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1.
3-Phosphoglycerate (PGA)-dependent O 2 evolution by mesophyll chloroplasts of the C 4 plant, Digitaria sanguinalis L. Scop. (crabgrass), was inhibited by micromolar levels of 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). As little as 1.8 micromolar DIDS added to the assay medium (containing 0.7 millimolar PGA) resulted in 80 to 100% inhibition of O 2 evolution. The extent of inhibition of O 2 evolution observed was dependent on various factors including: pH, concentration of DIDS to relative chlorophyll, concentration of PGA, and the time of addition of DIDS to the chloroplasts relative to addition of PGA. Preincubation of crabgrass chloroplasts with micromolar levels of DIDS, followed by washing to remove any nonirreversibly bound DIDS, inhibited PGA-dependent O2 evolution. Protection against this inhibition was afforded by preincubating the chloroplasts with various substrates before adding DIDS. For example, if the chloroplasts were first incubated with 8.3 millimolar PGA, phosphoenolpyruvate (PEP) or inorganic phosphate before adding 42 micromolar DIDS, the percentage of inhibition was decreased from 100% (without any substrate) to 0, 54, and 67%, respectively. 2-Phosphoglycerate caused a slight decrease in the inhibition (about 10%) and glucose-6-phosphate had no protective effect. If the chloroplasts were pretreated with DIDS initially, the inhibition could not be overcome by PGA, suggesting that DIDS acts as an irreversible inhibitor. Micromolar levels of DIDS also inhibited PGA dependent O2 evolution by isolated chloroplasts of the C3 plant barley. As with crabgrass, preincubation with PGA or inorganic phosphate resulted in a decrease in the DIDS inhibition, but PEP was very ineffective compared to the C4 chloroplasts. Oxalacetate-dependent O2 evolution and its stimulation by the uncoupler, NH4Cl, were unaffected by the addition of DIDS to crabgrass mesophyll chloroplasts. Furthermore, preincubation of the chloroplasts with DIDS (up to 65 micromolar) had no inhibitory effect on the extractable activity of NADP glyceraldehyde-3-P dehydrogenase and phosphoglycerate kinase. Inhibition by DIDS was interpreted to be at the substrate binding site of the phosphate translocator. The data further suggest that in C4 crabgrass chloroplasts, PEP is transported on a carrier which also transports PGA. 相似文献
2.
Summary A number of additives have been tested for their effects on o-diphenol: O 2 oxidoreductase activity of cane leaves. The most inhibitory compounds were thioglycollate, -mercaptoethanol, polyethylene glycol and bovine serum albumin. Sulphydryl (SH) compounds did not affect rates of photosynthetic CO 2 assimilation when used at concentrations below 10 -2 M. However, in the presence of Mn ++ ions they contributed to an O 2 consumption which masked photosynthetic O 2 evolution. Addition of SH compounds or of polymers to the grinding media increased rates of enzymic CO 2 assimilation in crude enzyme preparations from cane leaves, but did not affect rates of CO 2 assimilation in similar spinach preparations. Strong reducing agents, copper chelators, low O 2 tension and high pH were effective in reducing phenoloxidase activity, but presented problems in the isolation and assay of chloroplasts. The results are discussed in relation to (a) design of suitable media for preparation of active cane chloroplasts and (b) comparative studies of enzyme levels in plants of various genera. 相似文献
3.
Oxygen-evolution activity of spinach chloroplasts was investigatedby washing chloroplasts with 0.8M Tris buffer containing 20%acetone. This inactivitation was easily removed by two successivetreatments, dark- and light-reactivations. The first treatmentwas dark-reactivation step, rewashing inactivated chloroplastswith reduced DPIP (DPIP treatment). The second one was a light-reactivatedchloroplasts with incubating chloroplasts with Mn 2+, Ca 2+, dithiothreitoland bovine serum albumin under ilumination. Both light- and dark-reactivation treatments were required toregain oxygen-evolution activity of Tris-acetone-washed chloroplasts,which is characteristic of such chloroplasts. However, in Tris-washedchloroplasts considerable activity was recovered by dark-reactivationalone. Manganese and calcium contents of Tris-acetone-washed chloroplastswere compared with those of chloroplasts obtained by other preparations. Tris-acetone washing was presumed to inhibit the oxygen-evolutionsite of Photo-systetm II by affecting Mn, Ca and other substancesin chloroplasts. The inhibition site was estimated from a changein fluorescence yield of chlorophyll and the effect of artificialelectron donor specific for Photosystem II on NADP photoreductionactivity. (Received August 20, 1973; ) 相似文献
4.
