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1.
Chloroplast envelope membranes isolated from Pisum sativum seedlings have been found to contain a Mg-ATPase activity (specific activity 50-175 nanomoles per minute per milligram protein). The ATPase had a broad pH optimum between 7.0 and 9.5. The activity was not inhibited by oligomycin, N,N′-dicyclohexylcarbodiimide, ouabain, or antibodies directed against chloroplast coupling factor 1; nor was the activity stimulated by monovalent cations. However, the ATPase was inhibited by vanadate, molybdate, and adenylyl imidodiphosphate. The ATPase hydrolyzed a broad range of nucleoside triphosphates, but did not hydrolyze ADP, AMP, or pyrophosphate. The Km for Mg-ATP was determined to be 0.2 millimolar. The ATPase was found to be distinct from ADPase and pyrophosphatase activities also present in pea envelope membranes. The ATPase was determined to be located on the inner membrane of the envelope after resolution of inner and outer membranes by sucrose density gradient centrifugation. 相似文献
2.
Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore ( Acer pseudoplatanus L.) have been found to contain a Mg 2+-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg 2+, Mn 2+ and Ca 2+, whereas it was not affected by Sr 2+, K +, or Na +. The Km for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N′-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg 2+-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F 1 ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692). 相似文献
3.
Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells ( Lycopersicon esculentum Mill cv VF36). Suspension cultures were a source of large quantities of membranes from rapidly growing, undifferentiated cells. Proton transport activity was assayed as quench of acridine orange fluorescence. The activity of the proton translocating ATPase and of several other membrane enzymes was measured as a function of the cell culture cycle. The relative distribution of the enzymes between the 3,000, 10,000, and 100,000 g pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per gram fresh weight with time had a unique profile for each enzyme tested. Maximal yield of the proton translocating ATPase activity was obtained from cells in the middle logarithmic phase of growth, and from 50 to 90% of the activity was found in the 10,000 g pellet. The proton translocating ATPase activity was separable from NADPH cytochrome c reductase and cytochrome c oxidase on a sucrose gradient. Proton transport activity had a broad pH optimum (7.0-8.0), was stimulated by KCl with a Km of 5 to 10 millimolar, stimulation being due to the anion, Cl −, and not the cation, K +, and was not inhibited by vanadate, but was inhibited by NO 3−. The activity is tentatively identified as the tonoplast ATPase. 相似文献
4.
The tonoplast ATPase from corn coleoptile membranes was solubilized using a two-step procedure consisting of a pretreatment with 0.15% (w/v) deoxycholate to remove 60% of the protein, and 40 millimolar octyl-glucoside to solubilize the ATPase. During ultracentrifugation, the solublized ATPase entered a linear sucrose gradient faster than the majority of the protein, resulting in an 11-fold purification over the initial specific activity. The partially purified ATPase was almost completely inhibited by KNO 3 with an estimated Ki of 10 millimolar. The specific activity of the KNO 3-sensitive ATPase was increased 29-fold during purification. N,N′-Dicyclohexylcarbodiimide also completely inhibited the ATPase with half-maximal effects at a concentration of 4 micromolar. Neither vanadate nor azide inhibited enzyme activity. The purified ATPase was stimulated by Cl − and preferred Mg-ATP as substrate. Analysis of frations from the sucrose gradient by sodium dodecyl sulfate-polyacrylamide gel electrophoresis led to the identification of two major polypeptides at 72,000 and 62,000 daltons which were best correlated with ATPase activity. Several minor bands also appeared to copurify with enzyme activity, but were less consistent. Radiation inactivation experiments with intact membranes indicated that the functional molecular size of the tonoplast ATPase was nearly 400,000 daltons. This suggests that the ATPase is composed of several polypeptides, possibly including the 72,000- and 62,000-dalton proteins. 相似文献
5.
The effects of NO 3− and assay temperature on proton translocating ATPases in membranes of barley ( Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H +-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO 3 when assayed at 24°C or above and was tentatively identified as the tonoplast H +-ATPase. A smaller peak of H +-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO 3 and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H +-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO 3 inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO 3. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO 3 caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO 3 was explained by the temperature-dependent delay of the inhibition by KNO 3. The plasma membrane H +-ATPase maintained a pH gradient in the presence of KNO 3 for up to 30 minutes at 24°C. 相似文献
6.
