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1.
Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of
the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min–1 (mg protein)–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose,
and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present
in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete
glyoxylate cycle is not functional during symbiosis. The apparent K
m of isocitrate lyase for d,l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts
from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V
max nor the d,l-isocitrate K
m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to
facilitate operation of the glyoxylate cycle in B. japonicum.
Received: 10 October 1997 / Accepted: 16 January 1998 相似文献
2.
Jan R. Van der Ploeg Jaap Kingma Egbert J. De Vries Jos G.M. Van der Ven Dick B. Janssen 《Archives of microbiology》1996,165(4):258-264
Pseudomonas sp. GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is
toxic at low concentrations (1 mM). A muatnt that could grow on 2-bromoethanol with a growth rate of 0.034 h–1 at concentrations up to 5 mM was isolated and designated strain GJ1M9. Measurement of enzyme activities showed that mutant
and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was
threefold overexpressed in the mutant when grown on 2-chloroethanol, but only slightly overproduced when grown on 2-bromoethanol.
Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols. Haloacetate
dehalogenase levels were similar in the wild-type and the mutant. Activities of NAD-dependent aldehyde dehydrogenase were
only slightly higher in extracts of the mutant grown with 2-bromoethanol than in those of the wild-type grown with 2-chloroethanol.
SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant
from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2-chloroethanol.
The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa. The enzyme had low Km values for acetaldehyde and other non-halogenated aldehydes (0.8–4 μM), but much higher Km values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while Vmax values were similar for halogenated and non-halogenated aldehydes. Cultures that were pregrown on 2-chloroethanol rapidly
lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde
inactivates the enzyme. To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary
in order to compensate for the high Km for bromoacetaldehyde and for inactivation of the enzyme by bromoacetaldehyde.
Received: 31 August 1995 / Accepted: 4 December 1995 相似文献
3.
Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable
level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtilis. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway.
We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plasmid-encoded gbsA gene under T7φ10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium
sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on
phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested
that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could
use both NAD+ and NADP+ as cofactors, but NAD+ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine
revealed K
m values of 125 μM and 143 μM for its substrates glycine betaine aldehyde and NAD+, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic
conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated
the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was
highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction
product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress.
Received: 2 May 1997 / Accepted: 2 July 1997 相似文献
4.
Charles M. H. Hensgens Manny E. Nienhuis-Kuiper Theo A. Hansen 《Archives of microbiology》1994,162(1-2):143-147
Growth of Desulfovibrio gigas NCIMB 9332 in mineral, vitamin-supplemented media with ethanol as substrate was strongly stimulated by the addition of tungstate (optimal level approximately 10-7 M). At suboptimal tungstate concentrations, up to 1.0 mM acetaldehyde was detected in the culture supernatant and growth was slow. Omission of both tungstate and molybdate from the media prevented growth and ethanol utilization. Tungstate-deprived cultures that were grown on lactate had much lower aldehyde dehydrogenase (benzylviologen as acceptor; BV-AIDH) levels than tungstate-supplemented cultures. These data suggest that tungstate is required for the synthesis of active BV-AIDH. The characteristics of the enzyme activities in cell-free extracts show that the BV-AIDH activity present in tungstate-supplemented cultures is not due to the recently characterized molybdenum-containing aldehyde dehydrogenase of D. gigas. Out of 13 other strains of ethanol-oxidizing, gram-negative, sulfate-reducing bacteria tested, most strains grew well with either tungstate or molybdate supplementation. In contrast to a recent report, good growth on ethanol of two D. baculatus (Desulfomicrobium) strains (DSM 1741 and DSM 1743) was observed.Abbreviations
BV-AIDH
Benzylviologen-linked aldehyde dehydrogenase
-
DCPIP-AIDH
2,6-dichlorophenolindophenol-linked aldehyde dehydrogenase
-
DTT
dithiothreitol 相似文献
5.