Intact spinach ( Spinacia oleracea L.) chloroplasts, when pre-illuminated at 4 millimoles quanta per square meter per second for 8 minutes in a CO 2-free buffer at 21% O 2, showed a decrease (30-70%) in CO 2-dependent O 2 evolution and 14CO 2 uptake. This photoinhibition was observed only when the O 2 concentration and the quantum fluence rate were higher than 4% and 1 millimole per square meter per second, respectively. There was only a small decrease in the extent of photoinhibition when the CO 2 concentration was increased from 0 to 25 micromolar during the treatment, but photoinhibition was abolished when the CO 2 concentration was increased to 30 micromolar. Addition of small quantities of P-glycerate (40-200 micromolar) or glycerate (160 micromolar) was found to prevent photoinhibition. Other intermediates of the Calvin cycle (fructose-6-P, fructose-1,6-P, ribose-5-P, ribulose-5-P) also prevented photoinhibition to various extents. Oxaloacetate was not effective in preventing photoinhibition in these chloroplasts. The amount of O 2 evolved during treatments with 3-P-glycerate or glycerate was no more than 65% of that measured in the presence of low CO 2 concentrations (9-12 micromolar) which did not prevent photoinhibition. In all cases, the extent to which photoinhibition was prevented by these metabolites was not correlated to the amount of O 2 evolved during the photoinhibitory treatment. It is concluded that in these chloroplasts the prevention of the O 2-dependent photoinhibition of light saturated CO 2 fixation capacity is not linked to the dissipation of excitation energy via the photosynthetic electron transport nor to ATP utilization. The requirement of O 2 for photoinhibition of CO 2 fixation capacity in isolated chloroplasts may be explained by an effect of O 2 in allowing metabolic depletion of Calvin cycle intermediates. 相似文献
5.
Immobilized chloroplasts and Clostridium butyricum were employed for a photochemical energy conversion system. Spinach chloroplasts were immobilized in 2% agar gel. The optimum temperature of immobilized chloroplasts was 30°C. The maximum activity was obtained in the phosphate buffer solution (pH 8.0) containing 8μ M of ferredoxin under an N 2 bubbling condition. Hydrogen was evolved under illumination by immobilized chloroplasts and C. butyricum. Hydrogen produced by this system was applied to a hydrogen-oxygen fuel cell. Photoinduced current was obtained from this photochemical energy conversion system. A photocurrent of 0.4?1.5 mA was continuously obtained for 4 h. The conversion ratio from hydrogen to current was 80?100%. 相似文献
6.
The effects of phosphoenolpyruvate (PEP), inorganic phosphate (Pi), and ATP on 3-phosphoglycerate (PGA)-dependent O 2 evolution by chloroplasts of Digitaria sanguinalis (L.) Scop. (crabgrass) were evaluated relative to possible mechanisms of PEP transport by the C 4 mesophyll chloroplast. Crude and Percoll purified chloroplast preparations exhibited rates of PGA-dependent O 2 evolution in the range of 90 to 135 micromoles O 2 per milligram chlorophyll per hour, and up to 180 micromoles O 2 per milligram chlorophyll per hour at optimal Pi concentrations (approximately 0.2 millimolar at 9 millimolar PGA). Higher concentrations of Pi were inhibitory. PEP inhibited O 2 evolution (up to 70%) in both chloroplast preparations when the PEP to PGA ratio was high ( i.e. 9 millimolar PEP to 0.36 millimolar PGA). Usually no inhibition was seen when the PEP to PGA ratio was less than 2. PEP acted as a competitive inhibitor and, at a concentration of 9 millimolar, increased the apparent K m (PGA) from 0.15 to 0.53 millimolar in Percoll purified chloroplasts. A low concentration of PGA and high ratio of PEP to PGA, which are considered unphysiological, were required to detect any inhibition of O 2 evolution by PEP. Similar results were obtained from crude versus Percoll purified preparations. Neither the addition of Pi nor ATP could overcome PEP inhibition. As PEP inhibition was competitive with respect to PGA concentration, and as addition of ATP or Pi could not prevent PEP inhibition of PGA-dependent O 2 evolution, the inhibition was not due to PEP exchange of adenylates or Pi out of the chloroplast. Analysis of the effect of Pi and PEP, separately and in combination, on PGA-dependent O 2 evolution suggests interactions between PEP, Pi, and PGA on the same translocator in the C 4 mesophyll chloroplast. C 3 spinach chloroplasts were also found to be sensitive to PEP, but to a lesser extent than crabgrass chloroplasts. The apparent K i values (PEP) were 3 and 21 millimolar for crabgrass and spinach, respectively. 相似文献
7.