The membrane of anthocyanin containing Hippeatrum petal vacuoles was examined for protein and enzyme content after purification by equilibrium density centrifugation. Light scattering, protein, and a Mg 2+-dependent nucleotide specific ATPase were associated with membrane having a density of 1.08 to 1.12 grams per cubic centimeter. A small amount of acid phosphatase was also present in this region of the gradient, but this activity peaked at about 1.12 grams per cubic centimeter. A component of yeast tonoplast, α-mannosidase, was not significantly present. UDP-glucose, anthocyanidin-3- O-glucosyltransferase, thought to be a cytosol enzyme in Hippeastrum, was absent from tonoplast of vacuoles isolated by osmotic shock in 0.2 molar K 2HPO 4 or 0.35 molar mannitol. Vacuolar acid phosphatase was insensitive to ethylenediaminetetraacetate but was 80% inhibited by 10 millimolar KF, while ATPase was inactivated by 2 millimolar ethylenediaminetetraacetate and only 50% inhibited by 10 millimolar KF. Five major and about 9 minor polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein on 5 to 30 and 6 to 16% gradient gels. 相似文献
7.
An anion-sensitive H +-translocating ATPase was identified in membrane vesicles isolated from mature green tomato ( Lycopersicon esculentum) fruit. The H +-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by NO 3−, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H +-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The Km for the H +-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H +-ATPase are very similar to those described for a number of plant cell tonoplast H +-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation. 相似文献
8.
The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn ( Zea mays L. WF9 × MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat ( Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase. Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K+. K+-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K+ transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate. 相似文献
9.
Sealed microsomal vesicles were prepared from corn ( Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000 g microsomal fraction onto a 10% dextran T-70 cushion. The Mg 2+-ATPase activity of the sealed vesicles was stimulated by Cl − and NH 4+ and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO 3−, was insensitive to K +, and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide. Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg2+, was stimulated by Cl−, inhibited by NO3−, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg2+ ≥ Mn2+ > Co2+) and were inhibited by high concentrations of Ca2+. The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F1F0-ATPase and from those of the K+-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase. 相似文献
10.
Membranes from homogenates of growing and of dormant storage roots of red beet ( Beta vulgaris L.) were centrifuged on linear sucrose gradients. Vanadate-sensitive ATPase activity, a marker for plasma membrane, peaked at 38% to 40% sucrose (1.165-1.175 grams per cubic centimeter) in the case of growing material but moved to as low as 30% sucrose (1.127 grams per cubic centimeter) during dormancy. A band of nitrate-sensitive ATPase was found at sucrose concentrations of 25% to 28% or less (around 1.10 grams per cubic centimeter) for both growing and dormant material. This band showed proton transport into membrane vesicles, as measured by the quenching of fluorescence of acridine orange in the presence of ATP and Mg2+. The vesicles were collected on a 10/23% sucrose step gradient. The phosphate hydrolyzing activity was Mg dependent, relatively substrate specific for ATP (ATP > GTP > UTP > CTP = 0) and increased up to 4-fold by ionophores. The ATPase activity showed a high but variable pH optimum, was stimulated by Cl−, but was unaffected by monovalent cations. It was inhibited about 50% by 10 nanomolar mersalyl, 20 micromolar N,N′-dicyclohexylcarbodiimide, 80 micromolar diethylstilbestrol, or 20 millimolar NO3−; but was insensitive to molybdate, vanadate, oligomycin, and azide. Proton transport into vesicles from the 10/23% sucrose interface was stimulated by Cl−, inhibited by NO3−, and showed a high pH optimum and a substrate specificity similar to the ATPase, including some proton transport driven by GTP and UTP. The low density of the vesicles (1.10 grams per cubic centimeter) plus the properties of H+ transport and ATPase activity are similar to the reported properties of intact vacuoles of red beet and other materials. We conclude that the low density, H+-pumping ATPase of red beets originated from the tonoplast. Tonoplast H+-ATPases with similar properties appear to be widely distributed in higher plants and fungi. 相似文献
11.
