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M Gibbs

Publications and source records attributed to M Gibbs.

At least 91 records · Page 5Linked to original sources
PubMed1983

Photosynthetic Properties of Chloroplasts from Chlamydomonas reinhardii.

Chloroplasts isolated from synchronous cultures of the unicellular green alga Chlamydomonas reinhardii, SAG 11-32/b (-), fix CO(2) at rates between 25 and 50 micromoles per milligram chlorophyll per hour. The upper value is approximately half of the rate of the intact cell.During storage in the dark on ice, the chloroplast preparation loses 30 to 50% of its CO(2) fixing capability per hour. Under reducing conditions (+ 1 millimolar dithiothreitol), this loss of activity is about twice as fast. The same reducing conditions stimulate CO(2) fixation in the light.High concentrations of inorganic phosphate (>2 millimolar) inhibit CO(2) fixation. This inhibition is overcome by the addition of glycerate 3-phosphate. It is concluded that chloroplasts from C. reinhardii possess a higher plant type phosphate translocator. With respect to dependency upon light intensity, pH and Mg(2+) concentration, the results were similar to that reported for chloroplasts from higher plants. However, in contrast to higher plant chloroplasts, maximum CO(2) fixation is observed at the relatively low osmotic concentration of 0.12 molar mannitol in the reaction buffer.

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PubMed1983

Reduced osmotic potential inhibition of photosynthesis : site-specific effects of osmotically induced stromal acidification.

The effects of reduced reaction medium osmotic potential (0.67 molar sorbitol as compared to a control treatment with 0.33 molar sorbitol) on the enzymic steps of the photosynthetic carbon reduction cycle were investigated using isolated spinach (Spinacia oleracea L. var Longstanding Bloomsdale) chloroplasts. Reversal of reduced osmotic potential inhibition of photosynthetic rates by a stromal alkalating agent (NH(4)Cl) was associated with specific steps of the cycle. Low osmotic potential induced stromal acidification was found to be facilitated by osmotically induced chloroplast shrinkage. However, the action of the alkalating agent was found not to be associated with reversal of osmotically induced morphological changes of the stromal compartment.Labeled metabolite analyses indicated that the osmotic stress treatment caused the substrate for fructose 1,6-bisphosphatase (FBPase) to build up in the absence of NH(4)Cl, and the substrate for phosphoribulokinase to increase in the presence of NH(4)Cl. These data were interpreted as indicating that the most severe effect of osmotic stress on photosynthesis is at the site of FBPase, and that this inhibition is mediated by osmotically induced stromal acidification. Phosphoribulokinase activity inhibition at the low osmotic potential treatment was apparently less severe and not mediated by stromal acidification. A third site of osmotic inhibition, which was reversed by NH(4)Cl, and therefore was assumed to be mediated by stromal acidification, was at the step of ribulose 1,5-bisphosphate carboxylase.Additions of NH(4)Cl also enhanced the activity of the pH-insensitive phase of the photosynthetic carbon reduction cycle, 3-phosphoglyceric acid reduction, at the stress treatment. This effect was thought to be mediated by the removal of the block at FBPase. A model was proposed to outline the relative severity of osmotic stress effects at various sites of the photosynthetic carbon reduction cycle.

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PubMed1983

Stromal acidification mediates in vivo water stress inhibition of nonstomatal-controlled photosynthesis.

