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1.
GLYCEROL KINASE AND DIHYDROXYACETONE KINASE IN RAT BRAIN 总被引:4,自引:0,他引:4
—The enzymatic phosphorylation of glycerol and dihydroxyacetone by ATP to sn-glycerol-3-phosphate and dihydroxyacetone phosphate respectively in various subcellular fractions of rat brain was studied. A sensitive radiochemical assay was used where the labelled phosphorylated products were separated from the radioactive substrates by high voltage paper electrophoresis and the radioactivity in these compounds determined. Using this assay the glycerol kinase (EC 2.7.1.30) activity was found to be associated with the mitochondrial fraction of the brain. Under optimum conditions 2.45 nmol of glycerol was phosphorylated/min per mg of protein. The Km for glycerol was 70 μm at pH 7. This mitochondrial enzyme, like other glycerol kinases from different sources, also phosphorylated dihydroxyacetone. Under optimum conditions 1.7 nmol of dihydroxyacetone phosphate was formed/min per mg of mitochondrial protein. The Km for dihydroxyacetone was 0.6 mm . Glycerol kinase activity was also present in the cytoplasm of brain. However, the specific activity of this enzyme in cytosol is about 15% of the mitochondrial glycerol kinase. Compared to glycerol, dihydroxyacetone was phosphorylated by ATP in cytoplasm at a much higher rate. The pH optimum for this soluble dihydroxyacetone kinase was much lower (pH 6.5) than that of the soluble or mitochondrial glycerol kinase (pH 10.0). Using ammonium sulfate, brain cytoplasm was fractionated to yield a fraction in which the dihydroxyacetone kinase was enriched 2–3 fold with no glycerol kinase activity. Under optimum conditions 1.0 nmol of dihydroxyacetone was phosphorylated/min per mg protein. The Km for dihydroxyacetone was 60 μm . This cytosol fraction was also found to phosphorylate d -glyceraldehyde and l -glyceraldehyde at a rate of 30–40% to that of the dihydroxyacetone phosphorylation. The properties and the possible metabolic role of these enzymes in brain are discussed. 相似文献
2.
O. M. Neijssel S. Hueting K. J. Crabbendam D. W. Tempest 《Archives of microbiology》1975,104(1):83-87
Klebsiella aerogenes NCIB 418 assimilates glycerol via alternative pathways: one involves a glycerol kinase with a high affinity for glycerol (apparent K
m=1–2×10–6 M), and the second a glycerol dehydrogenase with a much lower affinity for its substrate (apparent K
m=2–4×10–2 M).In variously-limited chemostat cultures, one or the other pathway predominated. Thus, aerobic carbonlimited organisms contained only the glycerol kinase pathway whereas aerobic sulphate-limited or ammonia-limited organisms (grown on glycerol) used only the glycerol dehydrogenase pathway. Anaerobic cultures invariably contained glycerol dehydrogenase, and glycerol kinase was absent.Washed suspensions of aerobically-grown organisms oxidized glycerol with kinetics similar to that of the particular enzyme (the primary enzyme of the assimilatory pathway) which they possessed, thus indicating a close association between these two enzymes and the uptake process. But a supply of exogenous glycerol was not a prerequisite for the synthesis of either glycerol kinase or glycerol dehydrogenase, and nor was molecular oxygen the key factor in effecting modulation between the alternative pathways of glycerol metabolism, as had been previously suggested.The physiological significance of dual pathways of glycerol assimilation is discussed. 相似文献
3.
