Metabolism of Monoterpenes: Acetylation of (-)-Menthol by a Soluble Enzyme Preparation from Peppermint (Mentha piperita) Leaves

The essential oil from mature leaves of flowering peppermint (Mentha piperita L.) contains up to 15% (—)-menthyl acetate, and leaf discs converted exogenous (—)-[G-³H]menthol into this ester in approximately 15% yield of the incorporated precursor. Leaf extracts catalyzed the acetyl coenzyme A-dependent acetylation of (—)-[G-³H]menthol and the product of this transacetylase reaction was identified by radiochromatographic techniques. Transacetylase activity was located mainly in the 100,000g supernatant fraction, and the preparation was partially purified by combination of Sephadex G-100 gel filtration and chromatography on O-diethylaminoethyl-cellulose. The transacetylase had a molecular weight of about 37,000 as judged by Sephadex G-150 gel filtration, and a pH optimum near 9. The apparent K_m and velocity for (—)-menthol were 0.3 mm and 16 nmol/hr· mg of protein, respectively. The saturation curve for acetyl coenzyme A was sigmoidal, showing apparent saturation near 0.1 mm. Dithioerythritol was required for maximum activity and stability of the enzyme, and the enzyme was inhibited by thiol directed reagents such as p-hydroxymercuribenzoate. Diisopropylfluorophosphate also inhibited transacylation suggesting the involvement of a serine residue in catalysis. The transacylase was highly specific for acetyl coenzyme A; propionyl coenzyme A and butyryl coenzyme A were not nearly as efficient as acyl donors (11% and 2%, respectively). However, the enzyme was much less selective with regard to the alcohol substrate, suggesting that the nature of the acetate ester synthesized in mint is more dependent on the type of alcohol available than on the specificity of the transacetylase. This is the first report on an enzyme involved in monoterpenol acetylation in plants. A very similar enzyme, catalyzing this key reaction in the metabolism of menthol, was also isolated from the flowers of peppermint.     

Photosynthesis of Lipids from CO(2) in Spinacia oleracea

Young expanding spinach leaves exposed to ¹⁴CO₂ under physiological conditions for up to 20 minutes assimilated CO₂ into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the ¹⁴C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO₂in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H¹⁴CO₃ into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor.     

Fat Metabolism in Higher Plants: LVII. A Comparison of Fatty Acid-Synthesizing Enzymes in Chloroplasts Isolated from Mature and Immature Leaves of Spinach

Chloroplasts isolated from immature leaves of spinach (Spinacia oleracea) differ in enzyme levels from those isolated from mature leaves. On a chlorophyll basis, immature chloroplast preparations had 5- to 6-fold higher capacity to synthesize fatty acids from 2-¹⁴C-acetate compared to plastids isolated from mature leaves. This difference was correlated with higher activities for the enzymes, acetyl coenzyme A synthetase, malonyl coenzyme A synthetase, acetyl coenzyme A carboxylase, and oleyl coenzyme A transferase in plastid pressates obtained from immature leaves. Disrupted chloroplast preparations from both mature and immature leaves retained the ability to incorporate 2-¹⁴C-acetate into fatty acids in a pattern similar to that by isolated chloroplasts. 2-¹⁴C-Acetate, 2-¹⁴C-acetyl coenzyme A, 2-¹⁴C-malonate, and 1,3-¹⁴C malonyl coenzyme A were readily incorporated into a number of fatty acids. Moreover, the synthesis of oleate by chloroplast pressates from these substrates was strongly inhibited by KCN, flavin adenine mononucleotides and dinucleotides, and anaerobic conditions, while linolenic acid synthesis was unaffected by these compounds.     

Spinach leaf acetyl-coenzyme a synthetase: purification and characterization

Acetyl-coenzyme A (CoA) synthetase was purified 364-fold from leaves of spinach (Spinacia oleracea L.) using ammonium sulfate fractionation followed by ion exchange, dye-ligand, and gel permeation chromatography. The final specific activity was 2.77 units per milligram protein. The average M_r value of the native enzyme was about 73,000. The Michaelis constants determined for Mg-ATP, acetate, and coenzyme A were 150, 57, and 5 micromolar, respectively. The purified enzyme was sensitive to substrate inhibition by CoA with an apparent K_i for CoA of 700 micromolar. The enzyme was specific for acetate; other short and long chain fatty acids were ineffective as substrates. Several intermediates and end products of fatty acid synthesis were examined as potential inhibitors of acetyl-CoA synthetase activity, but none of the compounds tested significantly inhibited acetyl-CoA synthetase activity in vitro. The properties of the purified enzyme support the postulated role of acetyl-CoA synthetase as a primary source of chloroplast acetyl-CoA.     

Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica

Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PP_i-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PP_i were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PP_i displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.     

Identification and Characterization of Mitochondrial Acetyl-Coenzyme A Hydrolase from Pisum sativum L. Seedlings

Mitochondria from Pisum sativum seedlings purified free of peroxisomal and chlorophyll contamination were examined for acetyl-coenzyme A (CoA) hydrolase activity. Acetyl-CoA hydrolase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of the mitochondria. The products, acetate and CoA, were quantified by two independent methods and verified that the observed activity was an acetyl-CoA hydrolase. The pea mitochondrial acetyl-CoA hydrolase showed a K_m for acetyl-CoA of 74 micromolar and a V_max of 6.1 nanomoles per minute per milligram protein. CoA was a linear competitive inhibitor of the enzyme with a K_is of 16 micromolar. The sensitivity of the enzyme to changes in mole fraction of acetyl-CoA suggested that the changes in the intramitochondrial acetyl-CoA/CoA ratio may be an effective mechanism of control. The widespread distribution of mitochondrial acetyl-CoA hydrolase activity among different plant species indicated that this may be a general mechanism in plants for synthesizing acetate.     

Reduction of Nitrate via a Dicarboxylate Shuttle in a Reconstituted System of Supernatant and Mitochondria from Spinach Leaves

Substantial rates of nitrate reduction could be achieved with a reconstituted system from spinach leaves containing supernatant, mitochondria, NAD⁺, oxaloacetate (OAA), and an oxidizable substrate. Appropriate substrates were glycine, pyruvate, citrate, isocitrate, fumarate, or glutamate. The reduction of NO₃⁻ with any of the substrates could be inhibited by n-butyl malonate, showing that the transfer of reducing power from the mitochondria to the supernatant involved the malate exchange carrier. The addition of ADP to the reconstituted system decreased NO₃⁻ reduction and this decrease could be reversed by the addition of rotenone or antimycin A. The operation of the OAA/malate shuttle was achieved most quickly in the system when low concentrations (≤0.1 millimolar) of OAA were added. A corresponding increase in the lag time for the operation of the OAA/malate shuttle was observed when the OAA concentration was increased. Concentrations for half-maximal activity of OAA, glycine, NAD⁺, and NO₃⁻ in the reconstituted system were 42 micromolar, 0.5 millimolar, 0.25 millimolar, and 26 micromolar, respectively. The transfer of reducing power from the mitochondria to the soluble phase via the OAA/malate shuttle can not only provide NADH for cytoplasmic reduction but can also sustain oxidation of tricarboxylic cycle acids and the generation of α-ketoglutarate independently of the respiratory electron transport chain.     

Characterization of Citrate Synthase from Geobacter sulfurreducens and Evidence for a Family of Citrate Synthases Similar to Those of Eukaryotes throughout the Geobacteraceae

Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (k_cat = 8.3 s⁻¹; K_m = 14.1 and 4.3 μM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (K_i = 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K⁺ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.     

Rapid Modulation of Spinach Leaf Nitrate Reductase Activity by Photosynthesis : I. Modulation in Vivo by CO(2) Availability

It has been shown recently that in spinach leaves (Spinacia oleracea) net photosynthesis and nitrate reduction are closely linked: when net photosynthesis was low because of stomatal closure, rates of nitrate reduction decreased (WM Kaiser, J Förster [1989] Plant Physiol 91: 970-974). Here we present evidence that photosynthesis regulates nitrate reduction by modulating nitrate reductase activity (NRA, EC 1.6.6.1). When spinach leaves were exposed to low CO₂ in the light, extractable NRA declined rapidly with a half-time of 15 minutes. The inhibition was rapidly reversed when leaves were brought back to air. NRA was also inhibited when leaves were wilted in air; this inhibition was due to decreased CO₂ supply as a consequence of stomatal closure. The modulation of NRA was stable in vitro. It was not reversed by gel filtration. In contrast, the in vitro inhibition of nitrate reductase (NR) by classical inhibitors such as cyanide, hydroxylamin, or NADH disappeared after removal of free inhibitors by gel filtration. The negative modulation of NRA in —CO₂-treated leaves became manifest as a decrease in total enzyme activity only in the presence of free Mg²⁺ or Ca²⁺. Mg²⁺ concentrations required for observing half-maximal inhibition were about 1 millimolar. In the presence of EDTA, the enzyme activity was always high and rather independent of the activation status of the enzyme. NRA was also independent of the pH in the range from pH 7 to pH 8, at saturating substrate and Mg²⁺ concentrations. The apparent substrate affinities of NR were hardly affected by the in vivo modulation of NR. Only V_max changed.     

Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod

Cell-free extracts capable of converting [¹⁴C]-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [¹⁴C]-labeled GAs, prepared enzymically from [¹⁴C]mevalonic acid, were incubated with these extracts, and products were identified by gas chromatography-mass spectrometry. The following pathway was found to operate in extracts from spinach leaves grown under long day (LD) conditions: GA₁₂ → GA₅₃ → GA₄₄ → GA₁₉ → GA₂₀. The pH optima for the enzymic conversions of [¹⁴C]GA₅₃, [¹⁴C]GA₄₄ and [¹⁴C]GA₁₉ were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe²⁺, α-ketoglutarate and O₂ for activity, and ascorbate stimulated the conversion of [¹⁴C]GA₅₃ and [¹⁴C]GA₁₉. Extracts from plants given LD or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD. The results indicate that the activities of the enzymes oxidizing GA₅₃ and GA₁₉ are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing GA₄₄ remains high irrespective of light or dark treatment. This photoperiodic control of enzyme activity is not due to the presence of an inhibitor in plants grown in SD. These observations offer an explanation for the higher GA₂₀ content of spinach plants in LD than in SD.     

Characterization of biotin and 3-methylcrotonyl-coenzyme a carboxylase in higher plant mitochondria

Mitochondria from green pea (Pisum sativum) leaves were purified free of peroxisomes and chlorophyll contamination and examined for their biotin content. The bulk of the bound biotin detected in plant mitochondria was shown to be associated with the matrix space to a concentration of about 13 micromolar, and no free biotin was detected. Western blot analysis of mitochondrial polypeptides using horseradish peroxidase-labeled streptavidin revealed a unique biotin-containing polypeptide with a molecular weight of 76,000. This polypeptide was implicated as being the biotinylated subunit of 3-methylcrotonyl-coenzyme A (CoA) carboxylase. Fractionation of pea leaf protoplasts demonstrated that this enzyme activity was located largely in mitochondria. The 3-methylcrotonyl-CoA carboxylase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of mitochondria. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8.3 in the presence of Mg²⁺. Kinetic constants (apparent K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 0.05 millimolar; ATP, 0.16 millimolar; HCO₃⁻, 2.2 millimolar. The involvement of 3-methylcrotonyl-CoA carboxylase in the leucine degradation pathway in plant mitochondria is proposed.     

Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism

NAD-specific “malic” enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO₄²⁻, suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn²⁺, which cannot be replaced by NADP or Mg²⁺. For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn²⁺, and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 μmole/hr·mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO₂ for recycling during cessation of gas exchange in periods of severe drought.     

Plant dihydroxyacetone phosphate reductases : purification, characterization, and localization

A cytosolic form of dihydroxyacetone phosphate (DHAP) reductase was purified 200,000-fold from spinach (Spinacia oleracea L.) leaves to apparent electrophoretic homogeneity. The purification procedure included anion-exchange chromatography, gel filtration, hydrophobic chromatography, and dye-ligand chromatography on Green-A and Red-A agaroses. The enzyme, prepared in an overall yield of 14%, had a final specific activity of about 500 μmol of DHAP reduced min⁻¹ mg⁻¹ protein, a subunit molecular mass of 38 kD, and a native molecular mass of 75 kD. A chloroplastic isoform of DHAP reductase was separated from the cytosolic form by anion-exchange chromatography and partially purified 56,000-fold to a specific activity of 135 μmol min⁻¹ mg⁻¹ protein. Antibodies generated in rabbits against the cytosolic form did not cross-react with the chloroplastic isoform. The two reductases were specific for NADH and DHAP. Although they exhibited some dissimilarities, both isoforms were severely inhibited by higher molecular weight fatty acyl coenzyme A esters and phosphohydroxypyruvate and moderately inhibited by nucleotides. In contrast to previous reports, the partially purified chloroplastic enzyme was not stimulated by dithiothreitol or thioredoxin, nor was the purified cytosolic enzyme stimulated by fructose 2,6-bisphosphate. A third DHAP reductase isoform was isolated from spinach leaf peroxisomes that had been prepared by isopycnic sucrose density gradient centrifugation. The peroxisomal DHAP reductase was sensitive to antibodies raised against the cytosolic enzyme and had a slightly smaller subunit molecular weight than the cytosolic isoform.     

