W. Ronald Mills
Permanent URI for this collectionhttps://hdl.handle.net/10657.1/2408
Dr. Mills is a Professor of Biology and Chemistry at College of Science and Engineering in University of Houston-Clear Lake.
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Item Regulation of aspartate-family amino acid biosynthesis: studies using isolated pea chloroplasts(Kluwer Academic Publishers, 1993) Mills, RonaldItem Developmental variation in aspartate-family amino acid biosynthesis by isolated chloroplasts(Kluwer Academic Publishers, 1990) Mills, RonaldIn the last decade, it has become clear that the chloroplast is the main site, if not the sole site, for the biosynthesis of the nutritionally essential aspartate-derived amino acids (Fig. 1) in plant leaves [1,2]. For example, isolated intact chloroplasts carry out the light-driven synthesis of lysine, threonine, and isoleucine from labeled aspartic acid and malic acid [3]. This can be considered a photosynthetic process since ATP and NADPH produced in light are thought to be used directly (Fig. 1) to drive the synthetic reactions [3].Item DNA synthesis in chloroplasts. III. The DNA gyrase inhibitors nalidixic acid and novobiocin inhibit both thymidine incorporation into DNA and photosynthetic oxygen evolution in isolated chloroplasts(Journal of Experimental Botany, 1989) Mills, RonaldThe influence of two DNA gyrase inhibitors, nalidixic acid and novobiocin, on DNA synthesis in isolated pea chloroplasts was examined. Novobiocin at 1–5 mol m−3 markedly lowered [3H]thymidine incorporation into DNA (30–95% inhibition); while less effective, nalidixic acid at similar concentrations also diminished incorporation (25–35% inhibition). The inhibition of chloroplast DNA (ctDNA) biosynthesis by nalidixic acid and novobiocin was confirmed by autoradiography and densitometry. These data are consistent with the view that chloroplasts contain a DNA gyrase-like enzyme which is necessary for DNA replication. Despite this, interpretation of the results is not straightforward, as both nalidixic acid and novobiocin also inhibited photosynthetic activity. Each substance (at millimolar levels) reduced ferricyanide-dependent O2 evolution in isolated chloroplasts. However, at lower concentrations (0.05–0.3 mol m−3) they slightly enhanced photosynthetic electron flow; thus, these compounds may act as uncouplers of photophosphorylation as well as inhibitors of electron transport. Nalidixic acid and novobiocin at relatively low (0.1 mol m−3) concentrations also strongly reduced CO2-dependent O2 evolution (an index of CO2 photo-assimilation) in isolated plastids. Thus, caution must be exercised in assessing results from studies in which nalidixic acid and novobiocin are used with whole plants, cells, protoplasts or isolated chloroplasts.Item DNA synthesis in isolated chloroplasts(Cambridge University Press, 1988) Mills, RonaldItem DNA synthesis in chloroplasts. II. Developmental variation in TTP incorporation into DNA by plastid extracts(Journal of Experimental Botany, 1988) Mills, RonaldIncorporation of [3H]TTP into DNA by pea chloroplast extracts was highly dependent on the age of the tissue from which plastids were prepared. Catalytic activity was greatest in samples from 6- to 9-d-old plants; preparations from more mature tissues were much less effective. Moreover, activity was 3 to 10 times greater in younger tissues regardless of whether chlorophyll, protein or plastid number was used as the index of comparison. Enzymes from the first (oldest), second, third, and fourth (youngest) leaves of the same plants were also studied. Again, activity was 4 to 10 times greater in samples from the youngest tissues. When plastid extracts from older leaves were mixed with those from younger tissues, they did not reduce synthesis. Thus, the decline in activity does not appear to be due to the production of an inhibitor during plant development. One explanation for these data is that enzymes of ctDNA replication, such as DNA polymerase, vary in activity during leaf development; therefore changes in enzyme levels may be an important factor in controlling chloroplast DNA replication during development. We have also examined the incorporation of [3H]TTP into DNA by isolated intact pea chloroplasts; in general, labelled TTP was less readily incorporated into chloroplast DNA than was [3H]thymidine.Item DNA synthesis in chloroplasts. I. Characterization of and developmental variation in both light- and ATP-driven thymidine incorporation by isolated intact chloroplasts(Journal