Chlorophyll formation and glycine metabolism in laevulinic acid treated barley leaves

Laevulinic acid (LA) inhibited chlorophyll formation and δ-aminolaevulinic acid (ALA) accumulation in dark-grown barley leaves. Mole ratios (ALA: chlorophyll × 8) indicate that LA decreased ALA production by about 30%. The turnover of glycine-[¹⁴C] in 7-day-old leaves treated with LA was 70% slower than in control tissue and this resulted in an increase in endogenous glycine. Total amino acid also increased in LA treated leaves. The data indicate that any contribution made by glycine to ALA synthesis in LA-treated barley leaves would be significantly restricted.     

Effect of 2,2′-bipyridyl on porphyrin synthesis in etiolated and light-treated barley leaves

The effects of 2,2′-bipyridyl on porphyrin formation differed in illuminated and dark-treated barley leaves. In the dark, bipyridyl treatment increased photoconvertible protochlorophyllide (Pchlide, P650) and decreased the protohaem content. The increase in Pchlide could not be wholly accounted for by a diversion of ‘substrate’ from protohaem synthesis. The rate of Pchlide regeneration was slightly higher in chelator treated leaves which suggests increased δ-aminolaevulinic acid (ALA) synthesis. Only small quantities of Mg-protoporphyrinmonomethylester (Mg-protoME) were detected in etiolated leaves treated with bipyridyl in the dark. Protochlorophyll (P630) synthesis from exogenously supplied ALA was lower in the chelator treatments. The results suggest that only when substantial quantities of ALA are being utilized in dark-grown leaves does a ‘metal’ become limiting in the bipyridyl treated leaves. In the light, bipyridyl inhibited chlorophyll synthesis, again suggesting that when substantial amounts of ALA were being utilized a ‘metal’ becomes rate limiting. Bipyridyl treatment also inhibited ALA production in light-treated leaves. The incorporation of glycine-[¹⁴C] into ALA in the presence of bipyridyl was severely restricted compared to the incorporation of glutamate-[¹⁴C]. The data suggest two pathways for ALA synthesis; the classical ALA-synthetase which utilizes glycine and is operative in dark-grown leaves and a second enzyme system, which uses glutamate, and is of quantitative importance in the light.     

The effect of haem on chlorophyll synthesis in barley leaves

Exogenously supplied bovine haemin, fed to etiolated barley leaves, inhibited chlorophyll synthesis in leaves exposed to light. Haemin inhibited the regeneration of protochlorophyllide (P650) and the conversion of exogenously supplied δ-aminolaevulinate (ALA) to protochlorophyll (P630). The effect of haemin on chlorophyll production was overcome by incubating the leaves in water in the dark before light treatment, suggesting the operation of a rapid haem destruction mechanism in leaves. Protohaem turnover in dark-grown leaves was between 8 and 9 hr, based on the rate of degradation of erogenous haemin and the rate of protohaem breakdown in laevulinic acid (LA) treated leaves. The rate constant for haem destruction was 85 pmol/nmol/hr in the dark and 45 pmol/nmol/hr after 4 hr light. There was no evidence that light affects the synthesis of protohaem. It appears that the regulation of endogenous levels of protohaem is by breakdown and it is this mechanism which is under light control. Haem considerably decreased the incorporation of radioactivity from glycollate-[¹⁴C], glycine-[¹⁴C] and glutamate-[¹⁴C] into accumulated ALA in the presence of LA.     

The effects of levulinic Acid and 4,6-dioxoheptanoic Acid on the metabolism of etiolated and greening barley leaves

Application of levulinic acid (LA), a competitive inhibitor of δ-aminolevulinic acid (ALA) dehydratase, to greening plant tissues causes ALA to accumulate at the expense of chlorophyll. 4,6-Dioxoheptanoic acid (DA), which has been reported to be an effective inhibitor of this enzyme in animal systems, has a similar but more powerful effect on ALA and chlorophyll metabolism in greening leaves of Hordeum vulgare L. var. Larker. Both LA and DA also inhibit the uptake of [¹⁴C]amino acids into etiolated and greening barley leaves and reduce their incorporation into protein. Treatment of etiolated and greening leaves with these compounds results in the inhibition of ¹⁴CO₂ evolution from labeled precursors, including amino and organic acids. Inhibition of ¹⁴CO₂ evolution by these compounds is more effective in greening leaves than in etiolated leaves when [4-¹⁴C]ALA or [1-¹⁴C]glutamate are employed as precursors. Both LA and DA also inhibit the uptake and increase the incorporation of ³²Pi into organophosphorus by etiolated barley leaves. These results indicate that LA and DA have more far-reaching effects upon plant metabolism than was previously believed.     

Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves : II. Formation from Glutamate

Intact plastids from greening maize (Zea mays L.) leaves converted [¹⁴C]glutamate and [¹⁴C]2-ketoglutarate (KG) to [¹⁴C]5-aminolevulinic acid (ALA). Glutamate appeared to be the immediate precursor of ALA, while KG was first converted to glutamate, as shown by the effect of various inhibitors of amino acid metabolism. Plastids from greening leaves contained markedly higher activity as compared with etioplasts or chloroplasts. The synthesis of ALA by intact plastids was light dependent. The enzyme system resides in the stroma of plastids or may be lightly bound to membranes. The solubilized system showed maximal activity around pH 7.9 and required Mg²⁺, ATP, and NADPH although dependence on the latter was not clear-cut. A relatively high level of activity could be extracted from etioplasts. Maximal activity was obtained from plastids of leaves which had been illuminated for 90 minutes, after which activity declined sharply. The enzyme system solubilized from plastids also catalyzed the conversion of putative glutamate 1-semialdehyde to ALA in a reaction which was not dependent on the addition of an amino donor.

The system in maize greatly resembled the one which had been reported from barley. It is suggested that this system is the one responsible for the biosynthesis of ALA destined for chlorophyll formation.

     

The metabolic fate of [porphyrin-14C] cytochrome c in dogs.

[Porphyrin-¹⁴C] cytochrome $\text{c}$ (isolated from tissues of dogs injected with [¹⁴C] ALA) was given intravenously to one normal and one bile fistula dog. Essentially all of the injected ¹⁴C was excreted in the urine during the first six days. No (unchanged) cytochrome $\text{c}$ was detectable in the urine. Analysis of ¹⁴C and of light absorption at 400 nm in the successive eluates from Florisil columns showed that all ¹⁴C peaks coincided with pigment peaks, but some pigment peaks has no increase in ¹⁴C. The relative distribution of ¹⁴C in these pigment peaks changed markedly between the first and third days. Delayed excretion of some ¹⁴C suggested cellular uptake of cytochrome $\text{c}$ prior to the urinary excretion of its endogenous metabolites.     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

The occurrence of phosphatidyl choline exchange protein in leaves

The transfer of phosphatidyl choline between liposomes was stimulated by the protein fractions from spinach leaves, etiolated and greening leaves of $\text{Avena}$ seedlings. This is confirmed by the transfer of [¹⁴C]phosphatidyl choline or spin-labeled phosphatidyl choline between donor and acceptor liposomes. ESR spectrum changes also indicated that no spin-labeled phosphatidyl choline was released from donor liposomes by spinach leaf protein unless acceptor liposomes were present. [¹⁴C]phospholipids were transferred from liposomes to both spinach chloroplasts and $\text{Avena}$ etiochloroplasts by phosphatidyl choline exchange protein from germinated castor bean endosperms and further from liposomes to spinach chloroplasts by spinach leaf protein. These results support the view that phosphatidyl choline in the plastid is supplied from the synthesis site, the endoplasmic reticulum, by phospholipid exchange protein.     

Two Biosynthetic Pathways to delta-Aminolevulinic Acid in a Pigment Mutant of the Green Alga, Scenedesmus obliquus

Pigment mutant C-2A′ of the unicellular green alga Scenedesmus obliquus develops only traces of chlorophyll and has no detectable amount of δ-aminolevulinic acid (ALA) when grown in the dark. In light it develops ALA and in the presence of levulinic acid (LA), a competitive inhibitor of ALA dehydratase, it accumulates 0.18 mmoles of ALA per 10 microliters of packed cell volume per 12 hours. This amount could be increased up to 15 times by feeding precursors and cofactors.

Incubation with [U-¹⁴C]glutamate, [1-¹⁴C]glutamate, and [2-¹⁴C]glycine yielded significantly labeled ALA, whereas [1-¹⁴C]glycine did not label the ALA specifically. Thus, two pathways using either glycine/succinyl-coenzyme A or incorporating the whole C-5-skeleton of glutamate into ALA are present in this alga. The efficiency of the glycine/succinyl-coenzyme A pathway seems to be three times higher than that of the glutamate pathway. Incubation with [5-¹⁴C]2-ketoglutarate, which can serve both pathways as a precursor, resulted in radioactivity of ALA as high as the sum of both labeling with [1-¹⁴C]glutamate and [2-¹⁴C]glycine.

