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1.
Anthony Haystead 《Planta》1973,111(3):271-274
Summary A glutamine synthetase has been localised in the chloroplasts of Vicia faba. The enzyme has requirements for Mg2+ and ATP in the biosynthetic reaction and in addition will catalyse a -glutamyl transferase reaction in the presence of Mn2+ and arsenate. The enzyme is inhibited by AMP, CTP, glycine and alanine. These results are discussed in relation to the possible chloroplastic synthesis of nucleotide bases. Estimations of glutamine amide-2-oxoglutarate amino transferase (oxido-reductase) have demonstrated only low levels of activity in the chloroplast extracts. This enzyme is generally active in organisms where GS has an assimilary role. It is coneluded that glutamine synthetase has a biosynthetic and not an assimilatory role in the chloroplast. 相似文献
2.
Tadashi Araki 《Biochimica et Biophysica Acta (BBA)/General Subjects》1977,496(2):532-546
The inactivation of 2-oxoglutarate dehydrogenase complex by freeze-thawing was examined along with alterations of membrane phospholipids, in order to elucidate the mechanism of freezing injury in mitochondria.The dehydrogenase complex activity in slowly frozen and thawed mitochondria decreased to 70% as compared to intact mitochondria and further decreased during incubation. This inactivation during incubation was temperature dependent, i.e., at temperatures up to 25°C there was a slight decrease, while at higher temperatures there was a marked decrease in the dehydrogenase complex activity. Simultaneously, there was a significant accumulation of free fatty acids, generated from mitochondrial phospholipids, which inhibited 2-oxoglutarate dehydrogenase and subsequently enzyme complex activity. Oxoglutarate dehydrogenase activity in mitochondria was markedly inhibited by exogenous phospholipase A, and this inhibition was partially prevented with bovine serum albumin. Furthermore, when intrinsic phospholipase A was either inhibited or stimulated, there was a respective decrease or increase in the enzyme complex inactivation.The activity of the purified enzyme complex decreased slightly after slow freezing, but remained constant even when incubated at temperatures up to 32°C. However, the activity of this enzyme complex was markedly reduced when incubated either in the presence of venom phospholipase A or with exogenous fatty acid.The relationship between inactivation of the 2-oxoglutarate dehydrogenase complex, phospholipase A activation and production of free fatty acids in frozen and thawed mitochondria is discussed. 相似文献
3.
Using analytical subcellular fractionation techniques, 12% of the total L-alanine aminotransferase activity and 26% of the total L-aspartate aminotransferase activity was localized in enterocyte mitochondria. Alanine and aspartate were products from the oxidation of glutamine and glutamate by enterocyte mitochondria. At low concentrations, malate stimulated aspartate synthesis but was inhibitory at higher concentrations. The malate inhibition of aspartate synthesis, which increased in the presence of pyruvate, was accompanied by an increase in alanine synthesis. With glutamine as substrate in the presence of pyruvate and malate, alanine synthesis was increased by 127% on addition of purified L-alanine aminotransferase, in spite of large amounts of glutamate generated. It was concluded that when pyruvate is available the important route for glutamine or glutamate oxidation by transamination was via L-alanine:2-oxoglutarate aminotransferase and not via L-aspartate:2-oxoglutarate aminotransferase. Results suggested that mitochondria may account for 50% of alanine production from glutamine in the enterocyte despite the relatively low activity of L-alanine aminotransferase therein. 相似文献
4.
Sławomir Orzechowski Joanna Socha-Hanc Andrzej Paszkowski 《Acta Physiologiae Plantarum》1999,21(4):323-330
Alanine aminotransferase (AlaAT, EC 2.6.1.2) from leaves of 14-day-old maize seedlings was purified over 1600-fold to electrophoretical
homogeneity. Specific activity of the purified enzyme measured with L-alanine and 2-oxoglutarate as substrates was 2125 nkat·(mg
protein)−1 at 30 °C. The molecular weights of the native and sodium dodecyl sulfate — denatured AlaAT protein were 95 kDa and 50 kDa
respectively, indicating that the native enzyme is probably a homodimer. AlaAT almost exclusively catalyzed amino group transfer
from L-alanine to 2-oxoglutarate and the reverse reaction. The inhibitory experiments showed that pirydoxal phosphate is directly
involved in the enzymatic catalysis and the enzyme molecule contains essential SH groups. The use of phenylglyoxal demonstrated
the presence of arginine residue as anionic binding site in the active centre of AlaAT.