Stromal acidification has been reported to mediate reduced osmotic potential (psi(pi)) effects on photosynthesis in the isolated spinach chloroplast (Berkowitz, Gibbs 1983 Plant Physiol 72: 1100-1109). To determine if stromal acidification mediates osmotic dehydration inhibition of photosynthesis in vivo, the effects of a weak base (NH(4)Cl), which raises stromal pH, on CO(2) fixation of vacuum-infiltrated spinach leaf slices, Chlamydomonas reinhardii cells and Aphanocapsa 6308 cells under isotonic and dehydrating conditions were investigated. Five millimolar NH(4)Cl stimulated spinach leaf slice CO(2) fixation by 43% under stress (0.67 molar sorbitol) conditions, and had little effect on fixation under isotonic (0.33 molar sorbitol) conditions. Chlamydomonas cells were found to be more sensitive to reduced psi(pi) than spinach leaf slices. CO(2) fixation in the cells of the green alga Chlamydomonas reinhardii was 99 and 17 micromoles per milligram chlorophyll per hour, respectively, at 0.1 molar mannitol and 0.28 molar mannitol. Five millimolar NH(4)Cl stimulated CO(2) fixation of Chlamydomonas cells by 147% under stress (0.28 molar mannitol) conditions. Aphanocapsa 6308 cells (blue-green alga) were also found to be sensitive to reduced psi(pi), and inhibitions in photosynthesis were partially reversed by NH(4)Cl. These data indicate that in vivo water stress inhibition of photosynthesis is facilitated by stromal acidification, and that this inhibition can be at least partially reversed in situ.

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PubMed1982

Characterization of a Photosynthesizing Reconstituted Spinach Chloroplast Preparation : REGULATION BY PRIMER, ADENYLATES, FERREDOXIN, AND PYRIDINE NUCLEOTIDES.

A particulate preparation (MgP) capable of photosynthetic CO(2) assimilation without the addition of stromal protein was obtained by rupturing whole spinach (Spinacia oleracea var. America) chloroplasts in 15 millimolar MgCl(2) buffered with Tricine at pH 8.5. This CO(2) assimilation was dependent upon light, inorganic phosphate, ferredoxin, ADP, NAD or NADP, and primer. Excepting glycolate, the products of CO(2) fixation by MgP were similar to those found with whole chloroplasts.Glycerate-3-phosphate (PGA), fructose-1, 6-bisphosphate (FBP), and ribose-5-phosphate (R5P) but not fructose-6-P (F6P) functioned as primers. Concentrations of PGA and FBP but not of R5P higher than 2 millimolar were inhibitory to CO(2) fixation. A lag of CO(2) fixation was observed with PGA and FBP but not with R5P. This lag as well as inhibition by NADP, ADP, and ATP in the FBP-primed preparation was eliminated by an equimolar mixture of FBP plus F6P indicating FBPase as the sensitive site. NADP, ADP, and ATP also blocked CO(2) fixation by the PGA-fortified preparation but inhibition was even more sensitive than that observed when FBP was added. Inhibition by AMP in the PGA and FBP-primed preparations was not affected by the addition of F6P. When R5P was the starting primer, inhibition of CO(2) fixation was relatively insensitive to the adenylates and NADP. In contrast to the parent whole chloroplast, CO(2) fixation by MgP was insensitive to high (5 millimolar) inorganic phosphate. Depending upon the ferredoxin concentration, NAD was as effective as NADP in supporting CO(2) fixation.

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PubMed1982

Oxidation of reduced pyridine nucleotide by a system using ascorbate and hydrogen peroxide from plants and algae.

A NAD(P)H oxidizing system (NAAP) was detected and partially purified from leaves of spinach and Sedum praealtum, seeds and leaves of pea and cells of green and red algae which oxidized NAD(P)H in the presence of ascorbate and H(2)O(2).The partially-purified spinach system had substrate K(m) values of 5 micromolar for NADH, 50 micromolar for H(2)O(2), and 300 micromolar for l-ascorbic acid at the pH optimum of 6.8. NADH was a better electron donor than NADPH. Among other electron donors, isoascorbic acid had considerable activity but hydroquinone and resorcinol had only weak activities. The enzyme was inhibited by cyanide, alpha,alpha'-dipyridyl, and mono-and di-thiol reagents. Inhibition by thiol-reagents was partially restored by Fe(2+) as was enzymic activity lost following dialysis against buffer.Subcellular localization studies with spinach and S. praealtum leaves indicated that a portion of the cell's NAAP was in the chloroplast fraction. Photosynthetic conditions resulted in a decrease in this activity solubilized from spinach and S. praealtum chloroplasts. The presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or Fe(2+) in the incubation medium eliminated the light-mediated inhibition of NAAP.NAAP may function in the recycling of NAD(P)H generated in the dark within the chloroplast. Inasmuch as all preparations of NAAP contained ascorbate peroxidase activity, the data do not rule out the possibility that NAAP is the same protein as ascorbate peroxidase or, alternatively, a combination of ascorbate peroxidase and some other enzyme.

