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1.
E. Bolado-Martínez E. Acedo-Félix A. B. Peregrino-Uriarte G. Yepiz-Plascencia 《Applied Biochemistry and Microbiology》2012,48(5):444-451
Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri. 相似文献
2.
Homogeneous phosphoribulokinase (PRK; ATP: d-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was isolated from wheat leaves with a specific activity of 15 kat mg-1 protein. The purification included ammonium sulfate cuts, isoelectric precipitation, and hydrophobic and affinity chromatography on pentylagarose and Blue Sepharose CL 6B, respectively. Gel filtration of the purified enzyme yielded a 83000 Da protein. Subunits of about 42000 Da were estimated from sodium dodecyl sulfate-polyacrylamide gels. Wheat leaf PRK was stable for at least four weeks when stored at 4°C. Saturation curves for ribulose 5-phosphate (Ru5P) and ATP followed Michaelis-Menten kinetics (K
m values: K
m Ru5P=50–80 M; K
m ATP=70 M). The saturation curve for MgCl2 was sigmoidal (half-maximal velocity <0.5 mM). The affinity for Ru5P, ATP and Mg2+ was not affected by pH changes comparable to pH shifts in the stroma. In contrast to chloroplast fructose-bisphosphatase (Zimmermann et al. 1976, Eur. J. Biochem. 70, 361–367) the affinity for ligands remained unchanged in the dithiothreitol-activated and in the non-activated state. The activity of PRK was increasingly sensitive to inhibition by 3-phosphoglyceric acid with decreasing pH below pH 8.0.Abbreviations DTT
dithiothreitol
- EDTA
ethylenediamine-tetraacetic acid
- PRK
phosphoribulokinase
- Ru5P
ribulose-5-phosphate
- SDS-PAGE
sodium dodecyl sulfate-polyacryl-amide gel electrophoresis 相似文献
3.
Ulrich Nowitzki Ralf Wyrich Peter Westhoff Katrin Henze Claus Schnarrenberger William Martin 《Plant molecular biology》1995,29(6):1279-1291
Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.Abbreviations R5P3E
d-ribulose-5-phosphate 3-epimerase
- RPI
ribose-5-phosphate isomerase
- TKL
transketolase
- PRK
phosphoribulokinase
- GAPDH
glyceraldehyde-3-phosphate dehydrogenase
- FBP
fructose-1,6-bisphophatase
- FBP
fructose 1,6-bisphosphate
- G6PDH
glucose-6-phosphate dehydrogenase
- 6PGDH
6-phosphogluconate dehydrogenase
- OPPP
oxidative pentose phosphate pathway
- Rubisco
ribulose-1,5-bisphosphate carboxylase/oxygenase
- FBA
fructose-1,6-bisphophate aldolase
- IPTG
isopropyl -d-thiogalactoside
- GST
glutathione S-tranferase
- PBS
phosphate-buffered saline
- TPI
triosephosphate isomerase 相似文献
4.
Phosphoketolase (E.C. 4.1.2.9) was found in crude extracts of Thiobacillus novellus (ATCC 8093) with a specific activity of 0.070 mol triose formed min-1 (mg protein)-1. The pH optimum, 6.0, temperature optimum, 43° C, and K
m
, 4.27 mM, are in good agreement with values observed for phosphoketolase from other organisms.The level of phosphoketolase in lithotrophically grown T. novellus was observed to be much lower, 0.002 mol triose formed min-1 (mg protein)-1 than the level in heterotrophically grown cells.
T. thioparus, a lithotroph, and T. intermedius, a mixotroph, were examined and found not to contain phosphoketolase. T. A2, a mixotroph reported to be very similar to T. novellus, was examined and found to have about 20% of the level of phosphoketolase as that seen in heterotrophically grown T. novellus.Fructose-6-phosphate was examined as a possible alternate substrate but was found not to be enzymatically cleaved in our system.It therefore appears that T. novellus utilizes a previously unknown combination of a partially complete hexose monophosphate pathway, the phosphoketolase reaction, and a partially complete Embden-Meyerhof-Parnas pathway, in conjunction with the Krebs Cycle, for glucose catabolism. 相似文献
5.
