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1.
Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which
had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids
had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots
of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear.
The K
m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures
was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence
of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by
NADH was linear competitive with respect to acetyl-CoA (K
is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K
is = 56 ± 3.8 μM and K
ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD+ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when
the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment
of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely
to be relieved by NAD+ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle.
Received: 14 July 1999 / Accepted: 20 September 1999 相似文献
2.
CYP102A1 is an efficient medium- to long-chain fatty acid hydroxylase that is able to accept a wide range of non-natural substrates
which bear no resemblance to the natural ones. 4-Hexylbenzoic acid (HBA) and 4-nonyloxybenzoic acid (NOBA) were identified
as CYP102A1 substrates via screening studies using the BD Oxygen Biosensor System. Spectroscopic binding studies showed that
these two substrates bind in the active site of CYP102A1 with K
d values of 2.6 ± 0.1 μM for HBA and 1.9 ± 0.2 μM for NOBA. NADPH consumption rates in the presence of HBA and NOBA were 45 ± 1 min−1 and 61 ± 1 min−1, respectively. The coupling efficiency for NADPH was 57% for NOBA, while it was 77% for HBA. During whole-cell biotransformations,
HBA was converted into ω−1- and ω−2-hydroxyhexylbenzoic acid, whereas NOBA was oxidized to ω−2-hydroxynonyloxybenzoic acid
and ω−2,ω−4-dihydroxynonyloxybenzoic acid. HBA was used as a fatty acid mimic to compare whole-cell biotransformations with
cell-free extracts. Whole-cell biotransformations carried out in a biphasic system resulted in 86% conversion of 5 mM HBA,
producing 3.8 mM ω−2- and 0.5 mM ω−1-hydroxyhexylbenzoic acid in 4 h with a turnover number of 4.1 min−1, whereas 100% conversion of 5 mM HBA was obtained in 1 h with crude cell extracts and a cofactor regeneration system, giving
a turnover number of 10.5 min−1. 相似文献
3.
Weisstaub G Hertrampf E López de Romaña D Salazar G Bugueño C Castillo-Duran C 《Biological trace element research》2007,118(2):167-174
Zinc (Zn) deficiency and obesity can be observed together in some developing countries. Zn deficiency may enhance fat deposition
and decrease lean mass accrual, which in turn, appears to influence physical activity (PA), although this has not yet been
evaluated in obese children. The objective of the study was to find out the association between measurements of plasma Zn
and serum leptin, body composition, and PA in Chilean obese preschool children. Seventy-two 18- to 36-month-old obese children
[weight-for-length/height z score (WHZ) > 2.0 SD], belonging to low socioeconomic communities, participated in the study. Plasma Zn, serum leptin, weight,
waist circumference, height, total body water (TBW) assessed by deuterium isotopic dilution technique and daily activity,
measured by registering 48 h with an accelerometer, were evaluated. We found 82% of children with WHZ > 3 SD. The geometric
mean Zn intake was 6.2 ± 2.5 mg/day. The mean plasma Zn was 91.8 ± 11.4 μg/dL, with 10% of the children having levels <80 μg/dL.
No correlation was found between plasma Zn concentrations and either weight, WHZ, or waist circumference. Serum leptin was
lower in males than in females (2.9 ± 2.8 vs 6.8 ± 5.0 ng/mL, respectively; p < 0.001). TBW was different between males and females (56.2 ± 5.4 vs 52.8 ± 4.3% body weight, respectively; p = 0.004), but no significant association was found between TBW and plasma Zn. Moderate + intense PA, (as percentage of wake
time), was greater in males than in females (6.3 ± 3.1% vs 3.4 ± 2.3%, respectively; p < 0.001), but it was not significantly correlated to plasma Zn. In conclusion, plasma Zn was not associated with body composition
as assessed by TBW, serum leptin, or with the magnitude of physical activity in Chilean overweight preschool children. 相似文献
4.