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PubMed1982

Chloroplast Respiration : A MEANS OF SUPPLYING OXIDIZED PYRIDINE NUCLEOTIDE FOR DARK CHLOROPLASTIC METABOLISM.

A spinach (Spinacia oleracia var. America) chloroplast particle fortified with ferredoxin, fructose-1,6-bisphosphate, or ribose-5-phosphate and NADP has been shown to generate NADPH by the oxidation of glyceraldehyde-3 phosphate to glycerate-3-phosphate (PGA) and to reduce ferredoxin with the NADPH. The resulting reduced ferredoxin can reduce O(2) to H(2)O(2), nitrite to ammonia, or protons to H(2). Hydrogen production was the result of adding hydrogenase from Chlamydomonas reinhardii to the chloroplast preparation. The predicted stoichiometry of 1 PGA:1 O(2) in the absence of and 2 PGA:1 O(2) in the presence of catalase was observed indicating H(2)O(2) as the end product of O(2) reduction. The predicted stoichiometry of 3 PGA:1 nitrite:1 ammonia was also observed. A scheme is presented to account for a sustained generation of NADP and ATP necessary for the dissimilation of starch in the darkened chloroplast. The unifying term chloroplast respiration is introduced to account for those reactions in which reduced ferredoxin interacts with physiological acceptors other than NADP or nitrite, hydrogen, or O(2) respiration when nitrite, protons, or O(2) is the ultimate electron acceptor.

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PubMed1982

Oxidation of NAD(P)H in a Reconstituted Spinach Chloroplast Preparation Using Ascorbate and Hydrogen Peroxide.

The conversion of fructose-1,6-bisphosphate to glycerate-3-phosphate (PGA) was studied in a reconstituted spinach (Spinacia oleracea L.) chloroplast preparation to determine whether a chloroplast-localized NAB(P)H-oxidizing system (Kow, Smyth, Gibbs 1982 Plant Physiol 69: 72-76 with substrates of ascorbate, NAD(P)H, and H(2)O(2) could serve as a coupling enzyme in the recycling of NAD(P)H. The rate of PGA formation was monitored as an indicator of NAD(P) generation. With NAD as a cofactor, ascorbate enhanced PGA formation, and an additional increase resulted upon addition of glucose-glucose oxidase, a H(2)O(2)-generating enzyme. This increase in PGA formation due to H(2)O(2) was eliminated by the addition of catalase. With NADP and ferredoxin as cofactors, the recycling of NADP apparently was catalyzed both by ferredoxin-NADP reductase coupled to O(2) and by the NAD(P)H-oxidizing system.It was concluded that the oxidation of NAD(P)H by a system using ascorbate and H(2)O(2) can serve as a means of recycling NAD(P)H but that another reaction involving ascorbate and NAD(P)H may also function in the spinach chloroplast.

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PubMed1982

H(2) and CO(2) Evolution by Anaerobically Adapted Chlamydomonas reinhardtii F-60.