The uptake of 3H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium.
Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1
μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected:
(i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when
Na+ was replaced by Li+, K+ or Mg2+, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na+ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na+ concentration. Kinetic properties of the uptake of 0.1 μm
3H-choline in the presence and absence of medium Na+ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na+; a Na+-dependent portion (a mean of 0.52 of the total) had a K
m
for choline of 1.5 μm while K
m
in the absence of Na+ (Li+ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave Ki values of 1.7 μm and 5.0 μm HC-3 for the Na+-dependent and -independent fractions. The apparent K
m
of the Na+-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K
m
values reported for high-affinity, Na+-dependent choline uptake by neuronal cells.
Received: 18 February 1997/Revised: 7 December 1997 相似文献
6.
These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch
clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch
clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (KCa) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current
and the KCa activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V
m
) and 145 ± 13 pS at negative V
m
. Glibenclamide (50 μm), Ba2+ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50= 46 μm. The identity of this K+ channel with an ATP-sensitive K+ channel (KATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the KCa and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked KATP.
Received: 12 June 1997/Revised: 21 October 1997 相似文献
7.
Metabolism of EDTA and its metal chelates by whole cells and cell-free extracts of strain BNC1 总被引:5,自引:0,他引:5
The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts
of strain BNC1 was investigated. Metal-EDTA chelates with thermodynamic stability constants below 1012 were readily mineralized by whole cells with maximum specific turnover rates of 15 (MnEDTA) to 20 (Ca-, Mg-, and BaEDTA)
μmol g protein−1 min−1. With the exception of ZnEDTA, chelates with stability constants greater than 1012 were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded
after pretreatment by addition of calcium and magnesium salts in the pH range 9–11. The range of EDTA chelates converted by
cell-free extracts of strain BNC1 did not depend on their thermodynamic stabilities. The EDTA chelates of Ba2+, Co2+, Mg2+, Mn2+, and Zn2+ were oxidized whereas Ca-, Cd-, Cu-, Fe-, Pb-, and SnEDTA were not. The first catabolic enzyme appears to be an EDTA monooxygenase
since it requires O2, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded
via N,N′-ethylenediaminediacetate. The maximum specific turnover rate with MgEDTA, the favoured EDTA species, was 50–130 μmol g protein−1 min−1, and the K
m value was 120 μmol/l (K
s for whole cells = 8 μmol/l). Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues
of EDTA.
Received: 4 July 1997 / Received revision: 25 September 1997 / Accepted: 29 September 1997 相似文献
8.
Molecular characterization of phenylalanine ammonia lyase gene from <Emphasis Type="Italic">Cistanche deserticola</Emphasis> 总被引:1,自引:0,他引:1
Hu GS Jia JM Hur YJ Chung YS Lee JH Yun DJ Chung WS Yi GH Kim TH Kim DH 《Molecular biology reports》2011,38(6):3741-3750
We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes
from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein
exhibited Michaelis–Menten kinetics with a K
m of 0.1013 mM, V
max of 4.858 μmol min−1, K
cat of 3.36 S−1, and K
cat/K
m is 33,168 M−1 S−1. The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol−1 when l-Phenylalanine was used as a substrate; l-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results
showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min
resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl
aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid
(tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a K
i of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with K
i = 0.056 μM. 相似文献
9.
The Ca2+-activated maxi K+ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean
number of open channels (NP
o
) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the
stretch-activation of Ca2+-permeable cation channels, because the maxi K+ channel was not stretch-activated in both the cell-attached configuration using Ca2+-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K+ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K+ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K+ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV
t
) when luminal K+ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV
t
was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced
ΔV
t
to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV
t
to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV
t
was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV
t
=−7.2 ± 0.5 mV in control vs.ΔV
t
=−6.7 ± 0.6 mV in AA). These results suggest that the Ca2+-activated maxi K+ channel is responsible for flow-dependent K+ secretion by coupling with the stretch-activated Ca2+-permeable cation channel in the rabbit CNT.