Fructose-6-phosphate phosphoketolase was purified from type strains of two species of the genus Bifidobacterium: B. globosum and B. dentium. The first species has a preferred animal habitat, like feces of animals and rumen of cattle; the latter is harboured in human habitats, like feces and dental caries of man. Two electrophoretic types of phosphoketolase (F6PPK) were previously distinguished and called animal and human type according to the habitat of the bifid organism. The purified preparations of these two phosphoketolases displayed very different optimum pH range, metal activator and molecular weight; outstanding difference was found in the substrate specificity: the enzyme from B. globosum was able to split xylulose-5-P as well as fructose-6-P, whereas the phosphoketolase from B. dentium appeared to be specific for fructose-6-P. 相似文献
6.
André Morin Jean-Pierre Touzel Alain Lafond Danielle Leblanc 《Applied microbiology and biotechnology》1991,35(4):536-540
Summary Of 373 anaerobic microbial isolates screened for the enzymatic conversion of dihydrouracil to N-carbamyl--alanine, several strains of Clostridium spp., C. glycolicum, C. subterminale and Peptococcus anaerobius were positive. These Clostridium and Peptococcus strains produced also N-carbamyl-d-amino acids from the respective 5-monosubstituted hydantoins. The d-hydantoinase activity from whole cell suspensions of P. anaerobius strain CRDA 303 was characterized with regard to pH and temperature stability and activity by using dihydrouracil (DHU) and isopropylhydantoin (IPH) as substrates. The d-hydantoinase from P. anaerobius was optimal at 60°C and at pH 6.5–9.5 for the substrate DHU. It was stable up to 55°C and at pH 5.0–9.5 and could be stored at 4°C under an aerobic atmosphere for at least 14 days.
Offprint requests to: A. Morin 相似文献
7.
Lee JM Jeong DW Koo OK Kim OK Kim MJ Lee JH Chang HC Kim JH Lee HJ 《Biotechnology letters》2005,27(12):853-858
The gene encoding phosphoketolase, which is 2749 bp long and contains 814 amino acid polypeptides with a total molecular mass of 91.9 kDa, was cloned from Leuconostoc mesenteroides C7 (LMC7) and expressed in Escherichia coli. It exhibited a homology of >58% with phosphoketolases from other lactic acid bacteria. The phosphoketolase of LMC7 belongs to the xylulose 5-phosphate (X5P)/fructose 6-phosphate (F6P) phosphoketolase (Xfp) family, which is an enzyme with dual specificity for X5P and F6P. The members of this family contain typical thiamin pyrophosphate (TPP) binding sites as reported for other TPP-dependent enzymes, and several highly conserved regions as signature patterns for phosphoketolases. The plasmid pGPK containing the Xfp gene (xfp) exhibits phosphoketolase activity in E. coli. The specific activities of the enzyme from E. coli BL21 and E. coli EC101 harboring xfp were 0.28 and 0.14 units/mg, respectively. They both exhibited a 1.5-fold increase in the production of acetic acid from acetyl phosphate compared with their corresponding original strain.An erratum to this article can be found at 相似文献
8.
Shulami S Yaniv O Rabkin E Shoham Y Baasov T 《Extremophiles : life under extreme conditions》2003,7(6):471-481
3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase, catalyzes the aldol-type condensation between phosphoenolpyruvate (PEP) and d-arabinose-5-phosphate (A5P) to produce the unusual 8-carbon sugar KDO8P, and inorganic phosphate. A 15.5-kb segment containing the kdsA gene from the hyperthermophilic bacterium Aquifex pyrophilus was cloned from a genomic library and sequenced. The native kdsA gene lacks a typical ribosome binding site, but contains a conserved U,A-rich sequence upstream to the start codon. The purified kdsA gene product catalyzes the formation of KDO8P from its natural substrates, PEP and A5P, as determined by 1H NMR analysis. KDO8P synthase showed maximum activity at 80 °C and pH 5.5–6.0 at 10-min reaction assay. At temperatures of 70, 80, and 90 °C, the enzyme exhibited half-lives of 8.0, 2.25, and 0.5 h, respectively. The kinetic constants at 60 °C were KmA5P=70 M, KmPEP=290 M, and kcat=4 s–1. The isolated enzyme contained 0.19 and 0.26 mol iron and zinc, respectively, per mole of enzyme subunit. Treatment with metal chelators eliminated enzyme activity, and by the addition of several divalent metal ions, the activity was restored and even exceeded the original activity. These results indicate that A. pyrophilus KDO8P synthase is a metal-dependent enzyme. A C11A mutant of KDO8P synthase from A. pyrophulis retained less than 1% of the wild-type activity and was shown to be incapable of metal binding.Communicated by G. Antranikian 相似文献
9.