This work was supported by the State Committee for Scientific Research, a grant No. 5PO6A00510 相似文献
5.
Jaroslav Dršata Martin Beránek VladimÍr Palička 《Journal of enzyme inhibition and medicinal chemistry》2013,28(1):31-36
Incubation of 50 mM d -glucose with aspartate aminotransferase (AST, EC 2.6.1.1) preparations (purified pig heart enzyme or a rat liver 20,000 × g supernatant) at 25°C had no effect on enzyme activity. 50 mM d -fructose or d -ribose gradually inhibited pig heart AST under the same conditions to zero activity after 14 days. 50 mM dl -glyceraldehyde decreased enzyme activity to zero after 6 days of incubation. The inhibition of pig heart AST by 50 mM d -fructose or d -ribose was marked even at a temperature of 4°C but it was less pronounced than at 25°C. There was no effect of 0.5 mM 2-oxoglutarate on AST activity during incubation, while the presence of 25 mM l -aspartate decreased it rapidly. 0.5 mM 2-oxoglutarate partly prevented inhibition of AST by d -ribose or d -fructose, while an analogous experiment with 25 mM aspartate resulted in a rapid decline similar to that in the absence of sugars. 相似文献
6.
《Biochimica et Biophysica Acta (BBA) - Enzymology》1978,522(1):96-103
The reversible heat activation and cold inactivation of tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) of chick liver were investigated. When the enzyme obtained by gel filtration was preincubated at 37°C for 10 min with 50 μM pyridoxal 5′-phosphate (pryidoxal-5′-P), a 7-fold increase in enzyme activitiy was detected. When the preincubated enzyme was cooled to 0°C, it lost its activity. Furthermore, the dramatic cyclical changes in enzyme activity occurred by sequential heating at 37°C and cooling to 0°C of the enzyme, in the presence of pyridoxal-5′-P, over shorter periods of time without loss of enzyme activity. However, when α-ketoglutarate was added to the enzyme during cold exposure, no further decrease in activity was observed. This protective effect was seen at a concentration of 5 μM. 相似文献
7.
Sławomir Orzechowski Joanna Socha-Hanc Andrzej Paszkowski 《Acta Physiologiae Plantarum》1999,21(4):331-334
The intracellular distribution of alanine aminotransferase (AlaAT, EC 2.6.1.2) activity with L-alanine and 2-oxoglutarate
as a substrates in maize whole leaf extract and bundle sheath cells was studied. After isolation of the mitochondrial-peroxisomal
fraction, mitochondria and peroxisomes were separated by centrifugation on a linear 40–52 % (w/w) sucrose gradient. L-Alanine-2-oxoglutarate
transaminating activity of whole leaf extract showed two peaks: first distinctly higher associated with mitochondria and second
lower with peroxisomes. In bundle sheath cells only one peak of this activity was found. It corresponded to the mitochondrial
region of the gradient. It is proposed that mitochondrial L-alanine — 2-oxoglutarate activity was brought about by AlaAT.
Glycine aminotransferase (EC 2.6.1.4) could be responsible for the same activity in peroxisomes.
This work was supported by the State Committee for Scientific Research, a grant No. 5PO6A00510 相似文献
8.