In hansenula polymorpha glycerol is metabolized via glycerol kinase and NAD(P)-independent glycerol-3-phosphate (G3P) dehydrogenase, enzymes which hitherto were reported to be absent in this methylotrophic yeast. Activity of glycerol kinase was readily detectable when cell-free extracts were incubated at pH 7–8 with glycerol/ATP/Mg2+ and a discontinuous assay for G3P formation was used. This glycerol kinase activity could be separated from dihydroxyacetone (DHA) kinase activity by ion exchange chromatography. Glycerol kinase showed relatively low affinities for glycerol (apparent K
m=1.0 mM) and ATP (apparent K
m=0.5 mM) and was not active with other substrates tested. No inhibition by fructose-1,6-bisphosphate (FBP) was observed. Both NAD-dependent and NAD(P)-independent G3P dehydrogenases were present. The latter enzyme could be assayed with PMS/MTT and cosedimented with the mitochondrial fraction. Glucose partly repressed synthesis of glycerol kinase and NAD(P)-independent G3P dehydrogenase, but compared to several other non-repressing carbon sources no clear induction of these enzymes by glycerol was apparent. Amongst glycerolnegative mutants of H. polymorpha strain 17B (a DHA kinase-negative mutant), strains blocked in either glycerol kinase or membrane-bound G3P dehydrogenase were identified. Crosses between representatives of the latter mutants and wild type resulted in the isolation of, amongst others, segregants which had regained DHA kinase but were still blocked in the membrane-bound G3P dehydrogenase. These strains, employing the oxidative pathway, were only able to grow very slowly in glycerol mineral medium.Abbreviations DHA
dihydroxyacetone
- G3P
glycerol-3-phosphate
- EMS
ethyl methanesulphonate
- MTT
3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide
- PMS
phenazine methosulphate
- FBP
fructose-1,6-bisphosphate 相似文献
4.
Petrus Jakobus van Zyl Bernard Alexander Prior Stephanus Gouws Kilian 《Applied microbiology and biotechnology》1991,36(3):369-374
Summary Enzyme analyses indicated that the metabolism of glycerol by Zygosaccharomyces rouxii occurred via either glycerol-3-phosphate (G3P) or dihydroxyacetone (DHA). The route via DHA is significant in osmoregulation. The specific activities of glycerol dehydrogenase (GDHG) and DHA kinase, which metabolize glycerol via DHA, increased nine- and fourfold respectively during osmotic stress [0.960 water activity (aw) adjusted with NaCl] when compared to non-stressed conditions (0.998 aw). Both pathways are under metabolic regulation. Glycerol kinase, mitochondrial G3P dehydrogenase and DHA kinase are induced by glycerol while the latter is also repressed by glucose. Cells treated with cycloheximide prior to osmotic upshock showed significantly lower DHA kinase and GDHG levels and lower intracellular glycerol concentrations when compared to untreated control cells. Thus protein synthesis is essential for osmotic adaptation.
Offprint requests to: B. A. Prior 相似文献
5.
Yabing Duan Changyan Ge Shengming Liu Jianxin Wang Mingguo Zhou 《Molecular Plant Pathology》2013,14(7):708-718
Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high‐osmolarity glycerol mitogen‐activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H2O2‐induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real‐time polymerase chain reaction (qRT‐PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21‐activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild‐type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two‐component HKs in Sclerotinia. 相似文献
6.
The only species of fission yeasts capable of growing on glycerol or dihydroxyacetone were Schizosaccharomyces pombe and S. malidevorans. When growing on glycerol or grown on glucose until it was exhausted, these species contained glycerol:NAD+ 2-oxidoreductase and dihydroxyacetone kinase but no glycerol kinase, consistent with utilization of glycerol via dihydroxyacetone. When grown to exhaustion of glucose, S. octosporus, S. slooffiae and S. japonicus contained dihydroxyacetone kinase but no glycerol:NAD+ 2-oxidoreductase or glycerol kinase. Prior to exhaustion of glucose in the medium, all species contained dihydroxyacetone kinase, all species except S. japonicus contained glycerol:NADP+ 2-oxidoreductase, and only S. pombe and S. malidevorans contained glycerol:NAD+ 2-oxidoreductase. Possible roles for the glycerol:NAD+ 2-oxidoreductase, glycerol:NADP+ 2-oxidoreductase and dihydroxyacetone kinase in metabolism of glycerol and dihydroxyacetone are discussed.Non-standard abbreviations DHA
dihydroxyacetone
- DHAK
dihydroxyacetone kinase
- DHAP
dihydroxyacetone phosphate
- GK
glycerol kinase
- G2DH-NAD
glycerol
- NAD+
2-oxidoreductase
- G2DH-NADP
glycerol
- NADP+
2-oxidoreductase
- MEA
malt extract agar
- YEP
yeast extract phosphate medium 相似文献
7.