Activity of a pentulose (xylulose 5-phosphate) phosphoketolase was detected in 20 out of 25 yeasts examined. No significant activity was detected in any yeast grown with glucose, and the enzyme was induced by up to 70-fold when the yeasts were grown on xylose as sole carbon source. Biomass yields from xylose were greater than, or approximately equal to, those from glucose in 15 of the 19 yeasts which possessed phosphoketolase activity. The molar yield of C2 units from xylose, by metabolism via the pentose phosphate pathway, can be calculated to be insufficient to account for the high yields of biomass and ethanol obtained from xylose. We have shown that the presence of a phosphoketolase system can account for such yields by producing 2 mol C2 from 1 mol C5. This pathway must therefore be regarded as a major route of pentose dissimilation in such yeasts. 相似文献
10.
Yoke Y. Chiew Maxwell G. Shepherd Patrick A. Sullivan 《Archives of microbiology》1980,125(1-2):97-104
The synthesis of chitin during germ-tube formation in Candida albicans may be regulated by the first and last steps in the chitin pathway: namely l-glutamine-d-fructose-6-phosphate aminotransferase and chitin synthase. Induction of germ-tube formation with either glucose and glutamine or serum was accompanied by a 4-fold increase in the specific activity of the aminotransferase. Chitin synthase in C. albicans is synthesized as a proenzyme. N-acetyl glucosamine increased the enzymic activity of the activated enzyme 3-fold and the enzyme exhibited positive co-operativity with the substrate, UDP-N-acetylglucosamine. Although chitin synthase was inhibited by polyoxin D (K
i =1.2M) this antibiotic did not affect germination. During germ-tube formation the total chitin synthase activity increased 1.4-fold and the expressed activity (in vivo activated proenzyme) increased 5-fold. These results could account for the reported 5-fold increase in chitin content observed during the yeast to mycelial transformation.Non-Standard Abbreviations GlcNac
N-acetyl glucosamine
- UDP-GlcNac
UDP-N-acetyl glucosamine
- PMSF
phenylmethylsulphonylfluoride 相似文献
11.
Chappell Mark A. Bucher Theresa L. 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1987,157(1):129-136
Summary The effects of ambient temperature (T
a) on ventilation and gas exchange in chukar partridges (Alectoris chukar) were determined after acclimation to low and high altitute (LA and HA; 340 and 3,800 m, respectively).At both LA and HA, oxygen consumption (
) increased with decreasingT
a atT
a from 20 to –20°C. AtT
a of 35 to 40°C,
increased above thermoneutral values at HA but remained constant and minimal at LA. Water loss rates increased rapidly atT
a>30°C at both altitudes as birds began to pant. Ventilation rates (f) during panting were 5-to 23-fold greater than the minimalf at thermoneutralT
a.Increased
atT
a below thermoneutrality was supported by increased minute volume (V
i) at both altitudes. The change inV
i was primarily a function of changing tidal volume (V
t), althoughf increased slightly asT
a declined. Oxygen extraction (
) remained fairly constant atT
a below 20°C at both altitudes. BothV
t and
were considerably lower when birds were panting than at lowerT
a.Chukars showed few obvious ventilatory adaptations to HA. The 35% change in
between 340 and 3,800 m was accommodated by a corresponding change inV
i (btps), most of which was accomplished by increasedf at HA, along with a slight increase in
.Abbreviations and symbols
HA
high altitude
-
LA
low altitude
-
rate of evaporative water loss
-
oxygen extraction efficiency
-
f
respiratory frequency
-
V
t
tidal volume
-
V
i
minute volume
-
BMR
basal metabolic rate
-
MHP
metabolic heat production 相似文献
12.