of Experimental Botany, 1988) Mills, RonaldIntact chloroplasts from young pea leaves were able to incorporate [3H]thymidine into DNA at relatively high rates (50 pmol mg-1 chlorophyll h-1 or more), using light as the sole energy source. The intact plastids were also able to synthesize DNA in darkness, but only if ATP and MgCl2 (MgATP) were both present. The rates of MgATP-driven assimilation in darkness were equal to or greater than light-driven activity. Neither light nor dithiothreitol pretreatments enhanced thymidine incorporation in darkness, suggesting that enzymes of chloroplast DNA (ctDNA) biosynthesis are not regulated via a thioredoxin-type system. Although exogenous nucleosides (other than [3H]thymidine) were not an absolute requirement, dramatically elevated rates of incorporation (over 300 pmol mg-1 chlorophyll h-1) were seen when adenosine, cytidine, guanosine and thymidine were supplied in combination (500 mmol m-3 each). Radiolabelled DNA synthesized by the isolated chloroplasts was prepared using a new heat extraction method. After digestion by restriction endonucleases, ctDNA synthesized in organello was found to give typical autoradiography patterns for chloroplast DNA. Exonuclease III studies suggested that 5% to 15% of the newly synthesized DNA might be in a closed circular form. MgATP-driven synthesis in darkness was highly age-dependent. Chloroplasts from young (6 to 8-d-old) plants, or alternatively the youngest leaves of more mature plants, were 4—10 times more active than those from older tissues. Although these data do not establish conclusively that replication-type synthesis was occurring in the isolated chloroplasts, they are consistent with this suggestion.Item Synthesis of aspartate-family amino acids(CRC Press, 1987) Mills, RonaldItem DNA biosynthesis in chloroplasts and its regulation: Studies on isolated chloroplasts and chloroplast extracts(Progress in Photosynthesis Research, 1987) Mills, RonaldDuring leaf development, chloroplasts are formed by the binary fission of pre-existing plastids (1); thus, replication of chloroplast DNA (ctDNA) must occur to maintain an adequate amount in each organelle. The levels of ctDNA in plastids vary during plant development (2); the fraction of chloroplast DNA increases during early shoot development then stabilizes thereafter. The number of genome copies per plastid also increases early, however, they reach a maximum then decline as tissues mature. Such variation in the levels of ctDNA suggests that the capacity of chloroplasts to replicate their DNA changes during development. Here, we describe the use of both isolated intact pea chloroplasts and plastid extracts to study developmental changes in ctDNA biosynthesis.Item Synthesis of branched chain amino acids(CRC Press, 1987) Mills, RonaldItem Purification of chloroplasts using silica-sols(Academic Press, 1987) Mills, RonaldThis chapter describes procedures for the purification of chloroplasts using silica sols. Silica sols have two major advantages as media for density gradients—namely, low viscosity and negligible osmotic potential, allowing the preparation of less damaging isoosmotic gradients and more rapid sedimentation at lower gravitational forces. Use of silica sol media can allow rapid separation of highly intact and physiologically active chloroplasts; however, it should be recognized that the quality of the final preparation would depend on the quality of the initial homogenate. Good chloroplasts cannot be resurrected from poor starting material, and appropriate recipes and procedures must be adapted to the plant material and its primary disruption to give optimum results. A variety of centrifugation procedures can be used with silica sols. The procedures described in the chapter use Percoll, as it is readily available and relatively free from toxic effects. Removal of silica sol may be advantageous if there is any possibility of an effect on physiological function, or interaction (e.g., precipitation or gelling of the sol) with added reagents.Item DNA biosynthesis in intact, isolated chloroplasts(Clarendon Press, 1984) Mills, RonaldItem ß- alanine metabolism and high salinity stress in the sea anemone, Bunodosoma cavernata(Journal of Comparative Physiology B, 1984) Mills, RonaldDuring high salinity stress, β-alanine accumulates to high levels in the sea anemone,Bunodosoma cavernata. Following a salinity increase from 26‰ to 40‰ β-alanine increased 28-fold from 1.5 to 41.9 μmoles/g dry weight. Both whole animal studies and