The Relation Between Ion Absorption and Protein Synthesis in Beet Disks

Disks of red beet storage tissue were incubated under asepticconditions permitting the development of various metabolic processescommonly associated with aged disks, and the effects of chloramphenicoland puromycin on protein synthesis, on the development of invertaseactivity and ion absorption capacity, and on ion absorptionper se were determined. Low concentrations of chloramphenicoland puromycin inhibit the development of ion absorption capacitybut stimulate invertase development and protein synthesis, whilehigher concentrations inhibit all three processes. In contrastion absorption itself is unaffected by puromycin, but is sensitiveto quite low concentrations of chloramphenicol The D-threo andL-threo isomers of chloramphenicol have sharply contrasted effectson the development, as distinct from the utilization, of ionabsorption capacity. The D isomer inhibits the development ofion absorption capacity more effectively than the L isomer whichin turn inhibits absorption more effectively than the D isomer. A reappraisal is made of the hypothesis that ion absorptionis directly linked with protein turnover and to account forthe results a model is proposed in which D-threo-chloramphenicolis active both as an uncoupler of oxidative phosphoryalationand as an inhibitor of protein synthesis, while L-threo-chloramphenicolacts only in the former capacity and puromycin only in the latter.It is concluded that the inhibition of ion uptake by chloramphenicolcannot be attributed to a contemporaneous effect on proteinsynthesis. However, the results are consistent with the involvementof ATPase proteins in ion uptake.     

Plastid development in primary leaves of Phaseolus vulgaris: The light-induced development of the chloroplast cytochromes

Summary Etioplasts obtained from the primary leaves of dark-grown bean plants contained cytochromes f, b-559_LP and b-563 in a molar ratio of approximately 1.0:2.0:1.5. On illumination of the plants there was a lag of between 10 and 15 h before these cytochromes increased in amount, but after 48 h they had increased from 6- to 10-fold on a per plastid basis. The presence of cytochrome b-559_HP in the plastids was first detected after 15 h of illumination, which coincided with the commencement of grana formation and the onset of a number of photosynthetic reactions in the greening leaves. After 48 h of illumination the molar ratio for cytochromes f, b-559_HP, b-559_LP and b-563 was 1.0:1.2:2.8:2.6.Agranal chloroplasts formed by the exposure of dark-grown plants to intense light flashes contained high amounts of cytochromes f, b-559_LP and b-563 but cytochrome b-559_HP could not be detected.As the light-induced formation of cytochromes f, b-559_LP and b-563 was substantially inhibited by D-threo chloramphenicol, but not by the L-threo isomer, it seems likely that their formation was dependent on 70S ribosomes. Both chloramphenicol isomers gave plastids which lacked cytochrome b-559_HP.     

Subcellular Localization of a UDP-Glucose:Aldehyde Cyanohydrin beta-Glucosyl Transferase in Epidermal Plastids of Sorghum Leaf Blades

Epidermal and mesophyll protoplasts, prepared from leaf blades of 6-day-old light-grown Sorghum bicolor seedlings were separated by differential sedimentation and assayed for a number of enzymes. The epidermal protoplasts contained higher levels of NADPH-cytochrome c reductase (EC 1.6.2.4), triose phosphate isomerase (EC 5.3.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.31), and a UDP-glucose:cyanohydrin β-glucosyl transferase (EC 2.4.1.85), but lower levels of NADP⁺ triosephosphate dehydrogenase (EC 1.2.1.13) than did mesophyll protoplasts. When protoplast preparations were lysed and applied to linear sucrose density gradients, triosephosphate isomerase was found to be present in epidermal plastids. A significant fraction (41%) of the glucosyl transferase activity was also associated with the epidermal plastids.     