Using manometric and enzymic techniques, H(2) and CO(2) evolution in darkness and light has been studied in the green alga Chlamydomonas reinhardtii F-60. F-60 is a mutant strain characterized by an incomplete photosynthetic carbon reduction cycle but an intact electron transport chain.In the dark, starch was broken down, and H(2) and CO(2) was released. The uncoupler, carbonyl cyanide m-fluorophenylhydrazone with an optimum concentration of 5 to 10 micromolar, increased the rate of CO(2) release and starch breakdown but depressed H(2) formation. It was suggested that carbonyl cyanide m-fluorophenylhydrazone increased the rate of starch breakdown by making the chloroplast membrane permeable to H(+), removing a rate-limiting step, and leading to an altered fermentative pattern.Photoevolution of H(2) and CO(2), but not starch breakdown, was stimulated by acetate. Maximum stimulation occurred at concentrations from 1 to 10 millimolar. Carbonyl cyanide m-fluorophenylhydrazone stimulated starch breakdown and CO(2) and H(2) release in the light, but not to the extent of acetate. Inasmuch as the uptake and subsequent metabolism of acetate required ATP, it was suggested that acetate, like carbonyl cyanide m-fluorophenylhydrazone, stimulated H(2) photoproduction by removing ATP which limited the sequence of reactions. The contribution of photosystem II to the photoproduction of H(2), as judged from the effect of 10 micromolar 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, was at least 80%.CO(2) photoevolution increased linearly with time, but H(2) photoevolution occurred in two phases: a rapid initial phase followed by a second slower phase. The rate of H(2) release increased hyperbolically with light intensity, but the rate of CO(2) production tended to level off and decrease with increasing light intensity, up to 145 watts per square meter. It was proposed that a changing CO(2) and H(2) ratio is the result of interaction between the carbon and hydrogen metabolism and the photosynthetic electron transport chain.

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PubMed1982

Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : generation and use of reducing power.

The effect of increasing assay medium sorbitol concentration from 0.33 to 1.0 molar on the photosynthetic reactions of intact and broken spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts was investigated by monitoring O(2) evolution supported by the addition of glyceric acid 3-phosphate (PGA), oxaloacetic acid (OAA), 2,5-dimethyl-p-benzoquinone, and 2,6-dichlorophenolindophenol or as O(2) uptake with methyl viologen as acceptor.Uncoupled 2,6-dichlorophenolindophenol-supported whole chain electron transport (photosystems I and II) was inhibited from the 0.33 molar rate by 14% and 48.6% at 0.67 and 1.0 molar sorbitol in the intact chloroplast and by only 0.4% and 25.0% in the broken chloroplast preparation. Whole chain electron flow from water to other oxidants (OAA, methyl viologen) was also inhibited at increased osmoticum in intact preparations while electron flow from water to methyl viologen, ferricyanide, and NADP in broken preparations did not demonstrate the osmotic response. Electron transport to 2,5-dimethyl-p-benzoquinone (photosystem II) from H(2)O and to methyl viologen (photosystem I) from 3,3'-diaminobenzidine were found to be unaffected by osmolarity in both intact and broken preparations.The stress response was more pronounced (26-38%) with PGA as substrate in the presence of 0.67 molar sorbitol than the inhibition found with uncoupled and coupled linear electron flow. In addition, substrate availability and ATP generated by cyclic photophosphorylation evaluated by addition of Antimycin A were found not to be mediating the full osmotic inhibition of PGA-supported O(2) evolution. In a reconstituted (thylakoids plus stromal protein) chloroplast system to which a substrate level of PGA was added, O(2) evolution was only slightly (7.8%) inhibited by increased osmolarity (0.33-0.67 molar sorbitol) indicating that the level of osmotic inhibition above that contributed by adverse effects on electron flow can be attributed to the functioning of the photosynthetic carbon reduction cycle within the intact chloroplasts.

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PubMed1982

Hydrogen peroxide synthesis in isolated spinach chloroplast lamellae : an analysis of the mehler reaction in the presence of NADP reduction and ATP formation.