The oxygen requirements of yeasts for the fermentation of d-xylose and d-glucose to ethanol 总被引:1,自引:0,他引:1
Magdalena E. Ligthelm Bernard A. Prior James C. du Preez 《Applied microbiology and biotechnology》1988,28(1):63-68
Summary The effect of oxygen availability on d-xylose and D-glucose metabolism by Pichia stipitis, Candida shehatae and Pachysolen tannophilus was investigated. Oxygen was not required for fermentation of d-xylose or d-glucose, but stimulated the ethanol production rate from both sugars. Under oxygen-limited conditions, the highest ethanol yield coefficient (Ye/s) of 0.47 was obtained on d-xylose with. P. stipitis, while under similar conditions C. shehatae fermented d-xylose most rapidly with a specific productivity (qpmax) of 0.32 h-1. Both of these yeasts fermented d-xylose better and produced less xylitol than. P. tannophilus. Synthesis of polyols such as xylitol, arabitol, glycerol and ribitol reduced the ethanol yield in some instances and was related to the yeast strain, carbon source and oxygen availability. In general, these yeasts fermented d-glucose more rapidly than d-xylose. By contrast Saccharomyces cerevisiae fermented d-glucose at least three-fold faster under similar conditions.Nomenclature qpmax
maximum specific rate of ethanol production (g ethanol per g dry biomass per hour)
- Ye/s
ethanol yield (g ethanol per g substrate utilized)
- Yp/s
polyol yield (g polyol per g substrate utilized)
- Yx/s
biomass yield (g dry biomass per g substrate utilized)
- max
maximum specific growth rate (per hour) 相似文献
13.
A model of the C
3 photosynthetic system is developed which describes the sensitivity of the steadystate rate of carbon dioxide assimilation to changes in the activity of several enzymes of the system. The model requires measurements of the steady-state rate of carbon dioxide assimilation, the concentrations of several intermediates in the photosynthetic system, and the concentration of the active site of ribulose 1,5-bisphosphate carboxyalse/oxygenase (Rubisco). It is shown that in sunflowers (Helianthus annuus L.) at photon flux densities that are largely saturating for the rate of photosynthesis, the steady-stete rate of carbon dioxide assimilation is most sensitive to Rubisco activity and, to a lesser degree, to the activities of the stromal fructose, 6-bisphosphatase and the enzymes catalysing sucrose synthesis. The activities of sedoheptulose 1,7-bisphosphatase, ribulose 5-phosphate kinase, ATP synthase and the ADP-glucose pyrophosphorylase are calculated to have a negligible effect on the flux under the high-light conditions. The utility of this analysis in developing simpler models of photosynthesis is also discussed.Abbreviations
c
i
intercellular CO2 concentration
-
C
infP
supJ
control coefficient for enzyme P with respect to flux J
- DHAP
dihydroxyacetonephosphate
- E4P
erythrose 4-phosphate
- F6P
fructose 6-phosphate
- FBP
fructose 1,6-bisphosphate
- FBPase
fructose 1,6-bisphosphatase
- G3P
glyceraldehyde 3-phosphate
- G1P
glucose 1-phosphate
- G6P
glucose 6-phosphate
- Pi
inorganic phosphate
- PCR
photosynthetic carbon reduction
- PGA
3-phosphoglyceric acid
- PPFD
photosynthetically active photon flux density
-
R
n
J
response coefficient for effector n with respect to flux J
- R5P
ribose 5-phosphate
- Rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- Ru5P
ribulose 5-phosphate
- RuBP
ribulose 1,5-bisphosphate
- S7P
sedoheptulose 7-phosphate
- SBP
sedoheptulose 1,7-bisphosphate
- SBPase
sedoheptulose 1,7-bisphosphatase
- SPS
sucrose-phosphate synthase
- Xu5P
xylulose 5-phosphate
-
n
P
elasticity coefficient for effector n with respect to the catalytic velocity of enzyme P
This research was funded by an Australian Research Council grant to I.E.W. and was undertaken during a visity by K.A.M. to the James Cook University of North Queensland. The expert help of Glenys Hanley and Mick Kelly is greatly appreciated. 相似文献
14.
Among the several strains of halobacteria grown heterotrophically, ribulose bisphosphate carboxylase activity was detected in those which accumulate poly (-hydroxybutyrate), viz. Haloferax mediterranei, Haloferax volcanii and Halobacterium marismortui. In H. mediterranei, the activity was present in cell extracts prepared after growth on a variety of carbohydrates. The ribulose bisphosphate carboxylase activity in H. mediterranei was inhibited by carboxyarabinitol bisphosphate, and the enzyme cross-reacted with antibodies raised against the spinach enzyme. CO2 fixation by cell extract was stimulated by the addition of ATP and NADH. Preliminary data suggested that hydrogen could be a possible reductant.Abbreviations RuBP
ribulose bisphosphate
- Ru5P
ribulose 5-phosphate
- R5P
ribose 5-phosphate
- CABP
carboxyarabinitol bisphosphate
- PHB
poly (-hydroxybutyrate)
- DTT
dithiothreitol 相似文献
15.