Fifteen yeast strains of the genera Candida, Lodderomyces, Endomycopsis, Saccharomyces, Hansenula, Pichia and Torulopsis were investigated with respect to their ability to grow on methanol, glycerol and glucose as sole carbon and energy source. Eight of them can grow on both methanol and glycerol.Methanol is assimilated via triosephosphate (dihydroxyacetone) pathway. The dihydroxyacetone kinase is a key enzyme in methanol metabolism.The assimilation of glycerol can take place in bacteria via a phosphorylative or/and oxidative pathways. In general, the phosphorylative pathway is found in eucaryotes. In the present paper it is shown that in yeasts, which can utilize methanol and glycerol, too, glycerol is assimilated via an oxidative pathway, Dihydroxyacetone is a central intermediate in the assimilation of methanol as well as glycerol. It is metabolized by means of the dihydroxyacetone kinase.The enzyme formed during growth of Candida methylica on methanol does not differ from that of Candida valida H 122 after growing on glycerol as far as the regulatory properties are concerned. 相似文献
8.
Most microorganisms can metabolize glycerol when external electron acceptors are available (i.e. under respiratory conditions). However, few can do so under fermentative conditions owing to the unique redox constraints imposed by the high degree of reduction of glycerol. Here, we utilize in silico analysis combined with in vivo genetic and biochemical approaches to investigate the fermentative metabolism of glycerol in Escherichia coli. We found that E. coli can achieve redox balance at alkaline pH by reducing protons to H2, complementing the previously reported role of 1,2-propanediol synthesis under acidic conditions. In this new redox balancing mode, H2 evolution is coupled to a respiratory glycerol dissimilation pathway composed of glycerol kinase (GK) and glycerol-3-phosphate (G3P) dehydrogenase (G3PDH). GK activates glycerol to G3P, which is further oxidized by G3PDH to generate reduced quinones that drive hydrogenase-dependent H2 evolution. Despite the importance of the GK-G3PDH route under alkaline conditions, we found that the NADH-generating glycerol dissimilation pathway via glycerol dehydrogenase (GldA) and phosphoenolpyruvate (PEP)-dependent dihydroxyacetone kinase (DHAK) was essential under both alkaline and acidic conditions. We assessed system-wide metabolic impacts of the constraints imposed by the PEP dependency of the GldA-DHAK route. This included the identification of enzymes and pathways that were not previously known to be involved in glycerol metabolisms such as PEP carboxykinase, PEP synthetase, multiple fructose-1,6-bisphosphatases and the fructose phosphate bypass. 相似文献
9.
Yuzuru Sanemitsu Tamon Uematsu Satoru Inoue Katsutoshi Tanaka 《Bioscience, biotechnology, and biochemistry》2013,77(7):1927-1929
Enzyme activities involved in the initial step of glycerol metabolism were determined in cells of methylotrophic yeasts grown on glycerol, methanol or glucose. In Candida boidinii (Kloeckera sp.) No. 2201, the activities of glycerol kinase and dihydroxyacetone kinase were detected in cells grown on glycerol and methanol, respectively. The activity of NAD+-linked glycerol dehydrogenase of Hansenula polymorpha dl-1 was induced by glycerol and methanol, while that of Hansenula ofunaensis was induced by glycerol. The enzymes of both strains were subject to catabolite repression by glucose.The yeasts tested were divided into three groups as to the glycerol dissimilation patterns. Strains of the genera Candida, Saccharomyces, Pichia and Torulopsis had the phosphorylative pathway, in which glycerol is first phosphorylated. H. ofunaensis had the oxidative pathway, in which glycerol is first oxidized. H. polymorpha dl-1 had both the phosphorylative and oxidative pathways. 相似文献
10.
During growth on glycerol two marine Desulfovibrio strains that can grow on an unusually broad range of substrates contained high activities of glycerol kinase, NAD(P)-independent glycerol 3-phosphate dehydrogenase and the other enzymes necessary for the conversion of dihydroxyacetone phosphate to pyruvate. Glycerol dehydrogenase and a specific dihydroxyacetone kinase were absent. During growth on dihydroxyacetone, glycerol kinase is involved in the initial conversion of this compound to dihydroxyacetone phosphate which is then further metabolized. Some kinetic properties of the partially purified glycerol kinase were determined. The role of NAD as electron carrier in the energy metabolism during growth of these strains on glycerol and dihydroxyacetone is discussed.Glycerol also supported growth of three out of four classical Desulfovibrio strains tested. D. vulgaris strain Hildenborough grew slowly on glycerol and contained glycerol kinase, glycerol 3-phosphate dehydrogenase and enzymes for the dissimilation of dihydroxyacetone phosphate. In D. gigas which did not grow on glycerol the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase were absent in lactate-grown cells.Abbreviations DHA
dihydroxyacetone
- DHAP
dihydroxyacetone phosphate
- G3P
glycerol 3-phosphate
- GAP
glyceraldehyde 3-phosphate
- 3-PGA
3-phosphoglycerate
- 2-PGA
2-phosphoglycerate
- 2,3-DPGA
2,3-diphosphoglycerate
- PEP
phosphoenolpyruvate
- DH
dehydrogenase
- GK
glycerol kinase
- DHAK
dihydroxyacetone kinase
- TIM
triosephosphate isomerase
- PGK
3-phosphoglycerate kinase
- PK
pyruvate kinase
- LDH
lactate dehydrogenase
- DTT
dithiotreitol
- HEPES
4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid
- PIPES
piperazine-1,1-bis(2-ethane sulfonic acid)
- BV2+/BV+
oxidized/reduced benzylviologen
- PMS
phenazine methosulfate
- DCPIP
2,6-dichlorophenolindophenol
- MTT
3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide 相似文献
11.
Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial
glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (p
Gly
) was 2.3 ± 0.23 × 10−6 cm sec−1 with MIP vs. 0.92 ± 0.086 × 10−6 cm sec−1 in control oocytes. The p
Gly
of MIP was independent of concentration from 5 × 10−5 to 5 × 10−2
m, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mm Hg++, 0.2 mm DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mm Cu++, respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This
could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based
on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase
(0.12 ± 0.03 vs. 0.06 ± 0.01 pmol min−1 cell−1) without changing the binding of glycerol to the kinase (K
M
∼ 10 μm).
Received: 23 May 1997/Revised: 4 August 1997 相似文献
12.
JEAN GRUENBERG BERNARD SCHWENDIMANN PUKH RAJ SHARMA JACQUES DESHUSSES 《The Journal of eukaryotic microbiology》1980,27(4):484-491
SYNOPSIS. Under aerobic conditions, we have determined glycerol uptake in the long slender (LS) bloodstream form of Trypanosoma (Trypanozoon) brucei brucei by studying glycerophosphate accumulation in the parasites. The coupled enzyme theory applies to the permeation-phosphorylation sequence. Glycerol passage through the plasma membrane is asymmetric, the efflux process being favored over the influx process. No free diffusion of glycerol can be detected even under conditions under which free glycerol accumulates within the cells; most probably, glycerol permeation is mediated by a specific transport system. In the absence of respiratory activities, glycerol is known to be an end-product of T. brucei glycolysis; its production from glycerophosphate should allow ATP synthesis. The observed efflux of free glycerol following intracellular accumulation of glycerophosphate confirms the hypothesis that glycerol production occurs through reversal of glycerol kinase activity. We conclude that in vivo the role of the carrier-mediated asymmetric permeation process is to prevent inhibition of the reversal of the glycerol kinase-mediated reaction by removing free glycerol. 相似文献
13.
Role of the Sln1‐phosphorelay pathway in the response to hyperosmotic stress in the yeast Kluyveromyces lactis
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Miriam Rodríguez‐González Laura Kawasaki Nancy Velázquez‐Zavala Eunice Domínguez‐Martín Abraham Trejo‐Medecigo Natalia Martagón Emilio Espinoza‐Simón Araceli Vázquez‐Ibarra Laura Ongay‐Larios Dimitris Georgellis Eulàlia de Nadal Francesc Posas Roberto Coria 《Molecular microbiology》2017,104(5):822-836
The Kluyveromyces lactis SLN1 phosphorelay system includes the osmosensor histidine kinase Sln1, the phosphotransfer protein Ypd1 and the response regulator Ssk1. Here we show that K. lactis has a functional phosphorelay system. In vitro assays, using a heterologous histidine kinase, show that the phosphate group is accepted by KlYpd1 and transferred to KlSsk1. Upon hyperosmotic stress the phosphorelay is inactivated, KlYpd1 is dephosphorylated in a KlSln1 dependent manner, and only the version of KlSsk1 that lacks the phosphate group interacts with the MAPKKK KlSsk2. Interestingly, inactivation of the KlPtp2 phosphatase in a ΔKlsln1 mutant did not lead to KlHog1 constitutive phosphorylation. KlHog1 can replace ScHog1p and activate the hyperosmotic response in Saccharomyces cerevisiae, and when ScSln1 is inactivated, KlHog1 becomes phosphorylated and induces cell lethality. All these observations indicate that the phosphorelay negatively regulates KlHog1. Nevertheless, in the absence of KlSln1 or KlYpd1, no constitutive phosphorylation is detected and cells are viable, suggesting that a strong negative feedback that is independent of KlPtp2 operates in K. lactis. Compared with S. cerevisiae, K. lactis has only a moderate accumulation of glycerol and fails to produce trehalose under hyperosmotic stress, indicating that regulation of osmolyte production is different in K. lactis. 相似文献
14.