Since the newly synthesized chlorophyll was radioactive regardless of labeled substrate employed, both pathways culminate in chlorophyll formation.

     

Uteroplacental dysfunction and prostaglandin metabolism in zinc deficient pregnant rats

Relationships between perinatal mortality, disrupted uteroplacental function and prostaglandin metabolism have been studied in Zn-deficient rats. Uterine contractility $\text{in vitro}$, placental blood flow $\text{in viro}$, and uterine and placental prostaglandin synthesis from [1?¹⁴C] arachidonic acid $\text{in vitro}$ were investigated at day 22 of pregnancy. High amplitude uterine contractions were almost completely eliminated and utero-placental blood flow was decreased by 85% by Zn deficiency. Synthesis of [1?¹⁴C]-prostaglandin E₂, F_2α and 6-keto-F_1α from [1?¹⁴C] arachidonic acid decreased significantly in uterine tissue but increased in placentae. These possibly inter-related effects may contribute to the high perinatal mortality observed in Zn deficiency.     

Effects of desmethylimipramine and diphenylpropene derivatives on synthesis of rat brain lipids in vivo

The effects of antidepressant compounds on the synthesis of brain lipids from [1-¹⁴C] acetate $\text{in vivo}$ in 15 day old rats have been investigated. Compounds used included the drug desmethylimipramine (DMI), the tetrabenazine antagonist 3-methylamino-1:1-diphenylprop-1-ene (II) and the primary (I) and tertiary (III) amine analogues of (II). Compound (II), the most potent tetrabenazine antagonist in the diphenylpropene series, significantly increased lipogenesis, whereas the remaining compounds did not. The results from fractionation of the lipid extract from rats treated with (II) indicated that the incorporation of radioactivity from [1-¹⁴C] acetate increased proportionally in all neutral lipids and phospholipids. Tetrabenazine also increased brain lipogenesis $\text{in vivo}$ and altered the distribution within lipid classes of radioactivity from [1-¹⁴C] acetate. Using [¹⁴C] labelled compound, the concentration of (II) in the brain under the present experimental conditions has been determined.     

Aminoacyl transfer from phenylalanyl-tRNA microinjected into Xenopus laevis oocytes.

$\text{Xenopus laevis}$ oocytes were injected with [¹⁴C] phe-tRNA and the fate of the aminoacyl moiety was studied. The radioactive phenylalanine is gradually hydrolized off the tRNA once inside the cell. The rate of deacylation of the tRNA is not affected by inhibition of cellular protein synthesis by puromycin or cycloheximide. Part of the radioactive amino acid that leaves the tRNA (30 to 65%) is transferred directly into the oocyte nascent proteins as evidenced by the fact that its incorporation into proteins is not reduced by coinjection with a large excess of [¹²C] phenylalanine. Aminoacyl transfer from injected phe-tRNA into proteins is inhibited by puromycin and cycloheximide.     

Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves : I. Formation from 2-Ketoglutarate via 4,5-Dioxovaleric Acid

Cell-free extracts from greening maize (Zea mays L.) leaves catalyze the conversion of [¹⁴C]2-ketoglutarate (KG) to [¹⁴C]5-aminolevulinic acid (ALA) in a reaction which requires NADH and an amino donor and shows maximal activity around pH 6.5. The enzymic system is located in the cytosol. This cell fraction contains a low level of `KG dehydrogenase' activity and a transaminase which catalyzes the conversion of 4,5-dioxovaleric acid (DOVA) to ALA. The transaminase can use glutamate, aspartate, or alanine as amino donor. It is effectively inhibited by aminooxyacetate and ethylenediamine tetraacetate and shows maximal activity at pH 6.7. The activity of DOVA transaminase is only slightly affected by preillumination of leaves and can also be detected in green leaves and in roots.

DOVA was isolated from leaves and roots and determined as its benzoquinoxaline derivative. Significant amounts were found only in tissues in which ALA had accumulated or after it was exogenously supplied. DOVA was labeled in vivo by both [¹⁴C]ALA and [¹⁴C]KG. Small amounts were also formed from ALA in a cell-free system.