The nature and control of the tricarboxylate cycle in beetle flight muscle. 总被引:1,自引:1,他引:0
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The only exogenous substrates oxidized by mitochondria isolated from the flight muscle of the Japanese beetle (Popillia japonica) are proline, pyruvate and glycerol 3-phosphate. The highest rate of oxygen consumption is obtained with proline. The oxidation of proline leads to the production of more NH3 than alanine, indicating a functioning glutamate dehydrogenase (EC 1.4.1.2). Studies of mitochondrial extracts confirm the presence of a very active glutamate dehydrogenase, and this enzyme is found to be activated by ADP and inhibited by ATP. These extracts also show high alanine aminotransferase activity (EC 2.6.1.2) and a uniquely active "malic'' enzyme (EC 1.1.1.39). The "malic'' enzyme is activated by succinate and inhibited by ATP and by pyruvate. It is suggested that the input of tricarboxylate-cycle intermediate from proline oxidation is balanced by the formation of pyruvate from malate, and the complete oxidation of the majority of the pyruvate. Studies of the steady-state concentrations of mitochondrial CoASH and CoA thioesters during proline oxidation show a high succinyl (3-carboxypropionyl)-CoA content which falls on activating respiration with ADP. There is a concomitant rise in CoASH. However, the reverse transition, from state-3 to state-4 respiration, causes only very slight changes in acylation. The reasons for this are discussed. Studies of the mitochondrial content of glutamate, 2-oxoglutarate, malate, pyruvate, citrate and isocitrate during the same phases of proline oxidation give results consistent with control at the level of glutamate dehydrogenase and isocitrate dehydrogenase during proline oxidation, with the possibility of further control at "malic'' enzyme. During the oxidation of pyruvate all of the tricarboxylate-cycle intermediates and NAD(P)H follow the pattern of changes described in the blowfly (Johnson & Hansford, 1975; Hansford, 1974) and isocitrate dehydrogenase is identified as the primary site of control.?2OAuthor 相似文献
9.
1. Extracts of Pseudomonas sp. grown on butane-2,3-diol oxidized glyoxylate to carbon dioxide, some of the glyoxylate being reduced to glycollate in the process. The oxidation of malate and isocitrate, but not the oxidation of pyruvate, can be coupled to the reduction of glyoxylate to glycollate by the extracts. 2. Extracts of cells grown on butane-2,3-diol decarboxylated oxaloacetate to pyruvate, which was then converted aerobically or anaerobically into lactate, acetyl-coenzyme A and carbon dioxide. The extracts could also convert pyruvate into alanine. However, pyruvate is not an intermediate in the metabolism of glyoxylate since no lactate or alanine could be detected in the reaction products and no labelled pyruvate could be obtained when extracts were incubated with [1-14C]glyoxylate. 3. The 14C was incorporated from [1-14C]glyoxylate by cell-free extracts into carbon dioxide, glycollate, glycine, glutamate and, in trace amounts, into malate, isocitrate and α-oxoglutarate. The 14C was initially incorporated into isocitrate at the same rate as into glycine. 4. The rate of glyoxylate utilization was increased by the addition of succinate, α-oxoglutarate or citrate, and in each case α-oxoglutarate became labelled. 5. The results are consistent with the suggestion that the carbon dioxide arises by the oxidation of glyoxylate via reactions catalysed respectively by isocitratase, isocitrate dehydrogenase and α-oxoglutarate dehydrogenase. 相似文献
10.
High resolution gel chromatography of proteins 总被引:1,自引:0,他引:1
An assay for the determination of l-glutaminase in extracts and intact cells is described. The method is based on the stereospecific release of 3H2O from l-[2-3H]glutamine when l-glutaminase is coupled to l-aspartate:2-oxoglutarate amino transferase. The substrate, glutamine, and intermediate product, glutamate, are separated from the final reaction product, tritiated water, on a mixed-bed ion-exchange column which retains amino acids and organic acids but not water. The method has been adapted to determine the activity of l-glutaminase in cultured human diploid fibroblasts. 相似文献
11.
Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed. 相似文献
12.