Received: 21 August 1997/Revised: 20 March 1998 相似文献
10.
J R Kastner W J Jones R S Roberts 《Journal of industrial microbiology & biotechnology》1998,20(6):339-343
The kinetics of biomass formation, D-xylose utilization, and mixed substrate utilization were determined in a chemostat using the yeast Candida shehatae. The maximum growth rate of C. shehatae grown aerobically on D-xylose was 0.42 h−1 and the Monod constant, K
s, was 0.06 g L−1. The biomass yield, Y
{X/S}, ranged from 0.40 to 0.50 g g−1 over a dilution rate range of 0.2–0.3 h−1, when C. shehatae was grown on pure D-xylose. Mixtures of D-xylose and glucose (∼1 : 1) were simultaneously utilized over a dilution rate from 0.15 to 0.35 h−1 at pH 3.5 and 4.5, but pH 3.5 reduced μmax and reduced the dilution rate range over which D-xylose was utilized in the presence of glucose. At pH 4.5, μmax was not reduced with the mixed sugar feed and the overall or lumped K
s value was not significantly increased (0.058 g L−1
vs 0.06 g L−1), when compared to a pure D-xylose feed. Kinetic data indicate that C. shehatae is an excellent candidate for chemostat production of value added products from renewable carbon sources, since simultaneous
mixed substrate utilization was observed over a wide range of growth rates on a 1 : 1 mixture of glucose and D-xylose.
Received 21 August 1997/ Accepted in revised form 28 May 1998 相似文献
11.
ATP-sensitive K+ (KATP) channels have been characterized in pituitary GH3 cells with the aid of the patch-clamp technique. In the cell-attached configuration, the presence of diazoxide (100 μm) revealed the presence of glibenclamide-sensitive KATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mm) or sodium cyanide (300 μm) increased KATP channel activity, while nicorandil (100 μm) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of KATP channels in a concentration-dependent manner with an IC50 value of 30 μm. The activation of phospholipase A2 caused by mellitin (1 μm) was found to enhance KATP channel activity and further application of aristolochic acid (30 μm) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4′-dithiodipyridine (100 μm) also induced KATP channel activity. Diazoxide, mellitin and 4,4′-dithiodipyridine activated the KATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of
diazoxide (100 μm) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly
indicates that KATP channels similar to those seen in pancreatic β cells are functionally expressed in GH3 cells. In addition to the presence of Ca2+-activated K+ channels, KATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane
potential.
Received: 19 June 2000/Revised: 13 September 2000 相似文献
12.
Minoru Tanigawa Tomomitsu Shinohara Makoto Saito Katsushi Nishimura Yuichiro Hasegawa Sadao Wakabayashi Morio Ishizuka Yoko Nagata 《Amino acids》2010,38(1):247-255
Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. d-Amino acid dehydrogenase is a flavoenzyme that digests free neutral d-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 d-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial d-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to
d-proline. The enzyme mediated electron transport from d-proline to coenzyme Q1, thus distinguishing it from d-amino acid oxidase. The apparent K
m and V
max values were 40.2 mM and 25.0 μmol min−1 mg−1, respectively, for dehydrogenation of d-proline, and were 8.2 μM and 12.3 μmol min−1 mg−1, respectively, for reduction of Q1. The respective pH and temperature optima were 8.0 and 37°C. Enzyme activity was inhibited markedly by benzoate, and moderately
by SH reagents. DadA showed more similarity with mammalian d-amino acid oxidase than other bacterial d-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from d-proline to a c-type cytochrome was suggested spectrophotometrically. 相似文献
13.
Anaerobic degradation of α-resorcylate (3,5-dihydroxybenzoate) was studied with the denitrifying strain AR-1, which was assigned
to the described species Thauera aromatica. α-Resorcylate degradation does not proceed via the benzoyl-CoA, the resorcinol, or the phloroglucinol pathway. Instead,
α-resorcylate is converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by dehydrogenative oxidation and decarboxylation.