Properties and regulation of ribulose diphosphate carboxylase from Thiobacillus novellus 总被引:1,自引:0,他引:1
Ribulose-diphosphate carboxylase from Thiobacillus novellus has been purified to homogeneity as observed by polyacrylamide gel electrophoresis and U. V. light observation during sedimentation velocity analysis. The optimum pH for the enzyme with Tris-HCl buffers was about 8.2. Concentrations of this buffer in excess of 80 mM were inhibitory. The apparent K
m
RuDP was about 14.8 M with a Hill value of 1.5, for HCO
3
-
the apparent K
m
was about 11.7 mM with an n value of 1.18 and for Mg2+ about 0.61 mM. The enzyme was specific for this cation. Relatively high concentrations of either Hg2+ or pCMB were required before significant inhibition was observed. Activity declined slowly during a 4-hr incubation period in either 3.0 M or 8.0 M urea. Incubation for 12 hrs resulted in complete loss of activity which was not prevented by 10 mM Mg2+ and was not reversed by dialysis and subsequent addition of 10 mM cysteine. Polyacrylamide gel electrophoresis revealed a loss of the major band and the appearance of 2 new bands. SDS polyacrylamide gel electrophoresis gave an average M.W. of 73 500±2500 for the slower moving band and 12250 ±2500 for the faster moving. However, incubation in urea for up to 40 hrs revealed a decrease in the M.W. of the slower moving band to about 60000. The E
a
for the enzyme was calculated to be about 18.85 kcal mole-1, with the possibility of a break between 40 and 50°C. The Q
10 was 3.07 between 20 to 30°C whereas between 30 to 40°C it was 3.31. Only phosphorylated compounds caused significant inhibition of enzyme activity. They included ADP, FDP, F6P, G6P, PEP, 6PG, 2-PGA, R1P, R5P and Ru5P.Abbreviations ATP
adenosine-5-triphosphate
- FDP
fructose-1,6-diphosphate
- F6P
fructose-6-phosphate
- G6P
glucose-6-phosphate
- GPDH
glyceraldehyde-3-phosphate dehydrogenase
- NADH
nicotinamide adenine dinucleotide (reduced)
- OAA
oxalacetate
- pCMB
parachlormercuribenzoate
- PEP
phosphoenolpyruvate
- 6PG
6-phosphogluconate
- 2-PGA
2-phosphoglycerate
- 3-PGA
3-phosphoglycerate
- PGK
3-phosphoglyceric phosphokinase
- R1P
ribose-1-phosphate
- R5P
ribose-5-phosphate
- RuDP
ribulose-1,5-diphosphate
- Ru5P
ribulose-5-phosphate
- SDS
sodium dodecyl sulfate 相似文献
16.
The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO2) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor. In this study, [U-13C]glucose, [U-13C]fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses. In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively. Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive (K
i=180 M) and uncompetitive (Ki=350 M) inhibition by 6-phosphogluconate. Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively (K
i=1.3 M) with a low contribution of uncompetitive inhibition (Ki=13 M). The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 M) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 M). We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate. 相似文献
17.
d-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and d-mannitol dehydrogenase (EC 1.1.1.67) were estimated in a cell-free extract of the unicellular alga Platymonas subcordiformis Hazen (Prasinophyceae), d-Mannitol dehydrogenase had two activity maxima at pH 7.0 and 9.5, and a substrate specifity for d-fructose and NADH or for d-mannitol and NAD+. The K
m values were 43 mM for d-fructose and 10 mM for d-mannitol. d-Mannitol-1-phosphate dehydrogenase had a maximum activity at pH 7.5 and was specific for d-fructose 6-phosphate and NADH. The K
m value for d-fructose 6-phosphate was 5.5 mM. The reverse reaction with d-mannitol 1-phosphate as substrate could not be detected in the extract. After the addition of NaCl (up to 800 mM) to the enzyme assay, the activity of d-mannitol dehydrogenase was strongly inhibited while the activity of d-mannitol-1-phosphate dehydrogenase was enhanced. Under salt stress the K
m values of the d-mannitol dehydrogenase were shifted to higher values. The K
m value for d-fructose 6-phosphate as substrate for d-mannitol-1-phosphate dehydrogenase remained constant. Hence, it is concluded that in Platymonas the d-mannitol pool is derectly regulated via alternative pathways with different activities dependent on the osmotic pressure.Abbreviations Fru6P
d-fructose 6-phosphate
- Mes
2-(N-morpholino)ethanesulfonic acid
- MT-DH
d-mannitol-dehydrogenase
- MT1P-DH
d-mannitol-1-phosphate dehydrogenase
- Pipes
1,4-piperazinediethanesulfonic acid
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
18.
Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC. 1.4.3.5) has been purified from dry baker’s yeast to an apparent homogeneity on a polyacrylamide disc gel electrophoresis in the presence of 10 µm of phenylmethylsulfonyl fluoride throughout purification.1) The purified enzyme, obtained as holo-flavoprotein, has a specific activity of 27µmol/mg/hr for pyridoxamine 5′-phosphate at 37°C, and a ratio of pyridoxine 5′-phosphate oxidase to pyridoxamine 5′-phosphate oxidase is approximately 0.25 at a substrate concentration of 285 µm. Km values for both substrates are 18 µm for pyridoxamine 5′-phosphate and 2.7 µm for pyridoxine 5′-phosphate, respectively.2) The enzyme can easily oxidize pyridoxamine 5′-phosphate, but when pyridoxamine and pyridoxine 5′-phosphate are coexisted in a reaction mixture the enzyme activity is markedly suppressed much beyond the values expected from its high affinity (low Km) and low Vmax for the latter substrate.3) Optimum temperature for both substrates is approximately 45°C, and optimum pH is near 9 for pyridoxamine 5′-phosphate and 8 for pyridoxine 5′-phosphate.4) From the data obtained, the mechanism of regulation of this enzyme in production of pyridoxal 5′-phosphate and a reasonable substrate for the enzyme in vivo are discussed. 相似文献
19.
The distribution of the F420-reactive and F420-nonreactive hydrogenases from the methylotrophic Methanosarcina strain Gö1 indicated a membrane association of the F420-nonreactive enzyme. The membrane-bound F420-nonreactive hydrogenase was purified 42-fold to electrophoretic homogeneity with a yield of 26.7%. The enzyme had a specific activity of 359 mol H2 oxidized · min-1 · mg protein-1. The purification procedure involved dispersion of the membrane fraction with the detergent Chaps followed by anion exchange, hydrophobic and hydroxylapatite chromatography. The aerobically prepared enzyme had to be reactivated anaerobically. Maximal activity was observed at 80°C. The molecular mass as determined by native gel electrophoresis and gel filtration was 77000 and 79000, respectively. SDS gel electrophoresis revealed two polypeptides with molecular masses of 60000 and 40000 indicating a 1:1 stoichiometry. The purified enzyme contained 13.3 mol S2-, 15.1 mol Fe and 0.8 mol Ni/mol enzyme. Flavins were not detected. The amino acid sequence of the N-termini of the subunits showed a higher degree of homology to cubacterial uptake-hydrogenases than to F420-dependent hydrogenases from other methanogenic bacteria. The physiological function of the F420-nonreactive hydrogenase from Methanosarcina strain Gö1 is discussed.Abbreviations
transmembrane electrochemical gradient of H-
- CoM-SH
2-mercaptoethanesulfonate
- F420
(N-l-lactyl--l-glutamyl)-l-glutamic acid phospodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate
- F420H2
reduced F420
- HTP-SH
7-mercaptoheptanoylthreonine phosphate
-
Mb.
Methanobacterium
- PMSF
phenylmethyl-sulfonylfluoride
- Cl3AcOH
trichloroacetic acid 相似文献
20.
Marta E. Farías Ana M. Strasser de Saad Aída A. Pesce de Ruiz Holgado Dr. Guillermo Oliver 《Current microbiology》1991,22(4):205-211
l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM
l-cysteine in 50 mM phosphate buffer. Mr=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (Mr=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent Km forl-serine was 65 mM. Fe++ was required for the enzymatic activity, and the apparent Km value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit. 相似文献