Alanine aminotransferase (AlaAT, EC 2.6.1.2) and glycine aminotransferase (GlyAT, EC 2.6.1.4), two different enzymes catalyzing transamination reactions with L-alanine as the amino-acid substrate, were examined in maize in which alanine participates substantially in nitrogen transport. Preparative PAGE of a partially purified preparation of aminotransferases from maize leaves gave 6 fractions differing in electrophoretic mobility. The fastest migrating fraction I represents AlaAT specific for L-alanine as amino donor and 2-oxoglutarate as amino acceptor. The remaining fractions showed three aminotransferase activities: L-alanine-2-oxoglutarate, L-alanine-glyoxylate and L-glutamate-glyoxylate. By means of molecular sieving on Zorbax SE-250 two groups of enzymes were distinguished in the PAGE fractions: of about 100 kDa and 50 kDa. Molecular mass of 104 kDa was ascribed to AlaAT in fraction I, while the molecular mass of the three enzymatic activities in 3 fractions of the low electrophoretic mobility was about 50 kDa. The response of these fractions to: aminooxyacetate, 3-chloro-L-alanine and competing amino acids prompted us to suggest that five out of the six preparative PAGE fractions represented GlyAT isoforms, differing from each other by the L-glutamate-glyoxylate:L-alanine-glyoxylate:L-alanine-2-oxoglutarate activity ratio. 相似文献
13.
Preincubation of nitrate reductase (NR) extracted from wheat shoot tips with NADH in vitro, activated and stabilized activity at both O° and 25°. However, preincubation with potassium ferricyanide inactivated the NR in vitro. NADH also stabilized the NR activity in extracts from maize shoot tips. It was observed that NR from both wheat and maize was active at low temperatures. 相似文献
14.
Summary The presence of carnitine acetyl transferase (E.C.2.3.1.7) activity has been found for the first time in human platelets.
The enzymic activity was measured by a radiometric method based on the separation of labelled acetylcarnitine and carnitine
on a cation exchange column. Carnitine acetyl transferase activity closely paralleled the activity distribution of the mitochondrial
marker carnitine palmitoyl-transferase. Contrary to the marker enzyme, human platelet carnitine acetyl-transferase is rather
thermosensitive: 60% of its activity is lost after 10 min when kept at 37°C. 相似文献
15.
Mercury (0.01-1.0 mM) inhibited chlorophyll formation in greening maize leaf segments. However, supplementing incubation medium with 2-oxoglutarate, maintained substantially higher level of chlorophyll in absence of metal after an initial period of 8 hr. On preincubation of leaf segments with HgCl2, per cent inhibition of chlorophyll synthesis by metal was same in the presence and absence of 2-oxoglutarate. Supply of 2-oxoglutarate (0.1-10.0 mM) exerted concentration dependent effect on chlorophyll formation in absence or presence of metal. Increase in delta-amino levulinic acid dehydratase as well as NADH-glutamate synthase activity and decrease in NADH-glutamate dehydrogenase activity by 2-oxoglutarate in the presence of Hg suggested that glutamate for delta-amino levulinic acid synthesis could be made available from NH4+ assimilation via., glutamine synthetase/glutamate synthase pathway during mercury toxicity. 相似文献
16.
In kidney cortex tubules isolated from fed rabbits L-alanine is not utilized as glucose precursor, when added as a sole substrate. However, this amino acid decreases gluconeogenesis from low (up to 1 mM) 2-oxoglutarate concentrations and stimulates this process at higher (2.5-10 mM) ketoacid contents in the suspension medium. Aminooxyacetate, an inhibitor of aminotransferases, abolishes both inhibitory and stimulatory effects of L-alanine on glucose formation. The addition of 2-oxoglutarate increases the incorporation of L-[U-14C]alanine to glucose from 8- to 123-fold, depending upon the ketoacid and alanine concentrations used. In contrast, nonlabelled L-alanine decreases the incorporation of low [U-14C)2-oxoglutarate concentrations into glucose, while it does not affect contribution of 5 mM ketoacid to gluconeogenesis. The data indicate that (i) in the presence of 2-oxoglutarate L-alanine is utilized as glucose precursor in rabbit renal tubules and (ii) this amino acid may decrease the contribution of low extracellular concentrations of the ketoacid to gluconeogenesis. 相似文献
17.