Regulation of resynthesis of the CO2-acceptor in photosynthesis. Feedback inhibition of transketolase

The presence of ribulose-5-phosphate epimerase (EC 5.1.3.1, epimerase) in samples of ribose-5-phosphate isomerase (EC 5.3.1.6, isomerase) obtained from spinach ( Spinacea aleracea L. cv. Bloomsdale Long Standing) was determined using (i) a sampling procedure which measured the quantity of xylulose-5-phosphate formed in the reaction mixture and (ii) a coupled enzyme assay in which the rate of oxidation of NADH was measured after establishing steady-state concentrations of xylulose-5-phosphate, dihydroxacetonephosphate and glyceraldehyde-3-phosphate by the action of epimerase, transketolase (EC 2.2.1.1), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). In preparations where the ratio of isomerase to epimerase activities was less than 100, both assay procedures yielded valid indications of epimerase activity. The steady-state assay system was found, however, to seriously underestimate epimerase activity in enzyme preparations which were enriched in isomerase. Cross plots of epimerase activity determined by the sampling and steady-state procedures demonstrated that an inhibitor of the coupling enzyme mixture was formed in the presence of high relative concentrations of the isomerase. The inhibited coupling enzyme mixture was fully active with glycer-aldehyde-3-phosphate. Inhibition of the coupling enzyme mixture was attributed to transketolase. Feedback inhibition of transketolase is proposed to be of physiological significance in the photosynthesis cycle, operating to restrict resynthesis of CO₂-acceptor under conditions where high steady-state concentrations of the intermediates of the photosynthesis cycle are maintained.     

Inhibition of auxin-induced cell expansion in Jerusalem artichoke tuber slices by low concentrations of chloramphenicol

Summary D-threo chloramphenicol (CAP) at 5×10^-5 M, given continuously during a 24-hr aging period and subsequent post-age treatment with 2,4-dichlorophenoxyacetic acid (2,4-D)±kinetin markedly depressed cell expansion in Jerusalem artichoke (Helianthus tuberosus) tuber slices. Both the rate and total amount of expansion were reduced. An inhibitory effect of CAP could be detected at a concentration as low as 6.2×10^-6M with 2,4-D alone and 1.6×10^-6 M with 2,4-D+kinetin. CAP also inhibited if given with 2,4-D to unaged tissue, and partially inhibited growth of aged tissue when supplied only during or only after aging. Expansion was inhibited when IAA was used in place of 2,4-D. Growth of tissue slices free of detectable bacteria was depressed by CAP, eliminating a possible indirect action of the antibiotic through inhibition of beneficial bacteria. CAP also prevented appearance of pink and brown pigments which normally occur in association with auxin-treated tissues. L-threo CAP did not inhibit growth or pigment formation. Cell division in the tuber slices was not inhibited, and was possibly even stimulated, by D-threo CAP, even at a concentration of 2×10^-4 M. It is concluded that the use of CAP for bacterial control in plant cultures can be hazardous and needs careful checking. Presumably the inhibitory action of CAP results from inhibition of growth-dependant protein metabolism in mitochondria and/or plastids which occurs both during aging and post-aging growth. Partial suppression of metabolic changes during aging would maintain the tissue in a state favouring relatively high mitotic activity and slow growth in response to auxin.     

Adaptive Use of N2-Dimethyl-Substituted Pterins by Cultures of Crithidia fasciculata1

The compound 6-(L-erythro-1,2′,3′-trihydroxypropyl)pterin, at a concentration of 50 pg/ml (“L-erythro-neopteria”), supports half-maximal growth of Crithidia fasciculata; biopterin at a concentration of 30 pg/ml is shown to yield similar growth. N²-dimethyl-6-(L-erythro-1′,2′,3′-trihydroxypropyl)pterin (A) was inactive even at 100 ng/ml. Synergism was observed with the N²-dimethylamino derivative (A) in the presence of suboptimal biopterin, its activity then being of the order of L-erythro-neopterin. In contrast, the stereoisomeric N²-dimethyl-6-(D-erythro-1′,2′,3′-trihydroxypropyl)pterin (“dimethyl-D-erythro-neopterin”) and its 3′-mono-phosphate only slightly enhanced growth under similar conditions but both threo-isomers had no supplementary activity. Biopterin-induced growth was slowed by 6-(D-erythro1′,2′,3′-trihydroxypropyl)pterin (D-neopterin); the threo-isomers had no such effect. An adaptive demethylation capacity by growing cultures and competition of biopterin uptake by D-neopterin seems likely. The report of the occurrence in Euglena of N₂-dimethyl-6-(L-threo-1′,2′,3′-trihydroxypropyl)pterin and its 3′-mono-phosphate adds further interest to our observations.     