M. S. Piotrovskii T. A. Shevyreva I. M. Zhestkova M. S. Trofimova 《Russian Journal of Plant Physiology》2011,58(2):290-298
Five-day-old etiolated seedlings of maize (Zea mays L.) were used to study the kinetics of hydrogen peroxide formation upon lowering growth temperature from 25 to 6°C. The total
content of hydrogen peroxide in root and shoot tissues increased by 30–40% after 2-h cooling compared to the control level
but returned to the initial level or decreased even lower after 24-h cooling. In order to prove the involvement of plasma
membrane NADPH oxidase in changes of hydrogen peroxide content upon cooling, isolated plasma membranes were obtained from
untreated plants and from seedlings chilled at 6°C for 2 and 24 h. The NADPH-dependent generation of superoxide anion radical
in isolated plasma membranes was quantified by measuring the rate of formazan production from the tetrazolium salt XTT. The
activity of plasma membrane NADPH oxidase in shoots was 50 ± 9 nmol O2/(mg protein min), which was 1.5 times higher than the activity in roots. The enzyme activity in plasma membranes was inhibited
by low concentrations of diphenyleneiodonium. The effective concentration EC50 was 5.10 μM for shoots and 9.05 μM for roots. The activity of plasma membrane NADPH oxidase increased after 2-h cooling of
seedlings but reversed to the control level after 24-h cooling. This transient activation of NADPH oxidase upon cooling was
similar to the pattern of hydrogen peroxide formation in shoots and roots. Analysis of NADPH oxidase activity of plasma membrane
proteins after their separation in denaturing conditions followed by subsequent renaturation revealed four diphenyleneiodonium-sensitive
bands with mol wt of 130, 88, 51, and 48 kD. Western blot analysis of the reaction with antibodies against the catalytic domain
of phagocyte NADPH oxidase revealed the proteins with mol wt of only 88 and 48 kD. The properties of molecular organization
of plasma membrane NADPH oxidase are discussed in terms of its role in cell signaling. 相似文献
5.
6.
Soria F Ellenrieder G Oliveira GB Cabrera M Carvalho LB 《Applied microbiology and biotechnology》2012,93(3):1127-1134
α-l-Rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl
alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and
ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg
of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan
derivative (0.06 nkat/μg of protein). The activity–pH and activity–temperature profiles for all immobilized enzymes did not
show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C.
The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of
40–70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity
of the preparations did not appreciably decrease after ten successive reuses. Apparent K
m of α-l-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM)
were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better
performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides
(0.116 and 0.014 mg narirutin after 1 h, respectively). 相似文献
7.
K+-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring 86Rb efflux. In crypt cells, basal K+ efflux (rate constant: 0.24 ± 0.044 min−1, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba2+ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin,
iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K+ efflux, increasing the rate constant to 0.65 ± 0.007 min−1 and the span to 83 ± 3.2%. Ionomycin-induced K+ efflux was inhibited by clotrimazole (IC50 of 25 ± 0.4 μM) and charybdotoxin (IC50 of 65 ± 5.0 nM) and was insensitive to TEA, Ba2+, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca2+-activated intermediate-conductance K+ channels (IKca). Absence of extracellular Ca2+ did neither affect basal nor ionomycin-induced K+ efflux. However, intracellular Ca2+ depletion totally inhibited the ionomycin-induced K+ efflux, indicating that the activation of these K+ channels mainly depends on intracellular calcium liberation. K+ efflux was stimulated by intracellular Ca2+ with an EC50 of 1.1 ± 0.04 μM. In surface cells, K+ efflux (rate constant: 0.17 ± 0.027 min−1; span: 25 ± 3.4%) was insensitive to TEA and Ba2+. However, ionomycin induced K+ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present
IKCa channels but only crypt cells have TEA- and Ba2+-sensitive conductive pathways, which would determine their participation in colonic K+ secretion. 相似文献
8.
Marques RC de Sousa AF do Monte SJ Oliveira FE do Nascimento Nogueira N Marreiro DN 《Biological trace element research》2007,120(1-3):11-18
Studies have evidenced that zinc metabolism is altered in presence of Down syndrome, and zinc seems to have a relationship
with the metabolic alterations usually present in this syndrome. In this work, the Zn-related nutritional status of adolescents
with Down syndrome was evaluated by means of biochemical parameters and diet. A case–control study was performed in a group
of adolescents with Down syndrome (n = 30) and a control group (n = 32), of both sexes, aged 10 to 19 years. Diet evaluation was accomplished by using a 3-day dietary record, and the analysis
was performed by the NutWin program, version 1.5. Antropometric measurements were performed for evaluation of body composition.