Rhodopseudomonas acidophila strain 7050 can satisfy all its nitrogen and carbon requirements from l-alanine. Addition of 100 M methionine sulfoximine to alanine grown cultures had no effect on growth rate indicating that deamination of alanine via alanine dehydrogenase and re-assimilation of the released NH
4
+
by glutamine synthetase/glutamate synthase was an insignificant route of nitrogen transfer in this bacterium. Determination of aminotransferase activities in cell-free extracts failed to demonstrate the presence of direct routes from alanine to either aspartate or glutamate. The only active aminotransferase involving l-alanine was the alanine-glyoxylate enzyme (114–167 nmol·min–1·mg–1 protein) which produced glycine as end-product. The amino group of glycine was further transaminated to yield aspartate via a glycineoxaloacetate aminotransferase (117–136 nmol·min–1 ·mg–1 protein). No activity was observed when 2-oxoglutarate was substituted for oxaloacetate. The formation of glutamate from aspartate was catalysed by aspartate-2-oxoglutarate aminotransferase (85–107 nmol·min–1·mg–1 protein). Determinations of free intracellular amino acid pools in alanine and alanine+100 M methionine sulfoximine grown cells showed the predominance of glutamate, glycine and aspartate, providing further evidence that in alanine grown cultures R. acidophila satisfies its nitrogen requirements for balanced growth by transamination.Abbreviations ADH
alanine dehydrogenase
- GDH
glutamate dehydrogenase
- GS
glutamine synthetase
- GOGAT
glutamate synthase
- MSO
methionine sulfoximine
- GOT
glutamate-oxaloacetate aminotransferase
- GPT
glutamate-pyruvate amino-transferase
- AGAT
alanine-glyoxylate aminotransferase
- GOAT
glycine-oxaloacetate aminotransferase
- GOTAT
glycine-2-oxoglutarate aminotransferase
- AOAT
alanine-oxaloacetate aminotransferase 相似文献
18.
Homocitrate synthase from Penicillium chrysogenum. Localization, purification of the cytosolic isoenzyme, and sensitivity to lysine. 总被引:2,自引:0,他引:2
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Subcellular fractionation of cell-free extracts obtained by nitrogen cavitation showed that Penicillium chrysogenum Q176 contains a cytosolic as well as a mitochondrial homocitrate synthase activity. The cytosolic isoenzyme was purified about 500-fold, and its kinetic and molecular properties were investigated. Native homocitrate synthase shows a molecular mass of 155 +/- 10 kDa as determined by gel filtration and a pH of 4.9 +/- 0.1 as determined by chromatofocusing. The kinetic behaviour towards 2-oxoglutarate is hyperbolic, with Km = 2.2 mM; with respect to acetyl-CoA the enzyme shows sigmoidal saturation kinetics, with [S]0.5 = 41 microM and h = 2.6. The enzyme was inhibited strongly by L-lysine (Ki = 8 +/- 2 microM; 50% inhibition by 53 microM at 6 mM-2-oxoglutarate), competitively with 2-oxoglutarate, in protamine sulphate-treated and desalted cell-free extracts and in partially purified preparations. The extent of this inhibition was strongly pH-dependent. Both isoenzymes are equally susceptible to inhibition by lysine. The same inhibition pattern is shown by the enzyme from strain D6/1014A, which is a better producer of penicillin than strain Q176. 相似文献
19.
Extracts from cultured plant cells of spinach, maize and sycamore and from Lemna plants contain detectable glutathione peroxidase activity, using either hydrogen peroxide or t-butyl hydroperoxide as substrates. Using extracts from cultured maize cells, two peaks of glutathione peroxidase activity could be resolved by a combination of gel filtration and ion exchange chromatography. One peak was eluted along with glutathione transferase activity; the second was distinct from both glutathione transferase and ascorbic acid peroxidase, and was active with both hydrogen peroxide and organic hydroperoxides. It seems likely that at least two enzymes with glutathione peroxidase activity exist in higher plant cells. 相似文献
20.
Peroxidase activity was demonstrated in extracts from hazel cotyledons and the cytoplasmic enzyme was partially characterized. Levels of cytoplasmic and org@tnellar peroxidase were monitored in seeds stratified at 5” and incubated under moist conditions at 20°. Specific activities of the enzymes in both subcellular localizations remain constant throughout nine weeks stratification but show a steady rise in activity from seeds maintained at 20°. 相似文献