Some properties of hepatic glycerol kinase and their relation to the control of glycerol utilization 总被引:10,自引:8,他引:2
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1. Glycerol kinase (EC 2.7.1.30) is shown to catalyse a non-equilibrium reaction in rat liver; and, as it is the first enzyme in the pathway metabolizing glycerol, its properties may be pertinent to the metabolic regulation of glycerol uptake and utilization by this tissue. 2. The properties of hepatic glycerol kinase were studied by using a radiochemical technique to measure the enzyme activity. When the concentration of ATP is low the activity of glycerol kinase is inhibited by high concentrations of glycerol; but when the concentration of ATP is high there is no inhibition and the double-reciprocal plot is linear, providing a K(m) for glycerol of 3.16x10(-6)m. Glycerol kinase is activated by high ATP concentrations provided that the concentration of the second substrate (glycerol) is high; at low concentrations of glycerol ATP does not activate the enzyme so that the double-reciprocal plot is linear, providing a K(m) for ATP of 5.8x10(-5)m. It is suggested that these kinetics may be explained by a model similar to that described by Ferdinand (1966) for phosphofructokinase. 3. Hepatic glycerol kinase is inhibited by ADP and AMP, and raising the Mg(2+) concentration increases the inhibition by these two compounds; this suggests that ADP-Mg(2+) and AMP-Mg(2+) complexes are the inhibitory species. The physiological significance of these inhibitions may be to prevent phosphorylation of glycerol when the hepatic ATP concentration is low. It is suggested that this inhibition may provide an approach to the problem of measurement of rates of lipolysis by glycerol release in tissues that contain glycerol kinase (e.g. liver, kidney, muscle, adipose tissue). 4. Hepatic glycerol kinase is inhibited by l-3-glycerophosphate competitively with respect to glycerol. The physiological significance of this inhibition may be that factors that change the intracellular concentration of l-3-glycerophosphate could change glycerol uptake by the tissue. Thus it is suggested that thyroxine treatment or feeding rats on a diet high in glycerol, which increase the activity of glycerophosphate oxidase in liver and kidney cortex respectively, lead to an increased glycerol uptake through a decrease in the concentration of glycerophosphate in these tissues. It is known that ethanol administration decreases glycerol uptake by liver, and this can be explained by the increased concentration of l-3-glycerophosphate causing inhibition of glycerol kinase. 相似文献
15.
C. van der Drift R. A. M. M. Smits G. A. M. Michiels H. J. M. Op den Camp 《Archives of microbiology》1986,146(3):292-294
Bacillus fastidiosus was able to grow on glycerol as a carbon source when allantoin or urate was used as nitrogen source. The primary assimilatory enzyme for glycerol was glycerol kinase; glycerol dehydrogenase could not be detected. The glycerol kinase activity was increased 30-fold in allantoin/glycerol-grown cells as compared to alantoin-grown cells. Under both growth conditions high levels of glutamate dehydrogenase were found. Glutamine synthetase and glutamate synthase activities could not be demonstrated, while low levels of alanine dehydrogenase were present. It is concluded that B. fastidiosus assimilates ammonia by the NADP-dependent glutamate dehydrogenase.Abbreviations GS
glutamine synthetase
- GOGAT
glutamate synthase
- GDH
glutamate dehydrogenase
- ADH
alanine dehydrogenase 相似文献
16.