The Zn-related nutritional status of the groups was evaluated by means of zinc concentration determinations in plasma and
erythrocytes, and 24-h urinary zinc excretion, by using the method of atomic absorption spectroscopy. The diet of both groups
presented adequate concentrations of lipids, proteins, carbohydrates, and zinc. The mean values found for zinc concentration
in erythrocytes were 49.2 ± 8.5 μg Zn/g Hb for the Down syndrome group and 35.9 ± 6.1 μg Zn/g Hb for the control group (p = 0.001). The average values found for zinc concentration in plasma were 67.6 ± 25.6 μg/dL for the Down syndrome group and
68.9 ± 22.3 μg/dL for the control group. The mean values found for zinc concentration in urine were 244.3 ± 194.9 μg Zn/24 h
for the Down syndrome group and 200.3 ± 236.4 μg Zn/24 h for the control group. Assessment of body composition revealed overweight
(26.7%) and obesity (6.6%) in the Down syndrome group. In this study, patients with Down syndrome presented altered zinc levels
for some cellular compartments, and the average zinc concentrations were low in plasma and urine and elevated in erythrocytes. 相似文献
9.
Aldo Viarengo Graziella Mancinelli Bruno Burlando Isabella Panfoli Barbara Marchi 《Polar Biology》1999,21(6):369-375
Cell calcium is accumulated in intracellular stores by sarco-endoplasmic reticulum Ca2+ ATPases functionally interacting with the membrane lipid environment. Cold adaptations of membrane lipids in Antarctic Sea
organisms suggest possible adaptive effects also on sarco-endoplasmic reticulum Ca2+ ATPases. We investigated the SR Ca2+ ATPase of an Antarctic scallop, Adamussium colbecki, by characterising the enzyme activity and studying temperature effects. Ca2+ ATPase, assayed by following ATP hydrolysis, was thapsigargin- and vanadate-sensitive, showed maximum activity under 2 μM
Ca2+, 200 mM KCl and pH 7.2, and had a K
M for ATP of 22 ± 7 μM. Temperature effects showed an Arrhenius inversion between −1.8 and 0°C, indicating cold adaptation,
an Arrhenius break at 10°C, and a collapse above 20°C. A. colbecki accumulates high amounts of cadmium in the digestive gland; heavy metal effects on sarco-endoplasmic reticulum Ca2+ ATPases were therefore tested, finding an IC50 = 0.9 μM for Hg2+ and 3 μM for Cd2+. Finally, SDS-PAGE analysis showed a main band at about 100 kDa, which was identified as sarco-endoplasmic reticulum Ca2+ ATPase after trypsin digestion, and accounted for 60% total protein.
Accepted: 10 December 1998 相似文献
10.
Akula Ramakrishna Parvatam Giridhar Gokare Aswathanarayana Ravishankar 《In vitro cellular & developmental biology. Plant》2011,47(6):667-673
High-frequency somatic embryogenesis was achieved in Coffea canephora using calcium ionophore A23187, which influences the influx of calcium into a cell. With 100 μM calcium ionophore and 5 mM
calcium, 85% and 70% of cultures produced embryogenic tissue, with 105 ± 7 and 95 ± 8 primary embryos from each callus mass
respectively. Medium supplemented with 100 μM EGTA (calcium chelator) or 1 mM verapamil (calcium channel blocker) significantly
reduced somatic embryogenesis. Calcium imaging studies were done to determine the relationship between morphogenetic response
and the cellular calcium levels. The calcium ionophore/calcium treatment was very effective in driving cellular machinery
toward embryogenesis. The embryos were regenerated into plantlets when cultured on MS medium supplemented with 5 mM calcium/100 μM
calcium ionophore A23187. Somatic embryogenesis-derived plants were successfully transferred to soil and grown to maturity
in the field. 相似文献
11.
Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures
Summary. In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H2O2 is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was
shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H2O2. Superoxide dismutase (5 U/ml) induced an increase in H2O2 production by 22.2%. This indicates that at least part of the H2O2 is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene
iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H2O2 under copper stress for 90%. The influence of the pH on the H2O2 production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species
after copper stress.
Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003
RID="*"
ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan
171, 2020 Antwerp, Belgium. 相似文献
12.
B B Nepple J Kessi R Bachofen 《Journal of industrial microbiology & biotechnology》2000,25(4):198-203
Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and
anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic
cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic
interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration.
The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations
of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K
m of 15±1.3 μM CrO4
2− and a V
max of 420±50 μmol min−1 mg protein−1 was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO4
2− and 100±9.6 μmol CrO4
2− min−1 mg protein−1 for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203.
Received 05 January 2000/ Accepted in revised form 27 May 2000 相似文献
13.
Vanilla planifolia is a tropical orchid mainly known for the aromatic flavor of its cured pods. Callus cultures were initiated from leaf and
nodal explants of V. planifolia. Leaf explants showed better callus initiation than the nodal explants with callus biomass production maximal when cultured
on Murashige and Skoog (MS) basal medium containing 4.52 mM 2,4-dichlorophenoxy acetic acid and 2.22 mM benzyladenine (BAP).