Extraction of spinach ( Spinacia oleracea L.) chloroplasts with cholate-asolectin in the absence of Mg 2+ results in the rapid and selective inactivation of O 2 evolution and a partial (30 to 40%) loss of photosystem II (PSII) donor activity without extraction of thylakoid bound Mn (~5 to 6 Mn per 400 Chlorophyll). Inclusion of ethylene glycol in the extractions inhibits loss of O 2 evolution and results in quantitative and qualitative differences in proteins solubilized but does not significantly inhibit the partial loss of PSII donor activity. Similarly, in two stage experiments (extraction followed by addition of organic solvent and solubilized thylakoid protein), O 2 evolution ( V and Vmax) of extracted chloroplasts is enhanced approximately 2.5- to 8-fold. However, PSII donor activity remains unaffected. This reversal of cholate inactivation of O 2 evolution can be induced by solvents including ethanol, methanol, 2-propanol, and dimethyl sulfoxide. Such enhancements of O 2 evolution specifically required cholate-solubilized proteins, which are insensitive to NH 2OH and are only moderately heat-labile. NH 2OH extraction of chloroplasts prior to cholate-asolectin extraction abolishes reconstitutability of O 2 evolution. Thus, the protein(s) affecting reconstitution is unlike those of the O 2·Mn enzyme. The specific activity of the protein fraction effecting reconstitution of O 2 evolution is greatest in fractions depleted of the reported Mn-containing, 65-kilodalton, and the Fe-heme, 232-kilodalton (58-kilodalton monomer), proteins. Divalent (~3 millimolar) and monovalent (~30 millimolar) cations do not affect reconstitution of PSII donor activity but do affect reconstitution of O 2 evolution by decreasing the protein(s) concentration required for reconstitution of O 2 evolution in nonfractionated, cholate-asolectin extractions. The data indicate a reconstitution of the PSII segment linking the PSII secondary donor(s) to O 2-evolving centers. 相似文献
8.
Summary The ultrastructure of chloroplasts from two genera of coenocytic green algae, Codium and Caulerpa, were examined after suspension in hypotonic solution and in detergent at various concentrations. The capacity of the suspensions to carry out CO 2-dependent and ferricyanide-dependent O 2 evolution was measured under the same conditions of osmotic strength and detergent concentration.The chloroplasts in the preparations were in the form of cytoplasts and gave rates of O 2 evolution comparable with those expected from undamaged chloroplasts. Suspension in hypotonic solution depressed the rate of CO 2-dependent O 2 evolution in both species, but this was partially restored in the Codium chloroplasts when these were re-suspended in iso-osmotic solutions. Major structural changes were observed only after suspension in buffer when the Codium chloroplasts lost their outer envelope, most of their stroma, and the thylakoids became swollen. Caulerpa chloroplasts were more variable in their response and, even when suspended in buffer only, the proportion of the plastids which had lost all of their stroma and thylakoid swelling was never as common as in Codium chloroplasts. However, once suspended in hyper-osmotic medium below 700 mosmolar, Caulerpa chloroplasts could not regain their capacity for CO 2-dependent O 2 evolution.Detergent treatment removed the cytoplast membrane but not the cytoplasmic material adhering to the chloroplast envelope. High concentrations of detergent were needed to cause loss of the chloroplast envelope, loss of stromal contents and unstacking of the thylakoids. Caulerpa chloroplasts were less sensitive to detergent than those of Codium. There was no indication that specific structures such as the thylakoid organizing body were resistant to detergent action. The results show that exposure to hypotonic solutions and to detergent results in less damage to these chloroplasts than it would to those of higher plants. It is proposed that the basis of this unusual resistance is not due to the properties of the chloroplast membranes but to the presence of material which coats the organelles during isolation. This material is likely to be identical with the sulphated xylo-mannogalactan isolated from the vacuole contents of these algae and which has the visco-elastic properties essential to allow the organelles to resist disruption by osmotic forces and disintegration by detergents. 相似文献
9.