experiments with cell free homogenates indicate that under high salinity conditions an increase in the rate of β-alanine synthesis from aspartic acid as well as a decrease in the rate of β-alanine oxidation are responsible for the observed accumulation of β-alanine. The rate of aspartic acid decarboxylation to β-alanine is about 3 times greater in anemones acclimated to 40‰ than for those in normal salinity water (26‰). β-alanine oxidation to CO2 and acetyl-CoA proceeds 2.5 to 3 times slower in high salinity adaptedB. cavernata than in those acclimated to normal salinity. There is always a rapid degradation of uracil to β-alanine, but this does not change with salinity.Item Light-driven DNA biosynthesis in isolated pea chloroplasts(FEBS Letters, 1983) Mills, RonaldIsolated chloroplasts from Pisum sativum leaves catalyze [3H]thymidine incorporation into acid-insoluble material using light as the sole energy source. Neither ribonucleotides, deoxyribonucleotides nor deoxyribonucleosides (other than [3H]thymidine) are required. However, it is necessary that the plastids be intact and photosynthetically competent, as little synthesis occurs in lysed preparations or in the presence of photophosphorylation inhibitors. Thymidine incorporation is markedly reduced by the DNA synthesis inhibitors rifampicin, nalidixic acid, ethidium bromide and N-ethylmaleimide. Plant age is an important factor, since rates of synthesis are 3–10-times higher in plastids isolated from young plants (6–8 days old) than in chloroplasts from older ones (9–14 days old). The maximum rates using plastids from young leaves of 60–70 pmd/mg chlorophyll/h are 30–60-times greater than those previously reported [1].Item The assimilation of nitrogen and the synthesis of amino acids in chloroplasts and blue-green bacteria(Elsevier, 1982) Mills, RonaldItem Amino acid biosynthesis in chloroplasts(Balaban International Science Service, 1981) Mills, RonaldItem Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts(Plant Physiology, 1980) Mills, RonaldThe metabolism of 14C-labeled aspartic acid, diaminopimelic acid, malic acid and threonine by isolated pea (Pisum sativum L.) chloroplasts was examined. Light enhanced the incorporation of [14C] aspartic acid into soluble homoserine, isoleucine, lysine, methionine and threonine and protein-bound aspartic acid plus asparagine, isoleucine, lysine, and threonine. Lysine (2 millimolar) inhibited its own formation as well as that of homoserine, isoleucine and threonine. Threonine (2 millimolar) inhibited its own synthesis and that of homoserine but had only a small effect on isoleucine and lysine formation. Lysine and threonine (2 millimolar each) in combination strongly inhibited their own synthesis as well as that of homoserine. Radioactive [1,7-14C]diaminopimelic acid was readily converted into [14C]threonine in the light and its labeling was reduced by exogenous isoleucine (2 millimolar) or a combination of leucine and valine (2 millimolar each). The strong light stimulation of amino acid formation illustrates the point that photosynthetic energy is used in situ for amino acid and protein biosynthesis, not solely for CO2 fixation.Item A rapid method for isolation of purified physiologically active chloroplasts, used to study the intracellular distribution of amino acids in pea leaves(Planta, 1980) Mills, RonaldA procedure is described for the rapid (<5 min) isolation of purified, physiologically active chloroplasts from Pisum sativum L. Mitochondrial and microbody contamination is substantially reduced and broken chloroplasts are excluded by washing through a layer containing a treated silica sol. On average the preparations contain 93% intact chloroplasts and show high rates of (14)CO2 fixation and CO2-dependent O2 evolution (over 100 μmol/mg chlorophyll(chl)/h); they are also able to carry out light-driven incorporation of leucine into protein (4 nmol/mg chl/h). The amino-acid contents of chloroplasts prepared from leaves and from leaf protoplasts have been determined. Asparagine is the most abundant amino acid in the pea chloroplast (>240 nmol/mg chl), even thought it is proportionately lower in the chloroplast relative to the rest of the cell. The chloroplasts contain about 20% of many of the amino acids of the cell, but for individual amino acids the percentage in the chloroplast ranges from 8 to 40% of the cell total. Glutamic acid, glutamine and aspartic acid are enriched in the chloroplasts, while asparagine, homoserine and β-(isoxazolin-5-one-2-yl)-alanine are relatively