata ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Triton X ◽  
Sepharose 4B ◽  
Horse Cytochrome

Abstract A cytochrome oxidase (EC 1.9.3.1) was solubilized from the membrane fraction of aerobically grown cells of Rhodopseudomonas capsulata by treatment with Triton X-100. The enzyme was purified 160 fold by chromatography on DEAE-Sepharose CL-6B and affinity chromatography on cytochrome c-thiol activated Sepharose 4B.The purified enzyme has a pH-optimum at 8.5 and a temperature optimum at 35 °C. The ap­ parent Km for reduced horse cytochrome c is 24 μм (at pH 8 and 30 °C). The purified cytochrome oxidase was 50% inhibited by 1.5 μм KCN and 10 μм NaN3. The purified enzyme contained one polypeptide of mr 65,000 and 6-type cytochrome.

scholarly journals Soluble neutral and acid maltases in the suckling-rat intestine. The effect of cortisol and development

1974 ◽  
Vol 144 (2) ◽  
pp. 281-292 ◽  
Author(s):  
G Galand ◽  
G G Forstner
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Heat Sensitivity ◽  
Adult Rats ◽  
Ph Optimum ◽  
Maltase Activity ◽  
Acid Maltase ◽  
Membrane Bound ◽  
Sepharose 4B ◽  
The Relationship

The 100000g supernatants from 13-day-old suckling-rat intestinal homogenates contained 43.5% of the total intestinal maltase activity, compared with 7.1% in weaned adult rats aged 40 days. The soluble maltase activity was separated on Sepharose 4B into two quantitatively equal fractions at pH6.0, one containing a maltase with a neutral pH optimum and the other a maltase with an acid pH optimum. The neutral maltase was shown to be a maltase–glucoamylase identical with membrane-bound maltase–glucoamylase in molecular weight, heat-sensitivity, substrate specificity, Km for maltose and Ki for Tris. The soluble enzyme was induced by cortisol, but the ratio of the soluble to bound enzyme fell during induction. Solubility of the neutral maltase was not accounted for by the action of endogenous proteinases under the preparative conditions used. It is postulated that the soluble neutral maltase is a membrane-dissociated form of the bound enzyme and that the relationship between these two forms is modulated by cortisol. The acid maltase generally resembled acid maltase of liver, muscle and kidney. It was shown to be a maltase–glucoamylase with optimal activity at pH3.0, and molecular weight of 136000 by density-gradient centrifugation. At pH3.0 its Km for maltose was 1.5mm. It was inhibited by turanose (Ki=7.5mm) and Tris (Ki=5.5mm) but not by p-chloromercuribenzoate or EDTA. Some 55% of its activity was destroyed by heating at 50°C for 10min. The acid maltase closely resembled β-glucuronidase and acid β-galactosidase in its distribution in the intestine, response to tissue homogenization in various media, and decrease in activity with cortisol treatment and weaning, indicating that it was a typical lysosomal enzyme concentrated in the ileum.

scholarly journals Purification and characterization of pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis

1987 ◽  
Vol 246 (2) ◽  
pp. 529-536 ◽  
Author(s):  
K Williams ◽  
P N Lowe ◽  
P F Leadlay
Keyword(s):  
Trichomonas Vaginalis ◽  
Salting Out ◽  
Iron Protein ◽  
Ferredoxin Oxidoreductase ◽  
Membrane Bound ◽  
Pyruvate Ferredoxin Oxidoreductase ◽  
Bacterial Sources ◽  
Sepharose 4B ◽  
Thiamin Pyrophosphate

The pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis is an extrinsic protein bound to the hydrogenosomal membrane. It has been solubilized and purified to homogeneity, principally by salting-out chromatography on Sepharose 4B. Low recoveries of active enzyme were caused by inactivation by O2 and the irreversible loss of thiamin pyrophosphate. It is a dimeric enzyme of overall Mr 240,000 and subunit Mr 120,000. The enzyme contains, per mol of dimer, 7.3 +/- 0.3 mol of iron and 5.9 +/- 0.9 mol of acid-labile sulphur, suggesting the presence of two [4Fe-4S] centres, and 0.47 mol of thiamin pyrophosphate. The absorption spectrum of the enzyme is characteristic of a non-haem iron protein. The pyruvate: ferredoxin oxidoreductase from T. vaginalis is therefore broadly similar to the 2-oxo acid: ferredoxin (flavodoxin) oxidoreductases purified from bacterial sources, except that it is membrane-bound.

scholarly journals Adenosine 5′-tetraphosphate phosphohydrolase from yellow lupin seeds: purification to homogeneity and some properties