Light-dependent O(2) reduction concomitant with O(2) evolution, ATP formation, and NADP reduction were determined in isolated spinach (Spinacia oleracea L. var. America) chloroplast lamellae fortified with NADP and ferredoxin. These reactions were investigated in the presence or absence of catalase, providing a tool to estimate the reduction of O(2) to H(2)O(2) (Mehler reaction) concomitant with NADP reduction. In the presence of 250 micromolar O(2), O(2) photoreduction, simultaneous with NADP photoreduction, was dependent upon light intensity, ferredoxin, Mn(2+), NADP, and the extent of coupling of phosphorylation to electron flow.In the presence of an uncoupling concentration of NH(4) (+), saturating light intensity (>500 watts/square meter), saturating ferredoxin (10 micromolarity) rate-limiting to saturating NADP (0.2-0.9 millimolarity), and Mn(2+) (50-1000 micromolarity), the maxium rates of O(2) reduction were 13-25 micromoles/milligram chlorophyll per hour, while concomitant rates of O(2) evolution and NADP reduction were 69 to 96 and 134 to 192 micromoles/milligram chlorophyll per hour, respectively. Catalase did not affect the rate of NADPH or ATP formation but decreased the NADPH:O(2) ratios from 2.3-2.8 to 1.9-2.1 in the presence of rate-limiting as well as saturating concentrations of NADP.Photosynthetic electron flow at a rate of 31 micromoles O(2) evolved/milligram chlorophyll per hour was coupled to the synthesis of 91 micromoles ATP/milligram chlorophyll per hour, while the concomitant rate of O(2) reduction was 0.6 micromoles/milligram chlorophyll per hour and was calculated to be associated with an apparent ATP formation of only 2 micromoles/milligram chlorophyll per hour. Thus, electron flow from H(2)O to O(2) did not result in ATP formation significantly above that produced during NADP reduction.

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PubMed1982

Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : site-specific inhibition of the photosynthetic carbon reduction cycle.

The effects of reduced osmotic potential on the photosynthetic carbon reduction cycle were investigated by monitoring photosynthetic processes of spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts exposed to increased assay medium sorbitol concentrations. CO(2) assimilation was found to be inhibited at 0.67 molar sorbitol by about 60% from control rates at 0.33 molar sorbitol. This level of stress inhibition was greater than that affecting the reductive phase of the cycle; glycerate 3-phosphate reduction was inhibited at 0.67 molar by 27 to 40%. Sorbitol (0.67 molar) inhibited the rate of O(2) evolution at saturating and limiting concentrations of NaHCO(3), and extended the lag phase of O(2) evolution. This indicated that factors which are rate-limiting to the photosynthetic process are adversely affected by reduced osmotic potential.Analysis of photosynthetic products following CO(2) fixation in 0.33 molar sorbitol and 0.67 molar sorbitol indicated that reduced osmotic potential facilitated increases in the levels of fructose 1,6-bisphosphate and triose phosphates with reductions in glucose 6-phosphate and fructose 6-phosphate, implicating fructose 1,6-bisphosphatase as a site of osmotic stress. Osmotic inhibition of the reductive portion (glycerate 3-phosphate to triose phosphate) of the photosynthetic carbon reduction cycle was partially attributed to feedback inhibition by the product, triose phosphate, on glycerate 3-phosphate reduction. A saturating concentration of ribose 5-phosphate partially overcame osmotic inhibition of CO(2)-supported O(2) evolution, indicating another but apparently less severe site of stress inhibition in the sequence of ribose 5-phosphate to glycerate 3-phosphate.

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PubMed1982

Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts.

Photoassimilation of (14)CO(2) by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO(2) assimilation occurred.The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator.

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PubMed1981

Kinetics of the Oxyhydrogen Reaction in the Presence and Absence of Carbon Dioxide in Scenedesmus obliquus.