Intact chloroplasts were isolated from spinach leaves using media with either 330 mM sorbitol or 200 mM KCl as the osmoticum. Chloroplasts isolated in KCl exhibited higher rates of CO 2-dependent oxygen evolution in nine out of ten experiments, the average increase being 43%. Chloroplasts isolated in KCl routinely achieved rates of CO 2-dependent oxygen evolution of 200–300 mol·mg chlorophyll -1·hour -1 at 20°C. Intact chloroplasts were also isolated in media with 200 mM NaCl or choline chloride but the rates of CO 2 fixation were not superior to those isolated in sorbitol media. The K + content of chloroplasts isolated in KCl media was higher than for chloroplasts isolated in sorbitol. It is suggested that the use of KCl as an osmoticum prevents the loss of chloroplast K + which can occur during isolation in sorbitol media. Chloroplasts isolated in KCl lost, on average, 36% of the initial CO 2 fixation activity after storage for four hours on ice, compared to 24% loss of activity for chloroplasts isolated in sorbitol. This increased loss of activity was not observed if KCl was used in the grinding medium and sorbitol or glycinebetaine in the resuspension media. For measurement of the maximum photosynthetic capacity in vitro, the use of KCl in the grinding medium may be better than sorbitol.Abbreviations BSA
bovine serum albumin
- Chl
chlorophyll
- Pi
inorganic orthophosphate
- EDTA
ethlenediamine tetraacetic acid 相似文献
10.
Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C 4 plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C 4 subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O 2 evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O 2 evolution (29 to 58 micromoles O 2 evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide. 相似文献
11.
Summary Isolated higher plant chloroplasts with intact envelope membranes and bovine serum albumin were co-immobilized by treating the mixture with glutaraldehyde and then subjecting it to a freeze-thaw cycle. The immobilized chloroplasts are capable of photoinduced electron transport to lipophilic oxidants, but become compatible also with ionic oxidants after a transient hyposmotic shock.Abbreviations ASC
ascorbate
- Chl
chlorophyll
- DCIP
2,6-dichlorophenol indophenol
- DCMU
3-(3,4-dichlorophenyl)-1,1-dimethyl urea
- FeCN
K 3 Fe(CN) 6
- MV
methyl viologen
- PD ox
FeCN-oxidized p-phenylene diamine 相似文献
12.
A method of plant culture was developed for growing large leaves of glandless cotton on single stems. Chloroplasts isolated from these leaves actively reduced ferricyanide when assayed for the Hill reaction. Hill reaction activity increased 133% when the 0.5 m sucrose isolation medium was replaced with 10% (w/v) polyethylene glycol, both buffered at pH 7.6. The presence of 2 or 5% (w/v) bovine serum albumin in the sucrose buffer did not increase Hill activity. Ferricyanide reduction in the dark occurred in all assays, and the possibility of gossypol as the reductant is discussed. Half-life of the chloroplasts stored in 10% glycerol at -23 C was 23 days. The ammonium ion at 0.01 m enhanced Hill reaction activity up to 171%. Leaves containing chloroplasts with the highest Hill reaction activity were found near the 8th node below the apex. Leaf water potentials less than -28 bars reduced the activity about 50%. Daylight conditions during the winter months in the greenhouse reduced the activity about 30%. 相似文献
13.
A reconstituted spinach chloroplast system containing thylakoids, stroma and 0.1 mM NADPH supported O 2 evolution in the presence of oxidised glutathione (GSSG). The properties of the reaction were consistent with light-coupled GSSG-reductase activity involving H 2O as eventual electron donor. The reconstituted system also supported dehydroascorbate-dependent O 2 evolution in the presence of 0.6 mM reduced glutathione (GSH) and 0.1 mM NADPH with the concomitant production of ascorbate. The GSSG could replace GSH in which case the production of GSH preceded the accumulation of ascorbate. The data are consistent with the light-dependent reduction of dehydroascorbate using H 2O as eventual electron donor via the sequence H 2O→NADP→GSSG→dehydroascorbate. Approximately 30% of the GSH-dehydrogenase activity of spinach leaf protoplasts is localised in chloroplasts: this could not be attributed to contamination of chloroplasts by activity from the extrachloroplast compartment. Washed intact chloroplasts supported the uptake of ascorbate but the uptake mechanism had a very low affinity for ascorbate (K m approximately 20 mM). The rate of uptake of ascorbate was less than the rate of light-dependent reduction of dehydroascorbate and too slow to account for the rate of H 2O 2 reduction by washed intact chloroplasts. 相似文献
14.