Isozymes of the glycolytic and pentose-phosphate pathways in storage tissues of different oilseeds

Isozymes of hexose-phosphate isomerase (HPI; EC 5.3.1.9), pyruvate kinase (PK; EC 2.7.1.40) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) have been detected in the developing cotyledons of soybean (Glycine max (L.) Merr.), safflower (Carthamnus tinctorius L.) and sunflower (Helianthus annuus L.). In each seed there are two isozymes each of PK and HPI. The isozyme patterns of 6PGDH are more complex: soybean has two forms of the enzyme, safflower three, and sunflower six. In each tissue, at least 25% of the activity of each of the three enzymes is in the plastids. This supports the proposal that the glycolytic and pentose-phosphate pathways are operating in the plastids and that the plastids are the site of long-chain fatty-acid biosynthesis in developing oilseeds.Abbreviations HPI hexose-phosphate isomerase - 6PGDH 6-phosphogluconate dehydrogenase - PK pyruvate kinase     

The Development of Activities of Some Enzymes Concerned with Glycollate Metabolism in Greening Bean Leaves

The activities of phosphoglycollate phosphatase (EC 3.1.3.18 [EC] ),glycollate oxidase (EC 1.1.3.1 [EC] .). catalase (EC 1.11.1.6 [EC] ), theperoxisomal NADH-glyoxylate reductase (EC 1.1.1.26 [EC] ) which isconsidered to function as a hydroxypyruvate reductase in theperoxisomes, and the chloro-plastic NADPH-dependent glyoxylatereductaae, have been measured in extracts prepared from 14-d-olddark-grown bean leaves during the course of their greening inresponse to exposure to continuous illumination. All of theenzymes were found in the dark-grown leaves and on a per-leafbasis the activities increased from 6- to 12-fold with the exceptionof a 2–3-fold increase of NADPH-dependent glyoxylate reductaseduring 96-h greening, while the activities either remained constantor declined during similar periods in darkness. Initial lagperiods were evident before the illumination-induced increasesin enzyme activities. As D-threo-chloramphenicol did not affectthe increase in activity of any of these enzymes it would appearthat the increases were in no way dependent on protein synthesisby 70S ribosomes, or on the development of photosynthetic activity.     

NAD-dependent malate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase isoenzymes play an important role in dark metabolism of various plastid types

Chloroplasts isolated from spinach (Spinacia oleracea L.) leaves and green sweet-pepper (Capsicum annuum L. var. grossum (L.) Sendt.) fruits contain NADP-dependent malate dehydrogenase (MDH; EC 1.1.1.82) and the bispecific NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13). The NADP-dependent MDH and GAPDH are activated in the light, and inactive in the dark. We found that chloroplasts possess additional NAD-dependent MDH activity which is, like the NAD-dependent GAPDH activity, not influenced by light. In heterotrophic chromoplasts from red sweet-pepper fruits, the NADP-dependent MDH and the NAD(P)-GAPDH isoenzymes disappear during the developmental transition and only NAD-specific isoforms are found. Spinach chloroplasts contain both NAD/H and NADP/H at significant concentrations. Measurements of the pyridine dinucleotide redox states, performed under dark and various light conditions, indicate that NAD(H) is not involved in electron flow in the light. To analyze the contribution of NAD(H)-dependent reactions during dark metabolism, plastids from spinach leaves or green and red sweet-pepper fruits were incubated with dihydroxyacetone phosphate (DHAP). Exogenously added DHAP was oxidized into 3-phosphoglycerate by all types of plastids only in the presence of oxaloacetate, but not with nitrite or in the absence of added electron acceptors. We conclude that the NAD-dependent activity of GAPDH is essential in the dark to produce the ATP required for starch metabolism; excess electrons produced during triose-phosphate oxidation can selectively be used by NAD-MDH to form malate. Thus NADPH produced independently in the oxidative pentose-phosphate pathway will remain available for reductive processes inside the plastids. Received: 2 July 1997 / Accepted: 20 October 1997     

Changing Activities During Senescence and Sites of Synthesis of Photosynthetic Enzymes in Leaves of the Labiate, Perilla frutescens (L.) Britt.