It is suggested that DOVA may be an intermediate in the diversion of ALA to respiratory metabolism and that it is not involved in the biosynthesis of this porphyrin precursor.

     

In vivo biosynthesis of -linolenic acid in plants

[1-¹⁴C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of $\text{Chlorella}\text{pyrenoidosa}$ and $\text{Candida}\text{bogoriensis}$. In these systems the [¹⁴C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C_1–9 and the C_10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [¹⁴C] was localized in the C_1–9 fragment. While [1-¹⁴C]oleic acid was converted by whole cells of $\text{Chlorella}$ to [1-¹⁴C]linoleic and [1-¹⁴C]linolenic acids, [U-¹⁴C]oleic acid yielded [U-¹⁴C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid.     

Lanosterol 14α-demethylase. Similarity of the enzyme system from yeast and rat liver

The chemical synthesis of 24,25-dihydro[32-¹⁴C]lanosterol is described. The incubation of this material with a cell-free system from $\text{Saccharomvoes cerevisiae}$ or with a microsomal preparation from rat liver resulted in both cases in the release of [¹⁴C]formic acid. This result suggests that in the biosynthesis of ergosterol in yeast, as well as in that of cholesterol in higher animals, the 14α-methyl group of lanosterol is removed as formic acid. In both systems, the measurement of the rate of release of [¹⁴C]formic acid from 24,25-dihydro[32-¹⁴C]lanosterol provides a simple and direct assay of lanosterol 14α-demethylase. Carbon monoxide inhibited both yeast and liver 14α-demethylase.     

Control of plastidic glycolipid synthesis and its relation to chlorophyll formation

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv “Halo”) and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [¹⁴C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [¹⁴C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [¹⁴C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [¹⁴C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [¹⁴C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.     

Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.     

Biosynthesis of -linolenic acid by disrupted spinach chloroplasts

A disrupted spinach chloroplast preparation readily synthesized $\text{[}{}^{14}\text{C]α-linolenate}$ from [2-¹⁴C]acetate under anaerobic conditions. It can be shown by degradation data that [¹⁴C]oleate is not a precursor of [¹⁴C]linolenate and that $\text{cis}$ 7,10,13-hexadecatrienoic acid is the probable immediate precursor of the [¹⁴C]linolenate.     

Control of glycolysis and lipogenesis in the liver by glucagon at the phosphofructokinase-fructose 1,6-diphosphatase site

We have examined the effects of glucagon on lipogenesis from fasted-refed rats incubated under two conditions, either without added substrate or with 10 mml-lactate. Net glycolysis (from glycogen) occurs in the absence of glucagon. This glycolysis is inhibited by glucagon under conditions of no added lactate, and reversed by glucagon to a net gluconeogenesis in the presence of 10 mm lactate. Glucagon markedly inhibits fatty acid synthesis (estimated by incorporation of tritium from THO) in hepatocytes incubated without added substrate; but, in the presence of 10 mml-lactate, the inhibition of fatty acid synthesis is only about 10%. The inhibition of lipogenesis from endogenous glycogen is primarily caused by inhibition of glycolysis. Glucagon markedly lowers the $\text{C}\text{-4,5,6}\text{C}\text{-1,2,3}$ ratio in glucose produced from [1-¹⁴C]galactose, indicating a strong inhibition of phosphofructokinase flux. The $\text{C}\text{-1,2,3}\text{C}\text{-4,5,6}$ ratio in glucose from [1-¹⁴C]glycerol is only slightly less than 1, indicating an active fructose diphosphatase flux even under conditions of active net glycolysis. Glucagon increases this ratio only slightly, suggesting that an acute increase of fructose diphosphatase activity by glucagon may occur, but is of much less importance than the decrease of phosphofructokinase.     

Synthetic polynucleotides as model substrates for ribosomal RNA processing

A nuclear exoribonuclease from Novikoff ascites cells was used to study the hydrolysis of single-stranded heteropolymers containing [¹⁴C]adenylic acid and either uridylic acid or cytidylic acid and heteropolymers of [¹⁴C]adenylic acid and one of the corresponding 2′-$\text{O}$-methylated nucleotides. The results of these studies indicate that both the rate and extent of hydrolysis are greatly inhibited by the presence of 2′-$\text{O}$-methylated nucleotides. Restriction of exonuclease activity by 2′-$\text{O}$-methylated nucleotides provides a possible mechanism for rRNA processing.