lower. Leakage of amino acids from the chloroplast during preparation or repeated washing was ca. 20%. Some differences exist between the amino-acid composition of chloroplasts isolated from intact leaves and from protoplasts. In particular, γ-aminobutyric acid accumulates to high levels, while homoserine and glutamic acid decrease, during protoplast formation and breakage.Item Identification and measurement of homoserine by gas-liquid chromatography(Analytical Biochemistry, 1980) Mills, RonaldHomoserine has been analyzed quantitatively by gas-liquid chromatography of its N-heptafluorobutyryl isopropyl ester. The method was confirmed by analysis of the soluble amino acid fraction of pea leaves. The possible use of the method for analysis of methionine is discussed.Item Itracellular localization of aspartate kinase in spinach (Spinacea oleracea)(FEBS Letters, 1979) Mills, Ronald1. Enzyme distribution between chloroplasts and the nonchloroplast parts of green leaf cells of Spinacia oleracea, Nicotiana rustica, Vicia faba, and Phaseolus vulgaris have been investigated by use of the nonaqueous chloroplast isolation technique. Whereas pyruvate kinase and peroxidase were located only or mainly outside of the chloroplasts, the other enzymes studied, isocitric dehydrogenase, glutathione reductase, NAD- and NADP-dependent pyridine nucleotide quinone reductase, malic dehydrogenase, NAD- and NADP-dependent glyoxylate reductase, glutamate-oxaloacetate transaminase, NAD-dependent glutamic dehydrogenase, and NADP-dependent aspartic dehydrogenase were both inside and outside of the plastids. In contrast, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase is located only within the chloroplasts. 2. Intact isolated spinach chloroplasts incorporated only a very small amount of labeled carbon from 14CO2 into amino acids in the light. The addition of NH4Cl did not increase the amount of labeled amino acids and had no effect on the total amount of 14C fixed during short time photosynthesis. However, NH4⊕ caused changes in the pathway of carbon during photosynthesis. In the presence of NH4⊕, more 14C was incorporated into sugar monophosphates and phosphoglyceric acid than in the absence of NH4⊕. 3. 14C-labeled glycine and serine fed to intact isolated spinach chloroplasts were neither accumulated nor transformed into other compounds, but 14C-labeled glutamic acid was converted into glutamine. This transformation took place only in the light in chloroplasts containing an intact outer envelope. The addition of NH4⊕ and certain substrates and cofactors did not increase the rate of transformation. 4. The penetration of some amino acids and substrates through the outer envelope of the chloroplasts was investigated on aqueously isolated spinach plastids. It was found that a-ketoglutarate, oxaloacetate, pyruvate, aspartate, and alanine are able to penetrate the envelope although at least for some of these compounds the outer membrane of the chloroplasts acts as a partial barrier. 5. From the experiments reported here and in connection with the results published by other investigators it can be concluded that the most common amino acids such as glutamic acid, aspartic acid, alanine, glycine, and serine are able to penetrate through the outer envelope of the chloroplasts and the synthesis of these amino acids can occur in the leaf cells inside as well as outside of the chloroplasts.Item The isolation of a lysine sensitive aspartate kinase from pea leaves and its involvement in homoserine biosynthesis in isolated chloroplasts(FEBS Letters, 1979) Mills, RonaldHomoserine is an intermediate in the biosynthesis of the essential amino acids Thr and Met in microorganisms [I] and higher plants [2]. Pea seedlings synthesise massive amounts of Hse, mainly in the roots [3-51, although more recent studies suggest that Hse metabolism also takes place in the leaf [6]. Aspartate kinase (EC 2.7.2.4) catalyses the first reaction in Hse biosynthesis, and is subject to complex feedback re~lation by Lys, Thr and Met in microorganisms [7]. In various plants aspartate kinase is also inhibited by the same end products (reviewed [S I), but in pea seedlings the enzyme has only been shown to be inhibited by Thr [9]. This paper reports the light-dependent synthesis of Hse from Asp by isolated pea chloroplasts. The enzyme aspartate kinase has been isolated from pea leaves and chloroplasts and found to be sensitive to both Lys and Thr. We believe this is the first report of a Lys-sensitive aspartate kinase in legume plants.