Callus transferred to MS basal medium supplemented with 13.32 μM BAP, and 13.43 μM naphthaleneacetic acid (NAA) showed superior
growth response and produced 14.0 ± 1.0 shoots with an average length of 3.6 ± 0.1 cm after 40 d. Subsequent transfer of the
proliferated shootlets to MS basal medium supplemented with 8.88 μM BAP and 8.08 μM NAA produced 12.3 ± 0.14 plantlets with
an average height of 3.6 cm ± 0.10 cm. All plantlets produced profuse rooting within 35–40 d after transfer to half-strength
MS basal medium supplemented with NAA in combination with indole-3-acetic acid. Rooted plantlets were transferred for hardening,
with 80% of the plantlets becoming successfully established in the field. Potentially, more than 100,000 plantlets could be
produced within eight subcultures from callus obtained from leaf explant through the methods described here. 相似文献
14.
Release of an acid phosphatase activity during lily pollen tube growth involves components of the secretory pathway 总被引:2,自引:0,他引:2
Ibrahim H Pertl H Pittertschatscher K Fadl-Allah E el-Shahed A Bentrup FW Obermeyer G 《Protoplasma》2002,219(3-4):176-183
Summary. An acid phosphatase (acPAse) activity was released during germination and tube growth of pollen of Lilium longiflorum Thunb. By inhibiting components of the secretory pathway, the export of the acPase activity was affected and tube growth
stopped. Brefeldin A (1 μM) and cytochalasin D (1 μM), which block the production and transport of secretory vesicles, respectively,
inhibited the acPase secretion. The Ca2+ channel blocker gadolinium (100 μM Gd3+) also inhibited acPase secretion and tube growth, whereas 3 mM caffeine, another Ca2+ uptake inhibitor, stimulated the acPase release, while tube growth was inhibited. The Yariv reagent (β-D-glucosyl)3 Yariv phenylglycoside stopped tube growth by binding to arabinogalactan proteins of the tube tip cell wall but did not affect
acPase secretion. A strong correlation between tube growth and acPase release was detected. The secreted acPase activity had
a pH optimum at pH 5.5, a K
M of 0.4 mM for p-nitrophenyl phosphate, and was inhibited by zinc, molybdate, phosphate, and fluoride ions, but not by tartrate. In electrophoresis
gels the main acPase activity was detected at 32 kDa. The conspicuous correlation between activity of the secretory pathway
and acPase secretion during tube elongation strongly indicates an important role of the acPase during pollen tube growth and
the secreted acPase activity may serve as a useful marker enzyme assay for secretory activity in pollen tubes
Received July 25, 2001 Accepted January 15, 2002 相似文献
15.
NADH-stimulated, cyanide-resistant superoxide production in maize coleoptiles analyzed with a tetrazolium-based assay 总被引:9,自引:0,他引:9
Using the tetrazolium salt XTT (Na,3′-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid
hydrate) as a sensitive and physiologically compatible probe for the determination of superoxide (O2
·−) production in vivo, we have shown that maize (Zea mays L.) coleoptiles possess the capacity of generating O2
·− in the apoplastic space. Our results are in agreement with the notion that this activity is localized at the plasma membrane
and can be attributed to an O2
·−-synthesizing enzyme with catalytic and kinetic properties similar to that of the NADPH oxidase of mammalian phagocytes, with
the important exception that it utilizes NADH instead of NADPH as electron donor. When applied to the apoplastic space, NADH
strongly increased the O2
·−-producing activity of coleoptiles. The maize NADH-dependent O2
·−-synthase activity could clearly be differentiated from peroxidase-mediated O2
·−-synthesizing activity by its insensitivity to cyanide and azide, as well as by its much higher affinity to O2. Formation of O2
·−, and concomitantly appearing H2O2, was preferentially localized in the outer epidermis of the coleoptile. The physiological significance of O2
·− and H2O2 production in relation to the growth-controlling function of the epidermal cell wall is discussed.
Received: 14 January 2000 / Accepted: 2 May 2000 相似文献
16.