Nitrate, K3[Fe(CN)6], dichlorophenol indophenol, and the NAD+ analogue 3-acetylpyridine adeninedinucleotide were suitable electron acceptors for the oxidation reaction; NAD+ did not function as an electron acceptor. The oxidation reaction was strongly accelerated by the additional presence of the
redox carrier phenazine methosulfate, which could also be used as sole electron acceptor. Oxidation of α-resorcylate with
molecular oxygen in cell suspensions or in cell-free extracts of α-resorcylate- and nitrate-grown cells was also detected
although this bacterium did not grow with α-resorcylate under an air atmosphere. α-Resorcylate degradation to hydroxyhydroquinone
proceeded in two steps. The α-resorcylate-oxidizing enzyme activity was membrane-associated and exhibited maximal activity
at pH 8.0. The primary oxidation product was not hydroxyhydroquinone. Rather, formation of hydroxyhydroquinone by decarboxylation
of the unknown intermediate in addition required the cytoplasmic fraction and needed lower pH values since hydroxyhydroquinone
was not stable at alkaline pH.
Received: 8 July 1997 / Accepted: 20 October 1997 相似文献
14.
R. Wondergem M. Cregan L. Strickler R. Miller J. Suttles 《The Journal of membrane biology》1998,161(3):257-262
These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma
(HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and 3H-thymidine uptake. Glibenclamide added at 75 and 150 μm for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth
at concentrations as low as 1 μm (IC50= 73 μm) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range
in which glibenclamide decreased open probability of membrane KATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined
by flow cytometry using 10−5
m bromodeoxyuridine. Glibenclamide (100 μm) increased the percentage of cells in G0/G1 from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens
membrane KATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We
conclude that the sulfonylurea receptor and the corresponding membrane KATP channel are involved in mechanisms controlling HTB-9 cell growth. However, KATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle.
Received: 12 June 1997/Revised: 21 October 1997 相似文献
15.
Galdieria sulphuraria (Galdieri) Merola can grow heterotrophically on at least ten different polyols. We investigated their metabolic path to glycolysis/gluconeogenesis
and identified two NAD-dependent polyol dehydrogenases. Activity of other enzymes metabolizing mannitol or sorbitol could
not be detected. The two dehydrogenases had a broad substrate specificity and were termed xylitol dehydrogenase (EC 1.1.1.14;
substrate specificity: xylitol > d-sorbitol > d-mannitol > l-arabitol) and d-arabitol dehydrogenase (EC 1.1.1.11; substrate specificity: d-arabitol > l-fucitol > d-mannitol > d-threitol) according to the substrate with the lowest K
m value. The xylitol dehydrogenase was stable during purification. In contrast, the d-arabitol dehydrogenase was thermolabile and depended on divalent ions for stability and activity, preferentially Mn2+ and Ni2+. The molecular mass of the xylitol dehydrogenase was estimated to be 295 kDa by size-exclusion chromatography and 220 kDa
by rate-sedimentation centrifugation. The d-arabitol dehydrogenase had a molecular mass of 105 kDa as determined by rate-sedimentation centrifugation. The specific activity
of both enzymes increased about fourfold when cells were transferred from autotrophic to heterotrophic conditions regardless
of whether sugars or polyols were supplied as substrates. The significance of polyol metabolism in Galdieria sulphuraria with regard to the natural habitat of the alga is discussed.
Received: 15 January 1997 / Accepted: 12 February 1997 相似文献
16.