The changing activities of several regulatory enzymes of thephotosynthetic carbon reduction cycle accompanying ageing ofthe third leaf pair of Perilla frutescens fall into two distinctcategories: firstly, enzymes which reach maximum activity priorto the completion of leaf expansion followed by a rapid decline(phosphoribulokinase, ribulose-l,5-diphosphate carboxylase,and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase);secondly, enzymes which maintain high activity beyond completionof leaf expansion and decline only at a late stage in senescence(phosphoglycerate kinase, NADH-linked glyceraldehyde-3-phosphatedehydrogenase, alkaline fructose-1,6-diphosphatase, and ribose-5-phosphateisomerase). The introduction of the ribosomal inhibitors D-threochloramphenicol, lincomycin, D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, and cycloheximide to illuminated, detached shootsystems of 2-d-darkened Perilla plants has suggested that synthesisof ribulose diphosphate carboxylase, NADPH-dependent glyceraldehyde-3-phosphatedehydrogenase, and possibly phosphoribulokinase, is mediatedby 70 S-based chloroplastic ribosomes. A chloroplastic siteof synthesis of these three photosynthetic enzymes is consistentwith their early deterioration during leaf ageing.     

The location of nitrite reductase and other enzymes related to amino Acid biosynthesis in the plastids of root and leaves

Density gradient separation of plastids from leaf and root tissue was carried out. The distribution in the gradients of the activity of the following enzymes was determined: nitrite reductase, glutamine synthetase, acetolactate synthetase, aspartate aminotransferase, catalase, cytochrome oxidase, and triosephosphate isomerase. The distribution of chlorophyll was followed in gradients from leaf tissue. The presence of plastids that have retained their stroma enzymes was denoted by a peak of triosephosphate isomerase activity. Coincidental with this peak were bands of nitrite reductase, acetolactate synthetase, glutamine synthetase, and aspartate aminotransferase activity. The results suggest that most, if not all, the nitrite reductase and acetolactate synthetase activity of the cell is in the plastids. The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephosphate dehydrogenase activity in the cell. Some evidence was obtained for low levels of glutamate dehydrogenase activity in chloroplasts.     

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

Studies on {partial}-amino levulinic acid deydratase in radish cotyledons during chloroplast development

The subcellular localization and biosynthetic site of 8-aminolevulinic acid dehydratase [EC 4.2.1.24 [EC] , ALAD] were investigatedin relation to chloroplast development in radish cotyledons. ALAD was mainly located in the chloroplasts and cytoplasm. Mostof the ALAD in the chloroplasts was readily released by hypotonicshock. The enzyme was also found in the proplastids of etiolatedcotyledons. The normal increase in the activity of ALAD in the chloroplastsas well as the cytoplasm was inhibited by cycloheximide butunaffected by D-threo chloramphenicol and kanamycin during thegreening of radish cotyledons. We concluded that the ALAD inboth the cytoplasm and chloroplasts was synthesized on the cytoplasmic80S-ribosomes. This suggests that the ALAD formed on the 80S-ribosomesmight be incorporated into chloroplasts during their development. When etiolated radish seedlings were illuminated, ALAD in boththe cytoplasm and chloroplasts increased up to the point ofthe full development of the chloroplasts, and thereafter itdecreased. (Received August 20, 1975; )     

Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts

A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T₇ promoter. Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts. Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes. Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants.Abbreviations RPE ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - PGK phosphoglycerate kinase - FBP fructose-1,6-bisphosphatase - SBP sedoheptulose-1,7-bisphosphatase - OPPP oxidative pentose phosphate pathway - Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphosphate aldolase - IPTG isopropyl -d-thiogalactoside - TPI triosephosphate isomerase     

Glucose metabolism in Clostridium sporogenes and Clostridium sticklandii bacteria

Clostridium sporogenes 272 has a high rate of glucose fermentation. Its cell-free extract contains all glycolytic enzymes catalysing glucose degradation to pyruvate and shows the phosphoroclastic activity. C. sticklandii CSG has a low rate of glucose fermentation. Hence, the activity of the following enzymes is lower in this organism comparing to C. sporogenes: phosphohexoisomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12). Moreover, it is possible that the system of glucose transport into the cell is damaged in C. sticklandii.     

Measurement of the inorganic pyrophosphate in tissues of Pisum sativum L.

Purified pyrophosphate: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) was used to measure the inorganic pyrophosphate in unfractionated extracts of tissues of Pisum sativum L. The fructose 1,6-bisphosphate produced by the above enzyme was measured by coupling to NADH oxidation via aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). Amounts of pyrophosphate as low as 1 nmol could be measured. The contents of pyrophosphate in the developing embryo of pea, and in the apical 2 cm of the roots, were appreciable; 9.4 and 8.9 nmol g^-1 fresh weight, respectively. The possibility that pyrophosphate acts in vivo as an energy source for pyrophosphate: fructose 6-phosphate 1-phosphotransferase and for UDPglucose pyrophosphorylase (EC 2.7.7.9) is considered.     

Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source

Activities of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.3) were measured in cotyledons of sunflower (Helianthus annuus L. cv Peredovic) seedlings during germination and early growth under various external nitrogen sources. The presence of NO ₃ ^- in the medium promoted a gradual increase in the levels of NR and NiR activities during the first 7 d of germination. Neither NR nor NiR activities were increased in a nitrogen-free medium or in media with either NH ₄ ⁺ or urea as nitrogen sources. Moreover, the presence of NH ₄ ⁺ did not abolish the NO ₃ ^- -dependent appearance of NR and NiR activities. The increase of NR activity was impaired both by cycloheximide and chloramphenicol, which indicates that both cytoplasmic 80S and plastidic 70S ribosomes are involved in the synthesis of the NR molecule. By contrast, the appearance of NiR activity was only inhibited by cycloheximide, indicating that NiR seems to be exclusively synthesized on the cytoplasmic 80S ribosomes. Glutamine-synthetase activity was also strongly increased by external NO ₃ ^- but not by NH ₄ ⁺ or urea. The appearance of GS activity was more efficiently suppressed by cycloheximide than chloramphenicol. This indicates that GS is mostly synthesized in the cytoplasm. The cotyledons of the dry seed contain high levels of GDH activity which decline during germination independently of the presence or absence of a nitrogen source. Cycloheximide, but not chloramphenicol, greatly prevented the decrease of GDH activity.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

Enzymes of starch and sugar phosphate metabolism in achlorophyllous ribosome-deficient plastids from high-temperature-grown rye leaves

Several enzymes of non–photosynthetic sugar phosphate and starch metabolism were measured in gradient–purified chloroplasts from normal rye leaves ( Secale cereale L. cv. Halo) grown at 22°C and in the non-photosynthetic plastids isolated from 70S ribosome-deficient rye leaves grown at a non–permissive elevated temperature of 32°C. Activities of the enzymes phosphoglycerate kinase (EC 2.7.2.3), hexokinase (EC 2.7.1.1), phosphoglucose isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate de-hydrogenase (EC 1.1.1.46), ADPglucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21), and phosphorylase (EC 2.4.1.1) were present in ribosome-deficient plastids from 32°C-grown leaves indicating a cytoplasmic origin of the plastid-specific forms of these enzymes. While the photosynthetic marker enzyme NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) was considerably diminished, both the specific activities and the total activities per leaf of the plastid-specific forms of hexokinase, phosphoglucose isomerase and phosphoglucomutase were markedly increased in the ribosome–deficient plastids, relative to normal chloroplasts. The results demonstrate that after elimination of functional protein synthesis in the chloroplasts the supply of chloroplast–specific enzymes by the cytoplasm is not generally suppressed as observed for many enzymes and proteins involved in photosynthesis, but may even be increased in accord with changed metabolic demands.     

Changes in Enzyme Regulation during Growth of Maize: III. Intracellular Localization of Homoserine Dehydrogenase in Chloroplasts

Extracts of leaf tissue of Zea mays L. seedlings were fractionated on nonlinear sucrose gradients to separate subcellular organelles. Homoserine dehydrogenase (EC 1.1.1.3) was identified in those fractions containing intact chloroplasts, as judged by the presence of chlorophyll and triosephosphate isomerase activity. Neither enzyme activity was detected in fractions containing ruptured chloroplasts, mitochondria, or microbodies. Quantitative measurements of enzyme activity and chlorophyll, and electron microscopic analysis of plastid preparations support the conclusion that maize mesophyll chloroplasts contain a significant fraction of the total cellular content of homoserine dehydrogenase.     

Cloning of the amphibolic Calvin cycle/OPPP enzyme d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects

Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T₇ promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.Abbreviations R5P3E d-ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - PRK phosphoribulokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - FBP fructose-1,6-bisphophatase - FBP fructose 1,6-bisphosphate - G6PDH glucose-6-phosphate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - OPPP oxidative pentose phosphate pathway - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphophate aldolase - IPTG isopropyl -d-thiogalactoside - GST glutathione S-tranferase - PBS phosphate-buffered saline - TPI triosephosphate isomerase