Evidence was found for a Na +/H + antiport in tonoplast vesicles isolated from barley ( Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg 2+, and the pH gradient gradually dissipated. When 50 millimolar K + or Na + was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K + to vesicles from salt-grown roots. However, when 50 millimolar Na + was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K + and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na +/H + exchange. The Na +/H + exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na + concentration, with an apparent Km for Na + of 9 millimolar. The rate of Na +/H + exchange with 10 millimolar Na + was inhibited 97% by 0.1 millimolar dodecyltriethylammonium. 相似文献
12.
The (K +,Mg 2+)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N 2 without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co 2+ > Mg 2+ > Mn 2+ > Zn 2+ > Ca 2+) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg 2+, the enzyme was further activated by monovalent cations (K +, NH 4+, Rb + Na +, Cs +, Li +). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a Km of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K + approached simple Michaelis-Menten kinetics, with a Km of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K +, and preferred Mn 2+ to Mg 2+. The results demonstrate that the (K +,Mg 2+)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots. 相似文献
13.
Chloride transport, presumably via a Cl −-2H + co-transport system, was investigated in Chara corallina. At pH 6.5, the control influx (3.1 picomoles per centimeter 2 per second) was stimulated 4-fold by an 18-hour Cl − starvation. The stimulated influx was inhibited to 4.7 picomoles per centimeter 2 per second after a 60-minute pre-exposure to 0.5 millimolar 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). This compares with a nonsignificant inhibition of the control under similar conditions. At 2 millimolar DIDS, both stimulated and control influx were inhibited to values of 1.1 and 2.2 picomoles per centimeter 2 per second, respectively; in all cases, DIDS inhibition was reversible. Over the pH range 4.8 to 8.5, the control and DIDS-inhibited influx showed only slight pH sensitivity; in contrast, the stimulated flux was strongly pH dependent (pH 6.5 optimum). Inasmuch as changes in pH alter membrane potential, N-ethylmaleimide was used to depolarize the membrane; this had no effect on Cl − influx. A transient depolarization of the membrane (about 20 millivolts) was observed on restoration of Cl − to starved cells. The membrane also depolarized transiently when starved cells were exposed to 0.5 millimolar DIDS, but the depolarization associated with Cl − restoration was inhibited by a 40-minute pretreatment with DIDS. Exposure of control cells to DIDS caused only a small hyperpolarization (about 7 millivolts). DIDS may have blocked Cl − influx by inhibiting the putative plasmalemma H +-translocating ATPase. Histochemical studies on intact cells revealed no observable effect of DIDS on plasmalemma ATPase activity. However, DIDS application after fixation resulted in complete inhibition of ATPase activity. The differential sensitivity of the stimulated and control flux to inhibition by DIDS may reflect an alteration of transport upon stimulation, but could also result from differences in pretreatment. The stimulated cells were pretreated with DIDS in the absence of Cl−, in contrast to the presence of Cl− during pretreatment of controls. The differential effect could result from competition between Cl− and DIDS for a common binding site. Our histochemical ATPase results indicate that Cl− transport and membrane ATPase are separate systems, and the latter is only inhibited by DIDS from the inside of the cell. 相似文献
14.
Segments of oat ( Avena sativa L.) roots which had been exposed to 1 millimolar CdSO 4 in quarter-strength Hoagland No. 1 solution exhibited decreased respiratory rates, ATP levels, membrane-bound ATPase activity, and reduced K + fluxes. Respiration and ATP levels were decreased after a 2-hour treatment with 1 millimolar CdSO 4 to 65 and 75%, respectively, of control rates. A membrane-bound, Mg 2+-dependent, K +-stimulated acid ATPase was rapidly inhibited to 12% of control activity in the presence of 1 millimolar CdSO 4. Potassium uptake into root segments was inhibited to 80% of control values after 30 minutes in the presence of CdSO 4. A 2-hour pretreatment of root segments with CdSO 4 inhibited K + uptake to 15% of control values. Cytoplasmic K + efflux was inhibited with 1 millimolar CdSO 4. The rates and the degree of Cd2+ inhibition of the parameters listed above suggest that one of the first sites of Cd2+ action is the plasmalemma K+ carrier (ATPase) in oat roots. 相似文献
15.
The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent Km-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; Vmax app was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 × 10 6 vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H +-translocating enzyme activities revealed Km app values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Δ μH + while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity. 相似文献
16.