Clostridium acetobutylicum strain P262 utilized lactate at a rapid rate [600 nmol min–1 (mg protein)–1], but lactate could not serve as the sole energy source. When acetate was provided as a co-substrate, the growth rate was
0.05 h–1. Butyrate, carbon dioxide and hydrogen were the end products of lactate and acetate utilization, and the stoichiometry was
1 lactate + 0.4 acetate → 0.7 butyrate + 0.6 H2 + 1 CO2. Lactate-grown cells had twofold lower hydrogenase than glucose-grown cells, and the lactate-grown cells used acetate as
an alternative electron acceptor. The cells had a poor affinity for lactate (Ks = 1.1 mM), and there was no evidence for active transport. Lactate utilization was catabolyzed by an inducible NAD-independent
lactate dehydrogenase (iLDH) that had a pH optimum of 7.5. The iLDH was fivefold more active with d-lactate than l-lactate, and the K
m
for d-lactate was 3.2 mM. Lactate-grown cells had little butyraldehyde dehydrogenase activity, and this defect did not allow the
conversion of lactate to butanol.
Received: 17 October 1994 / Accepted: 30 January 1995 相似文献
17.
Bacteria that produced NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among l-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified
in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an
isoelectric point of 5.8 and a relative molecular weight (M
r) of approximately 330,000. The enzyme is composed of eight identical subunits with an M
r of approximately 41,000. The apparent K
m values for l-phenylalanine and NAD+ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55° C
for 10 min.
Received: 30 July 1997 / Accepted: 4 November 1997 相似文献
18.
O. Pines S. Shemesh E. Battat I. Goldberg 《Applied microbiology and biotechnology》1997,48(2):248-255
Saccharomyces cerevisiae accumulates l-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate
dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression
of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a
6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in l-malic acid accumulation in the production medium. The high apparent K
m of MDH2 for l-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function
of the enzyme and differs from the previously published K
m of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be
a limiting factor, thus providing a system for further metabolic engineering of l-malic acid production. The overexpression of MDH2 activity also causes an elevation in the accumulation of fumaric acid and
citric acid. Accumulation of fumaric acid is presumably caused by high intracellular l-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing
possibility that cytosolic l-malic acid is a direct precursor of citric acid in yeast.
Received: 22 January 1997 / Received revision: 14 April 1997 / Accepted: 19 April 1997 相似文献
19.
Cell-free extracts (CFEs) of chromium-resistant bacterium Bacillus sphaericus AND 303 isolated from serpentine soil of Andaman, India reduced Cr(VI) in in vitro condition, and the reductase activity was solely localized in the soluble cell-fractions (S12, S32, and S150). The enzyme was constitutive as the CFEs from cells grown in Cr(VI)-free and Cr(VI)-containing media reduced a more or less
equal amount of Cr(VI). Optimum Cr(VI) reductase activity was obtained at an enzyme (S150) concentration equivalent to 4.56 mg protein/mL, 300 μM Cr(VI) and pH 6.0 after 30 min incubation at 30°C. The enzyme was heat labile; 80% of its activity was lost when exposed
at 70°C for 15 min. Kinetics of Cr(VI) reductase activity fit well with the linearized Lineweaver-Burk plot and showed a Vmax of 1.432 μmol Cr(VI)/mg protein/min and Km of 158.12 μM Cr(VI). The presence of additional electron donors accelerated Cr(VI) reductase activity of CFE, and an increase of 28% activity
over control was recorded with 1.0 μM NADH. Heavy metal ions such as Ni(II), Cu(II), and Cd(II) were strong inhibitors of Cr(VI) reductase unlike that of 100 μM Co(II), which retained 93% activity over control. 相似文献
20.
A K+ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid
bilayers. This channel was identified as an ATP-sensitive K+ channel (KATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na2. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0
mV and with a 5/250 KCl gradient, the main conductance of the KATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil
stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels.
In general, KATP channels were characterized by brief openings followed by long closings (open probability, P
o
≈ 0.02); only occasionally (3 out of 12 experiments) did the KATP channels have a high open probability (P
o
≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl2 to the internal side of the channel.
Received: 7 November 1995/Revised: 10 June 1996 相似文献