Abstract A simple mechanical method for the rapid isolation of chloroplasts with high rates of photosynthesis from young leaves of oat ( Avena sativa L.) was described. The photosynthetic activity of these chloroplasts was stable for at least 2 h with rates of CO 2-dependent O 2 evolution of 30–40 μmol g 1 Chl s 1. The photosynthetic properties of these chloroplasts were similar to those reported for spinach and pea chloroplasts isolated by mechanical disruption. The pH optimum for photosynthetic O 2 evolution was pH 7.6. The induction time was 0.5–2 min. Maximal rates of photosynthetic O 2 evolution in these chloroplast preparations were obtained in the absence of both divalent cations and EDTA. Addition of divilent cations strongly inhibited photosynthesis which could be partially restored by the subsequent addition of EDTA. But when these cations were not present in the assay medium the addition of EDTA greater than 1 mol m 3 decreased photosynthetic activity. The optimal orthophosphate concentration required for photosynthesis in these chloroplast preparations was 0.2–0.3 mol m 3. In contrast, the addition of pyrophosphate either in the light or dark inhibited photosynthesis. In a comparative study, chloroplasts were also isolated from oat and wheat ( Triticum aestivum L., cultivar Hybrid C306) protoplasts. These chloroplast preparations were found to have properties similar to those determined for oat chloroplasts isolated by the mechanical method reported above. 相似文献
15.
This study examines the effect of antimycin A and nitrite on 14CO 2 fixation in intact chloroplasts isolated from spinach ( Spinacia oleracea L.) leaves. Antimycin A (2 micromolar) strongly inhibited CO 2 fixation but did not appear to inhibit or uncouple linear electron transport in intact chloroplasts. The addition of small quantities (40-100 micromolar) of nitrite or oxaloacetate, but not NH 4Cl, in the presence of antimycin A restored photosynthesis. Antimycin A inhibition, and the subsequent restoration of photosynthetic activities by nitrite or oxaloacetate, was observed over a wide range of CO 2 concentration, light intensity, and temperature. High O 2 concentration (up to 240 micromolar) did not appear to influence the extent of the inhibition by antimycin A, nor the subsequent restoration of photosynthetic activity by nitrite or oxaloacetate. Studies of O 2 exchanges during photosynthesis in cells and chloroplasts indicated that 2 micromolar antimycin A stimulated O 2 uptake by about 25% while net O 2 evolution was inhibited by 76%. O 2 uptake in chloroplasts in the presence of 2 micromolar antimycin A was 67% of total O 2 evolution. These results suggest that only a small proportion of the O 2 uptake measured was directly linked to ATP generation. The above evidence indicates that cyclic photophosphorylation is the predominant energy-balancing reaction during photosynthesis in intact chloroplasts. On the other hand, pseudocyclic O 2 uptake appears to play only a minimal role. 相似文献
16.
Brief saturating light flashes were used to probe the mechanism of inactivation of O 2 evolution by Tris in chloroplasts. Maximum inactivation with a single flash and an oscillation with period of four on subsequent flashes was observed. Analyses of the oscillations suggested that only the charge-collecting O 2-evolving catalyst of photosystem II (S 2-state) was a target of inactivation by Tris. This conclusion was supported by the following observations: ( a) hydroxylamine preequilibration caused a three-flash delay in the inactivation pattern; ( b) the lifetimes of the Tris-inactivable and S 2-states were similar; and ( c) reagents accelerating S 2 deactivation decreased the lifetime of the inactivable state. Inactivation proved to be moderated by F, the precursor of Signal II s, as shown by a one flash delay with chloroplasts having high abundance of F. Evidence was obtained for cooperativity effects in inactivation and NH 3 was shown to be a competitive inhibitor of the Tris-induced inactivation. S 2-dependent inactivation was inhibited by glutaraldehyde fixation of chloroplasts, possibly suggesting that inactivation proceeds via conformational changes of the S 2-state. 相似文献
17.