The oxyhydrogen reaction in the presence and absence of CO(2) was studied in H(2)-adapted Scenedesmus obliquus by monitoring the initial rates of H(2), O(2), and (14)CO(2) uptake and the effect of inhibitors on these rates with gas-sensing electrodes and isotopic techniques. In the presence of 0.02 atmosphere O(2), the pH(2) was varied from 0 to 1 atmosphere. Whereas the rate of O(2) uptake increased by only 30%, the rate of H(2) uptake increased severalfold over the range of pH(2) values. At 0.1 atmosphere H(2) and 0.02 atmosphere O(2), rates for H(2) and O(2) uptake were between 15 and 25 micromoles per milligram chlorophyll per hour. As the pH(2) was changed from 0 to 1 atmosphere, the quotient H(2):O(2) changed from 0 to roughly 2. This change may reflect the competition between H(2) and the endogenous respiratory electron donors. Respiration in the presence of glucose and acetate was also competitive with H(2) uptake. KCN inhibited equally respiration (O(2) uptake in the absence of H(2)) and the oxyhydrogen reaction in the presence and absence of CO(2). The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone accelerated the rate of respiration and the oxyhydrogen reaction to a similar extent. It was concluded that the oxyhydrogen reaction both in the presence and absence of CO(2) has properties in common with components of respiration and photosynthesis. Participation of these two processes in the oxyhydrogen reaction would require a closely linked shuttle between mitochondrion and chloroplast.

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PubMed1981

In vivo enamel fluoride uptake from and caries inhibition by topical fluoride agents.

Acidulated phosphate fluoride (APF), Duraphat or Fluor Protector, was applied to the molar teeth of rats fed a cariogenic diet. The first maxillary molar teeth were subjected to a microbiopsy procedure for fluoride analysis and the mandibular molars scored for caries. All three topical fluoride agents produced a significant increase in the fluoride content of the outermost 5.0 micron of enamel. Only APF produced a significant reduction in caries incidence at all sites.

Acidulated Phosphate Fluoride↗
PubMed1980

Influence of Hydrogen Peroxide upon Carbon Dioxide Photoassimilation in the Spinach Chloroplast: I. HYDROGEN PEROXIDE GENERATED BY BROKEN CHLOROPLASTS IN AN "INTACT" CHLOROPLAST PREPARATION IS A CAUSAL AGENT OF THE WARBURG EFFECT.

Photosynthesis and the Warburg effect (O(2) inhibition of photosynthesis) were evaluated in preparations of intact spinach chloroplasts enriched with varying amounts of lysed chloroplasts. Increasing the ratio of broken to intact plastids resulted in decreased rates of CO(2) assimilation.Hydrogen peroxide when added at 10 or more micromolar also inhibited photosynthesis in these preparations. Inhibition of the photosynthetic rate by both factors was eliminated by addition of catalase. These findings indicate that H(2)O(2) presumably generated by the broken chloroplasts was the causal agent of this inhibition.The Warburg effect also became more pronounced by increasing the level of broken to intact chloroplasts. This effect was examined as a function of added catalase, pH, and O(2) concentration. At 21% O(2) and 0.44 to 0.68 millimolar CO(2), catalase relieved the effect almost completely at pH 7.5, but at pH 8.3, the rate was restored only to about half or less of the control. At pH 7.6, 0.44 millimolar CO(2), and 100% O(2), the effect was only slightly overcome by catalase.A rise in glycolate synthesis by the isolated spinach chloroplast has been shown previously to be coupled to an increase in pH and O(2) (Kow, Robinson, Gibbs 1977 Plant Physiol 60: 492-495; Robinson, Gibbs, Cotler 1977 Plant Physiol 59: 530-534). At 21% O(2), glycolate synthesis was not affected by the addition of catalase at pH 7.5 or 8.3. It is proposed that at 21% O(2) and without some means of removing H(2)O(2), that portion of the Warburg effect attributed to glycolate synthesis has been overestimated at pH values in the order of 7.5. In contrast, that portion of the Warburg effect which was, at alkaline pH, insensitive to catalase represented the stress placed upon the photosynthetic carbon reduction cycle which resulted from an enhanced synthesis of glycolate. At 100% O(2) aeration and pH 7.5 to 8.5, the Warburg effect may also represent O(2)-mediated inhibition of a Calvin cycle enzyme within the intact plastid.