Physical and Kinetic Properties and Regulation of the NAD Malic Enzyme Purified from Leaves of Crassula argentea

The NAD malic enzyme has been purified to near homogeneity from the leaves of Crassula argentea Thunb. The enzyme has two subunits, one of 59,000 daltons, and one of 62,000 daltons. In native gels stained for activity, the enzyme appears to exist in the dimeric, tetrameric, and predominantly the octameric forms.

The enzyme uses either Mg²⁺ or Mn²⁺ as the required divalent cation, and utilizes NADP at a rate less than 20% of that with NAD. With Mn²⁺ the K_m for malate²⁻ is lower than with Mg²⁺, but V_max is lower than with Mg²⁺. In the forward (malate-decarboxylating) direction with NAD, the kinetic parameters are essentially like those observed for the enzyme from C₃ plants. In the reverse reaction, run with Mn²⁺, the activity is 1.5% of that in the forward reaction. The equilibrium constant is 1.1 × 10⁻³ molar.

The kinetic mechanism of the reaction, at least in the forward direction, is sequential, with apparently random binding of all reaction components. Product inhibition patterns confirm this.

The enzyme displays a strong hysteretic lag, which is shortened by high enzyme concentrations, high substrate concentrations, and the presence of the product NADH.

The enzyme is activated by coenzyme A with K_a = 4 micromolar. AMP also shows competitive activation, with K_a = 24 micromolar. The activation by coenzyme A and AMP is additive, implying separate sites for their binding. Phosphoenolpyruvate activates the reaction at low (micromolar) concentrations, but higher concentrations of phosphoenolpyruvate cause deactivation. Fumarate²⁻ is a strong activator, with K_a = 0.3 millimolar. Fructose-1,6-bisphosphate activates the enzyme, but its most pronounced effect is in shortening the lag. Citrate is a competitive inhibitor of malate, with K_i = 4.9 millimolar.

     

Cytosolic Phosphofructokinase from Spinach Leaves : II. Affinity for Mg and Nucleoside Phosphates

Cytosolic ATP-phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was inhibited by submillimolar concentrations of free Mg²⁺. The free Mg²⁺ concentration required for 50% inhibition of PFK activity was 0.22 millimolar. Inhibition by free Mg²⁺ was independent of the MgATP²⁻ concentration. Inorganic phosphate (Pi) reduces the inhibition of PFK activity by Mg²⁺. Free ATP (ATP⁴⁻) also inhibits PFK activity. For free ATP the inhibition of PFK activity was dependent on the MgATP²⁻ concentration. Fifty percent inhibition of PFK activity requires 1.2 and 3.7 millimolar free ATP at 0.1 and 0.5 millimolar MgATP²⁻, respectively. It was proposed that free ATP competes for the MgATP²⁻ binding site, whereas free Mg²⁺ does not. Pi diminished the inhibitory effect of free ATP on PFK activity. Free ATP and Pi had substantial effects on the MgATP²⁻ requirement of cytosolic PFK. For half-maximum saturation of PFK activity 3 and 76 micromolar MgATP²⁻ was required at 0.007 and 0.8 millimolar free ATP in the absence of Pi. At 5 and 25 millimolar Pi, half-maximum saturation was achieved at 9 and 14 micromolar MgATP²⁻. PFK activity was inhibited by Ca²⁺. The inhibition by Ca²⁺ depends upon the total Mg²⁺ concentration. Fifty percent inhibition of PFK activity required 22 and 32 micromolar Ca²⁺ at 0.1 and 0.2 millimolar Mg²⁺, respectively. At physiological concentrations of about 0.5 millimolar free Mg²⁺, Ca²⁺ would have little effect on cytosolic PFK activity from spinach leaves. PFK is not absolutely specific for the nucleoside 5′-triphosphate substrate. Besides MgATP²⁻, MgUTP²⁻, MgCTP²⁻, and MgGTP²⁻ could be used as a substrate. All four free nucleotides inhibit PFK activity. The physiological consequences of the regulatory properties of cytosolic PFK from spinach leaves will be discussed. A model will be introduced, in an attempt to describe the complex interaction of PFK with substrates and the effectors Mg²⁺ and Pi.     