Summary
Saccharomyces
anamensis having -galactosidase activity, has been immobilized in calcium alginate gel matrix that retained 78.6% enzyme activity to that of native cells. Optimum pH(7.0) was negligibly affected by immobilization. K m values for immobilized and native cells were 119 mM and 102 mM respectively. Protective agents like dithioerythritol, bovine serum albumin, enhance the enzyme activity when added prior to immobilization. Immobilized cells can be stored in refrigeration(4°C) for 42 days without a significant loss of enzyme activity. 相似文献
18.
The estimated light emission spectrum was determined for a singlet oxygen ( 1O 2)-producing system, NaOCl + H 2O 2, alone and in the presence of tryptophan and bovine serum albumin. Tryptophan and bovine serum albumin caused a decrease in the red emission of 1O 2 and an increase in the amount of shorter wavelength light. This effect was due to chemiluminescence rather than fluorescence. Arachidonic acid caused a similar spectral shift, while guanosine demonstrated a late chemiluminescent reaction of predominantly short wavelength light in the presence of 1O 2. 相似文献
19.
Summary The variations and characteristics of o-diphenoloxidase activity (O-diphenol-O 2-oxidoreductase EC 1.10.3.1) were examined in aging, isolated spinach chloroplasts to determine whether this activity, measured in the presence of 4-methylcatechol as substrate, could be responsible for the inhibition of O 2 evolution during aging of these organelles in dark and light.The rate of the Hill reaction (oxygen evolution and the corresponding photoreduction of ferricyanide) during aging in the dark was inhibited at pH 8.0 and stimulated at pH 6.5. This difference did not depend on the nature of the buffer used (Tris-HCl or phosphate). Furthermore, the pH optimum for the ferricyanide-Hill reaction was shifted to lower values (from pH 8.0 to 6.5) on aging of chloroplasts. This phenomenon is probably due to uncoupling during aging. In the light, the Hill reaction was markedly inhibited. However, the ratio moles O 2 evolved/moles ferricyanide reduced diminished slowly in darkness and rapidly when the chloroplasts were aged in the light.Aging of chloroplasts in darkness was accompanied by a slow decrease in the latent period which precedes the initiation of the oxidation, followed by an increase in O-diphenoloxidase activity. Light-aged chloroplasts showed an initial stimulation and then a smaller increase in enzyme activity compared with that of the dark-aged chloroplasts. This latter phenomenon was probably due to secondary reactions caused by photo-inactivation. Under light conditions, the latent period decreased rapidly and disappeared after one hour.This latent period varied considerably with the season and was reduced or obliterated by treatments with light, fatty acids, Triton-X, hypotonic medium and increasing concentrations of substrate: that is by treatments which generally enhance chloroplast swelling. Thus it appears that the latent period is not a characteristic of O-diphenoloxidase but depends on the integrity of chloroplast structure.The enzyme activity was characterized by a stoichiometry of about 1 moles O 2 consumed per 1.2 moles substrate oxidized, indicating that oxidation was probably proceeding further than conversion of O-diphenol to O-diquinone. The latter compound could be used as a Hill oxidant and it permitted measurement of O 2 evolution in the same reaction mixture in the presence of light. Under these experimental conditions, O 2 evolution (a DCMU sensitive reaction) was first stimulated in dark-aged chloroplasts and rapidly inhibited in light-aged chloroplasts.At appropriate concentrations, KCN, a potent inhibitor of oxidases, enhanced O 2 evolution, suggesting that O-diphenoloxidase activity interferes with O 2 evolution. This possibility is discussed in view of our previous findings on chloroplast aging in vitro. 相似文献
20.
A mixture of glycosidases from the liver of the gastropod Turbo cornutus was co-immobilized with bovine serum albumin and glutaraldehyde, and then cast as membranes. The properties of immobilized N-acetyl-β-d-hexosaminidase were studied. The recovery of N-acetyl-β-d-hexosaminidase after immobilization was unaffected by increasing the concentration of glutaraldehyde, but was decreased by increasing the bovine serum albumin concentration. The immobilized enzyme showed enhanced resistance towards proteolytic and thermal inactivation. While the pH optimum for the soluble enzyme was 4.0, a bimodal pH curve with optima at 3.4 and 5.0 was observed after insolubilization. This bimodality was abolished when the immobilized enzyme was assayed in the presence of M NaCl. The Km values, for p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside, of the immobilized isoenzymes of N-acetyl-β-d-hexosaminidase were larger than those of their soluble counterparts. No loss of activity could be detected in the membrane after using it for 24 consecutive assays or after storage for at least 50 days at 4°. 相似文献
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