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PubMed1980

Carbon dioxide fixation and related properties in sections of the developing green maize leaf.

Light and dark (14)CO(2) assimilation, pulse-chase ((14)CO(2) followed by (12)CO(2)) labeling experiments both in the light and in the dark, photorespiratory activity and some enzymes (ribulose 1,5-bisphosphate (RuBP) carboxylase, phosphoenolpyruvate (PEP) carboxylase, and NADP-malic enzyme) were followed in sections of 2.5 centimeters from the base (younger tissue) to the tip (oldest tissue) of the green maize leaf. Tissue was taken from the third leaf of 12- to 16-day-old plants consisting of sections 0 to 2.5 centimeters (base), 4.5 to 7.0 centimeters (center) and 9.0 to 11.5 centimeters (top) measured from the base. Some of these properties were also determined in the intact leaves of 4-day-old maize plants.Electron microscopy indicated a Kranz anatomy in all sections. Differentiation into mesophyll granal chloroplasts and bundle sheath agranal chloroplasts had taken place only in the center and top pieces.All of the sections contained PEP carboxylase, RuBP carboxylase, and NADP-malic enzyme. The ratio of PEP:RuBP carboxylase increased from 3.03 (top) to 4.66 (base) whereas the PEP carboxylase:NADP-malic enzyme ratio rose from 2.87 (top) to 9.57 (base).Under conditions of light or dark, the majority of the newly incorporated (14)CO(2) was found in malate and aspartate in all sections and in 4-day-old leaves. The (14)C-labeling pattern typical of C(4) plants was present in the center and top sections and to a lesser extent in the 4-day-old leaves. In the base tissue, the percentage of radioactivity in malate and aspartate remained relatively constant both during photosynthesis and pulse-chase experiments. In contrast, radioactivity in glycerate-3-phosphate decreased with time coupled to an increase in sugar phosphates. To account for the isotopic pattern in the base tissue, parallel fixation by PEP carboxylase and RuBP carboxylase was proposed with the photosynthetic carbon reduction cycle functioning to some extent independently within the bundle sheath chloroplasts. The apparent lack of cooperation between the mesophyll and bundle sheath cells may have been due to inadequate levels of NADP-malic enzyme required for shuttling carbon as CO(2) from the PEP carboxylase products to the Calvin cycle.

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PubMed1979

Inactivation of Hydrogenase in Cell-free Extracts and Whole Cells of Chlamydomonas reinhardi by Oxygen.

O(2) irreversibly inactivates hydrogenase from Chlamydomonas reinhardi. The mechanism for the inactivation involves the reaction of one molecule of hydrogenase with one molecule of O(2) (or two oxygen atoms) in the transition complex of the rate-limiting step. The second order rate constant for this reaction is 190 atmospheres(-1) minute(-1) (1.4 x 10(5) molar(-1) minute(-1)). At levels above 0.01 atmosphere O(2), the increased numbers of O(2) molecules may compete for the site of inactivation hindering the proper orientation for inactivation of any one O(2) molecule and resulting in lowered rates of inactivation.CO is a reversible inhibitor of hydrogenase acting competitively against H(2). The K(i) for CO is 0.0010 atmosphere. CO antagonizes O(2) inactivation. In a period when complete inactivation by O(2) would usually occur, the presence of CO greatly reduces the inactivation rate.After 3 hours of adaptation in whole cells, the presence of H(2) lowers the rate of deadaptation of hydrogenase. Inasmuch as H(2) promotes increased O(2) uptake the cellular concentration of O(2) is likely to be lower. After 48 hours of adaptation O(2) uptake is reduced even when H(2) is present and the pattern of deadaptation under O(2) with and without H(2) and CO is qualitatively the same as observed for the inactivation of cell-free hydrogenase. The mechanism of inactivation of cell-free hydrogenase by O(2) may be the same as the mechanism for loss of hydrogenase during deadaptation in whole algal cells.

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