1997 ◽  
Vol 328 (1) ◽  
pp. 257-262 ◽  
Author(s):  
Andrzej GURANOWSKI ◽  
Elżbieta STARZYŃSKA ◽  
Paul BROWN ◽  
G. Michael BLACKBURN
Keyword(s):  
Divalent Cation ◽  
Polypeptide Chain ◽  
Lupinus Luteus ◽  
Nucleoside Triphosphate ◽  
Yellow Lupin ◽  
Ph Optimum ◽  
Single Polypeptide Chain ◽  
Lupin Seeds ◽  
Single Polypeptide ◽  
Hydrolysis Of

Adenosine 5ʹ-tetraphosphate phosphohydrolase (EC 3.6.1.14) has been purified to homogeneity from the meal of yellow lupin (Lupinus luteus) seeds. The enzyme is a single polypeptide chain of 25±1 kDa. It catalyses the hydrolysis of a nucleoside 5ʹ-tetraphosphate to a nucleoside triphosphate and orthophosphate, and hydrolysis of tripolyphosphate but neither pyrophosphate nor tetraphosphate. A divalent cation, Mg2+, Co2+, Ni2+ or Mn2+, is required for these reactions. The pH optimum for hydrolysis of adenosine 5ʹ-tetraphosphate (p4A) is 8.2, Vmax is 21±1.7 μmol/min per mg of protein and the Km for p4A is 3±0.6 μM. At saturating p4A concentrations, the rate constant for the reaction is 8.5±0.7 s-1 [at 30 °C, in 50 mM Hepes/KOH (pH 8.2)/5 mM MgCl2/0.1 mM dithiothreitol]. p4A and guanosine 5ʹ-tetraphosphate are hydrolysed at the same rate. Adenosine 5ʹ-pentaphosphate (p5A) is degraded 1/200 as fast and is converted into ATP and two molecules of orthophosphate, which are liberated sequentially. This contrasts with the cleavage of p5A by the lupin diadenosine tetraphosphate hydrolase (EC 3.6.1.17), which gives ATP and pyrophosphate. Zn2+, F- and Ca2+ ions inhibit the hydrolysis of p4A with I50 values of 0.1, 0.12 and 0.2 mM respectively.

Biochemical Characterization of Chloromethane Emission from the Wood-Rotting Fungus Phellinus pomaceus

1998 ◽  
Vol 64 (8) ◽  
pp. 2831-2835 ◽  
Author(s):  
Deepti Saxena ◽  
Saleh Aouad ◽  
Jihad Attieh ◽  
Hargurdeep S. Saini
Keyword(s):  
Atmospheric Chemistry ◽  
Biochemical Characterization ◽  
Active Form ◽  
Methyl Chloride ◽  
Bound State ◽  
Ph Optimum ◽  
Methyl Donors ◽  
Membrane Bound

ABSTRACT Many wood-rotting fungi, including Phellinus pomaceus, produce chloromethane (CH3Cl). P. pomaceus can be cultured in undisturbed glucose mycological peptone liquid medium to produce high amounts of CH3Cl. The biosynthesis of CH3Cl is catalyzed by a methyl chloride transferase (MCT), which appears to be membrane bound. The enzyme is labile upon removal from its natural location and upon storage at low temperature in its bound state. Various detergents failed to solubilize the enzyme in active form, and hence it was characterized by using a membrane fraction. The enzyme had a sharp pH optimum between 7 and 7.2. Its apparent Km for Cl− (ca. 300 mM) was much higher than that for I− (250 μM) or Br− (11 mM). A comparison of theseKm values to the relative in vivo methylation rates for different halides suggests that the realKm for Cl− may be much lower, but the calculated value is high because the CH3Cl produced is used immediately in a coupled reaction. Among various methyl donors tested, S-adenosyl-l-methionine (SAM) was the only one that supported significant methylation by MCT. The reaction was inhibited by S-adenosyl-l-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings advance our comprehension of a poorly understood metabolic sector at the origin of biogenic emissions of halomethanes, which play an important role in atmospheric chemistry.

Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes

1982 ◽  
Vol 2 (1) ◽  
pp. 76-81
Author(s):  
M Gottlieb ◽  
D M Dwyer
Keyword(s):  
Acid Phosphatase ◽  
Leishmania Donovani ◽  
Surface Membrane ◽  
Extracellular Enzyme ◽  
Growth Cycle ◽  
Growth Media ◽  
Ph Optimum ◽  
Membrane Bound ◽  
Extracellular Acid Phosphatase

An extracellular acid phosphatase was detected in the growth media of Leishmania donovani promastigotes. The enzyme was released at all stages of the growth cycle and in amounts which accounted for 90% of the total amount of this enzyme in the culture. The exoenzyme exhibited a pH optimum of 4.5 to 5.0 and was active with a variety of organic phosphates. The enzymatic activity was excluded from Sephacryl S-300 and was retained by ultrafilters with nominal molecular weight cutoffs of up to 300,000. The results of comparative studies indicated that the extracellular enzyme was distinct from a surface membrane-bound acid phosphatase of L. donovani promastigotes which has been previously described.

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