A Mg 2+-dependent, cation-stimulated ATPase was associated with plasma membranes isolated from corn leaf mesophyll protoplasts. Potassium was the preferred monovalent cation for stimulating the ATPase above the Mg 2+-activated level. The enzyme was substrate-specific for ATP, was inhibited by N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzoate, and orthovanadate, but was insensitive to oligomycin or sodium azide. A Km of 0.28 millimolar Mg 2+-ATP was determined for the K +-ATPase, and the principal effect of potassium was on the Vmax for ATP hydrolysis. Since potassium stimulation was not saturated at high concentrations, a nonspecific role was proposed for potassium stimulation. A nonspecific phosphatase was also found to be associated with corn leaf plasma membranes. However, it could not be determined positively whether this activity represented a separate enzyme. 相似文献
17.
A uracil-DNA glycosylase activity has been purified about 750-fold from the chloroplasts of light-grown Zea mays seedlings. This report represents the first direct demonstration of a DNA-glycosylase repair activity in chloroplasts. The activity, in part, was associated with a chloroplast Triton X-100 sensitive membrane. Its apparent Km was 1.0 micromolar for a poly(dA-dT/U) substrate, and its molecular weight, as determined by gel filtration, was 18,000. The enzyme exhibited optimal activity at pH 7.0 with an atypically narrow pH tolerance. Activity was inhibited greater than 60% by 10 millimolar NaCl, 5 millimolar MgCl 2, or 5 millimolar EDTA. Enzyme activity was inhibited 80% by 10 millimolar N-ethylmaleimide, a sulfhydryl group-blocking agent. The activity removed uracil more rapidly from single-stranded DNA than from double-stranded DNA. With this report, uracil-DNA glycosylase activity has now been attributed to all three DNA-containing organelles of eucaryotic cells. 相似文献
18.
Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by “aging” in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. “Aged” or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO 3, which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or “aged” submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO 3−, but inhibited by Cl −, indicating that Cl − stimulation is a distinguishing property of the pea mitochondrial ATPase complex. 相似文献
19.
Because glyoxylate inhibits CO 2 fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization. The fixation of CO2 by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO2 fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO2 fixation seems to be a general effect of salts of weak acids. Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP+) from intact chloroplast preparations had an apparent Km (glyoxylate) of 140 micromolar and a Vmax of 3 micromoles per minute per milligram chlorophyll. 相似文献
20.
A dihydroxyacetone phosphate (DHAP) reductase has been isolated in 50% yield from Dunaliella tertiolecta by rapid chromatography on diethylaminoethyl cellulose. The activity was located in the chloroplasts. The enzyme was cold labile, but if stored with 2 molar glycerol, most of the activity was restored at 30°C after 20 minutes. The spinach ( Spinacia oleracea L.) reductase isoforms were not activated by heat treatment. Whereas the spinach chloroplast DHAP reductase isoform was stimulated by leaf thioredoxin, the enzyme from Dunaliella was stimulated by reduced Escherichia coli thioredoxin. The reductase from Dunaliella was insensitive to surfactants, whereas the higher plant reductases were completely inhibited by traces of detergents. The partially purified, cold-inactivated reductase from Dunaliella was reactivated and stimulated by 25 millimolar Mg 2+ or by 250 millimolar salts, such as NaCl or KCl, which inhibited the spinach chloroplast enzyme. Phosphate at 3 to 10 millimolar severely inhibited the algal enzyme, whereas phosphate stimulated the isoform in spinach chloroplasts. Phosphate inhibition of the algal reductase was partially reversed by the addition of NaCl or MgCl 2 and totally by both. In the presence of 10 millimolar phosphate, 25 millimolar MgCl 2, and 100 millimolar NaCl, reduced thioredoxin causes a further twofold stimulation of the algal enzyme. The Dunaliella reductase utilized either NADH or NADPH with the same pH maximum at about 7.0. The apparent Km (NADH) was 74 micromolar and Km (NADPH) was 81 micromolar. Apparent Vmax was 1100 μmoles DHAP reduced per hour per milligram chlorophyll for NADH, but due to NADH inhibition highest measured values were 350 to 400. The DHAP reductase from spinach chloroplasts exhibited little activity with NADPH above pH 7.0. Thus, the spinach chloroplast enzyme appears to use NADH in vivo, whereas the chloroplast enzyme from Dunaliella or the cytosolic isozyme from spinach may utilize either nucleotide. 相似文献
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