Isolation of dihydroxyacetone phosphate reductase from dunaliella chloroplasts and comparison with isozymes from spinach leaves

A dihydroxyacetone phosphate (DHAP) reductase has been isolated in 50% yield from Dunaliella tertiolecta by rapid chromatography on diethylaminoethyl cellulose. The activity was located in the chloroplasts. The enzyme was cold labile, but if stored with 2 molar glycerol, most of the activity was restored at 30°C after 20 minutes. The spinach (Spinacia oleracea L.) reductase isoforms were not activated by heat treatment. Whereas the spinach chloroplast DHAP reductase isoform was stimulated by leaf thioredoxin, the enzyme from Dunaliella was stimulated by reduced Escherichia coli thioredoxin. The reductase from Dunaliella was insensitive to surfactants, whereas the higher plant reductases were completely inhibited by traces of detergents. The partially purified, cold-inactivated reductase from Dunaliella was reactivated and stimulated by 25 millimolar Mg²⁺ or by 250 millimolar salts, such as NaCl or KCl, which inhibited the spinach chloroplast enzyme. Phosphate at 3 to 10 millimolar severely inhibited the algal enzyme, whereas phosphate stimulated the isoform in spinach chloroplasts. Phosphate inhibition of the algal reductase was partially reversed by the addition of NaCl or MgCl₂ and totally by both. In the presence of 10 millimolar phosphate, 25 millimolar MgCl₂, and 100 millimolar NaCl, reduced thioredoxin causes a further twofold stimulation of the algal enzyme. The Dunaliella reductase utilized either NADH or NADPH with the same pH maximum at about 7.0. The apparent K_m (NADH) was 74 micromolar and K_m (NADPH) was 81 micromolar. Apparent V_max was 1100 μmoles DHAP reduced per hour per milligram chlorophyll for NADH, but due to NADH inhibition highest measured values were 350 to 400. The DHAP reductase from spinach chloroplasts exhibited little activity with NADPH above pH 7.0. Thus, the spinach chloroplast enzyme appears to use NADH in vivo, whereas the chloroplast enzyme from Dunaliella or the cytosolic isozyme from spinach may utilize either nucleotide.     

The origin of chloroplastic acetyl coenzyme A

Pyruvic dehydrogenase activity has been examined in a number of highly purified leaf organelles. In spinach leaf cell, the major activity is in the mitochrondrion with low activity in isolated chloroplasts. The major source of CO₂ derived from pyruvic acid metabolism in the isolated chloroplast is via the acetolactic synthase reaction localized in the chloroplast. Evidence is presented that the leaf mitochondrion contains both the pyruvic acid dehydrogenase and an acetyl coenzyme A hydrolase. It is suggested that free acetic acid is generated in the mitochrondrion and then moves to the chloroplast where acetyl coenzyme synthetase converts it from the metabolically inert acid to the very metabolically active acetyl coenzyme A.     

Prolindehydrogenase aus keimenden farnsporen

A soluble enzyme which converts proline to glutamic acid using NAD as coenzyme was isolated from young prothallia and spores of the fern Anemia phyllitidis. The purification was about 36-fold. The pH optimum is between 10·2 and 10·7; the K_m for proline is 4·6 × 10⁻⁴ M and for NAD 3·4 × 10⁻⁴ M. There are no multiple forms of this enzyme, as proved by gel electrophoresis.     

Characterization of Fatty Acid biosynthesis in isolated pea root plastids

Fatty acid biosynthesis from Na[1-¹⁴C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl₂, 1 millimolar each of MnCl₂ and glycerol-3-phosphate, 15 millimolar KHCO₃, 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35°C. At the standard incubation temperature of 25°C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [¹⁴C]acetate into fatty acids. On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.     

The role of sulfhydryl groups in the ribulose- 1,5-bisphosphate carboxylase and oxygenase reactions.

Ribulose-l,5-bisphosphate carboxylase (E.C. 4.1.1.39) isolated from Chromatium strain D contains 64 free cysteinyl -SH groups per mol (M_r 5.11 × 10⁵) as determined using three different titrants: p-[¹⁴C]chloromercuribenzoate, the Ellman reagent, and [¹⁴C]iodoacetamide.Distribution of -SH groups in the two constituent subunits (A and B) isolated from spinach and Chromatium ribulose-1,5-bisphosphate carboxylases was determined to be for spinach, 9 in A and 3 in B; and for Chromatium, 7 in A and 1 in B.The relationship between the numbers of -SH groups blocked vs residual activities of both the ribulose-1,5-bisphosphate carboxylase and oxygenase reactions was examined by titration with p-chloromercuribenzoate. In both spinach and Chromatium enzymes, antisigmoidal curves were obtained for the degree of the enzyme activity loss in relation to the numbers of -SH groups masked. However, at alkaline pH the Chromatium enzyme shows a sharp decline in both carboxylase and oxygenase activities, apparently due to the alkali dissociation of the enzyme molecule accompanied by its structural deformation. The functional role of -SH groups in the ribulose-1,5-bisphosphate carboxylase molecule is discussed in relation to two constituent enzyme reactions, and it is concluded that in both enzyme sources the active sites are probably the same for the two reactions.