Casimiro-Lopes G de Oliveira-Junior AV Portella ES Lisboa PC Donangelo CM de Moura EG Koury JC 《Biological trace element research》2009,127(2):109-115
The purpose of this study was to compare plasma leptin, plasma zinc, and plasma copper levels and their relationship in trained
female and male judo athletes (n = 10 women; n = 8 men). Blood samples were obtained 24 h after training to measure plasma zinc, copper, and leptin levels. Subjects presented
similar values to age (22 ± 2 years old), body mass index (24 ± 1 kg/m2), plasma zinc (17.2 ± 2 μmol/L), copper (12.5 ± 2 μmol/L), and leptin (5.6 ± 1.3 μg/L). However, height, total body mass,
lean mass, fat mass, and sum of ten-skinfold thickness were higher in male than female. Plasma leptin was associated with
sum of ten skinfolds in male (r = 0.91; p < 0.001) and female athletes (r = 0.84; p < 0.003). Plasma zinc was associated with leptin in males (r = 0.82; p < 0.05) while copper was associated with plasma leptin in females (r = 0.66; p < 0.05). Our results suggest that young judo athletes lost sex-related differences in leptin levels. Plasma zinc, plasma
copper, and energy homeostasis may be involved in regulation of plasma leptin. 相似文献
17.
K. S. Lam S. W. Mamber E. J. Pack S. Forenza P. B. Fernandes D. M. Klaus 《Applied microbiology and biotechnology》1998,49(5):579-583
The effect of space flight on the production of the antibiotic monorden on two types of agar media, T8 and PG, by Humicola fuscoatra WC5157 was examined on board the US Space Shuttle mission STS-77 in May 1996. Paired space-flight and ground control samples
were prepared using identical hardware, protocol, media, and inoculum. Inoculation occurred simultaneously for both groups
2.5 h after launch. The flight and ground samples were allowed to grow for the entire 10-day mission in a dark, thermally
controlled (22 °C) environment. Post-flight HPLC analysis of the flight and ground sample extracts indicated that the production
of monorden by H. fuscoatra WC5157 in the flight samples was higher than in the ground samples in both agar media. In the T8 medium, the production of
monorden in the flight and ground samples was 11.6 ± 3.5 μg and 8.9 ± 1.1 μg respectively (30% increase). In the PG medium,
the production of monorden in the flight and ground samples was 23.8 ± 3.3 μg and 8.2 ± 2.2 μg respectively (190% increase).
The production of monorden in the flight and ground control samples was confirmed by HPLC-MS analysis.
Received: 30 September 1997 / Received revision: 23 December 1997 / Accepted: 2 January 1998 相似文献
18.
P. Wahl P. Walser-Volken K. Laumen M. Kittelmann O. Ghisalba 《Applied microbiology and biotechnology》1999,53(1):12-18
Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed
at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic
acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific
activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation.
The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn2+ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of
the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure.
Received: 8 February 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999 相似文献
19.
Cell-free preparations of Tetrahymena thermophila catalyze the direct desaturation of cholesterol to Δ7-dehydrocholesterol (provitamin D3). The activity was isolated in the microsomal fraction from Tetrahymena homogenates. Δ7-Desaturase activity was stimulated fivefold by the addition of 6 mM ATP. Other cofactors assayed, including NAD, NADP, NADH
or NADPH, had no significant effect. The activity was found in microsomes prepared from stationary-phase cultures of the ciliate,
grown either with or without added cholesterol, thus indicating that it is constitutively expressed in T. thermophila cells.
Received: 17 May 1999 / Accepted: 24 September 1999 相似文献
20.
Tomaskova Z Gaburjakova J Brezova A Gaburjakova M 《Journal of bioenergetics and biomembranes》2007,39(4):301-311
The objective of this work was to characterize in more detail the inhibition effect of diisothiocyanatostilbene-2′,2-disulfonic
acid (DIDS) on anion channels isolated from the rat heart mitochondria. The channels reconstituted into a planar lipid membrane
displayed limited powers of discrimination between anions and cations and the ion conductance measured under asymmetric (250/50 mM
KCl, cis/trans) and symmetric (150 mM KCl) conditions was ∼100 pS. DIDS caused a dramatic decrease in the channel activity (IC50 = 11.7 ± 3.1 μM) only when it was added to the cis side of a planar lipid membrane. The inhibition was accompanied by the significant prolongation of closings and the shortening
of openings within the burst as well as gaps between bursts were prolonged and durations of bursts were reduced. The blockade
was complete and irreversible when concentration of DIDS was increased up to 200 μM. Our data indicate that DIDS is an allosteric
blocker of mitochondrial anion channels and this specific effect could be used as a tool for reliable identification of anion
channels on the functional level. 相似文献