W. de Koning R. A. Weusthuis W. Harder L. Dijkhuizen 《Applied microbiology and biotechnology》1990,32(6):693-698
Summary Various factors controlling dihydroxyacetone (DHA) and glycerol production from methanol by resting cell suspensions of a mutant of Hansenula polymorpha, blocked in DHA kinase and glycerol kinase, were investigated. The presence of methanol (250mM) and an additional substrate (0.5%, w/v) to replenish the xylulose-5-phosphate required for the assimilation reaction (DHA synthase) was essential for significant triose production by this double mutant. A number of sugars were tested as additional substrates and C5 sugars gave the highest triose accumulation (ca. 20mM after 45h). Glucose was the poorest additional substrate and triose production only started after its exhaustion, which occurred in the first few hours. Other sugars were metabolized at a much lower rate and accumulation of trioses began right at the start of the experiments and gradually increased with time. The production rate of total trioses increased, and the relative amount of glycerol diminished with higher oxygen supply rates. The data suggest that conversion of DHA into glycerol, catalysed by reduced nicotine adenine dinucleotide (NADH)-dependent DHA reductase, is partly regulated via intracellular NADH levels. Further support for this hypothesis was obtained in experiments with antimycin A, an inhibitor of the electron transport chain. Addition of higher amounts of methanol and xylose, either by increasing the initial concentrations or by repeated addition of these substrates, resulted in considerably enhanced productivity and a switch towards glycerol formation. After reaching a level of approximately 25mM the DHA concentration remained constant while the glycerol level gradually increased with time. After an incubation period of 350 h, a total of 3.9 M methanol and 0.62 M xylose had been converted, which resulted in accumulation of 0.76 M trioses, mostly glycerol.Offprint requests to: L. Dijkhuizen 相似文献
17.
18.
Summary Glycerol kinase activity found in the epididymal adipose tissue of lean litter-mates of hyperglycemic obese mice exhibits two distinct Km values. The Km(s) obtained graphically by a Hoftsee plot are 40 and 637 m. The glycerol kinase of obese mice showed 29 times more total activity per fat pad or 9 times more activity per mg of protein as compared to that of the lean controls. The increased glycerol kinase activity found in the obese mice predominantly exhibited low Km value. Increase in the activity with the high Km value was minimal. The apparent molecular weight of adipose glycerol kinase is approximately 60,000–65,000. A higher activity of glycerol kinase in the epididymal adipose tissue of the obese mice (ob/ob) has been reported by Treble and Mayer
1. The significance of this increased enzyme in obesity2 has been investigated by many investigators3–6, using either whole epididymal fat pad or isolated fat cells as the source of glycerol kinase. However, little is known regarding the properties of this enzyme, and even the characterization of the liver glycerol kinase is not yet complete7, 8.We have recently shown that adipose glycerol kinase of Sprague-Dawly rat and of Swiss mice exhibit two Km values, and the apparent molecular weight is approximately 55,000–60,0009. The present study was undertaken in order to establish whether the enzyme in ob/+ mice also exhibits two Km values and if so, whether the activity associated with both Km(s) is increased. We found that the specific conditions for enzyme activity determination and the basis on which the activity is expressed are quantitatively important in clarifying the difference in glycerol kinase activity of ob/ob and ob/+ mice. The increased activity in ob/ob mice is predominantly the low Km enzyme, and the apparent molecular weight is approximately 60,000–65,000.Supported in part by the Juvenile Diabetes Foundation. We thank Mr. CHARLES ROSENTHAL for technical assistance. 相似文献
19.
The BRAF mutation confers sensitivity of thyroid cancer cells to the BRAF inhibitor PLX4032 (RG7204)
Joanna Xing Ruixin Liu Mingzhao Xing Barry Trink 《Biochemical and biophysical research communications》2011,(4):617
Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAFV600E, as a result of the BRAFT1799A mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAFV600E-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAFT1799A mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAFT1799A mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC50 values (0.115–1.156 μM) in BRAFV600E mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC50 values (56.674–1349.788 μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAFT1799A mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAFT1799A mutation-selective therapeutic agent for thyroid cancer. 相似文献
20.
The Saccharomyces cerevisiae DIS2S1/GLC7 gene encodes a type 1 protein phosphatase indispensable for cell proliferation. We found that introduction of a multicopy DIS2S1 plasmid impaired growth of cells with reduced activity of the cAMP-dependent protein kinase. In order to understand further the interaction between the two enzymes, a temperature-sensitive mutation in the DIS2S1 gene was isolated. The mutant accumulated less glycogen than wild type at the permissive temperature, indicating that activity of the Dis2s1 protein phosphatase is attenuated by the mutation. Furthermore, the dis2s1
ts mutation was shown to be suppressed by a multicopy plasmid harboring PDE2, a gene for cAMP phosphodiesterase. These results indicate that the Ras-cAMP pathway interacts genetically with the DIS2S1/GLC7 gene. 相似文献