Ferredoxin-dependent Nitrite Reductase from Spinach Leaves

A ferredoxin-dependent nitrite reductase from Spinacea oleracea was purified approximately 180-fold, with a specific activity of 285 units/mg protein. This purified enzyme also had methyl viologen-dependent nitrite reductase activity, with a specific activity of 164 units/mg protein. After disc electrophoresis with polyacrylamide gel, the purified enzyme showed one major and one minor protein band.

The molecular weight of the enzyme was estimated to be 86,000 from Ultrogel filtration. This purified enzyme in oxidized form had absorption peaks at 278, 390, 573 and 690 nm. The absorbance ratios, A₃₉₀: A₂₇₈ and A₆₇₃: A₃₉₀ were 0.61 and 0.37, respectively.

By applying the purified enzyme to DEAE-Sephadex A–50 column chromatography, the ferredoxin-dependent nitrite reductase activity was selectively decreased. However, the methyl viologen-dependent nitrite reductase activity was increased, with a specific activity of 391 units/mg protein. This modified enzyme was homogeneous by disc electrophoresis with polyacrylamide gel.     

Ferredoxin-dependent Sulfite Reductase from Spinach Leaves

The intermediate of the aromatization of 4-oxocyclohexanecar-boxylic acid (OHA) to 4-hydroxybenzoic acid (HA) by Coryne-bacterium cyclohexanicum was identified as (+)-4-oxocyclohex-2-enecarboxylic acid (O2A) using a combined system of gas-liquid chromatography (GLC) and a mass spectrometer and polarimeter.     

ATP-Dependent Proteolytic Activity from Spinach Leaves

Spinach (Spinacia oleracea CV Bloomsdale Long Standing) leaf cytoplasmic starch phosphorylase and rabbit muscle phosphorylase a were inactivated by incubation with partially purified leaf extract in the presence of ATP and Mg²⁺. The inactivating factor(s) were heat stable and susceptible to protease attack. Phosphorylase inactivation was prevented by incubation in the presence of p-aminobenzamidine and phenylboronic acid, or prolonged treatment with phenylmethylsulfonyl fluoride or leupeptin for the ATP-stimulated inhibitory activity. Mg²⁺ -dependent inactivation was prevented by incubation with leupeptin, phenylmethylsulfonyl fluoride, p-aminobenzamidine, or 5′-adenylate. ATP-mediated inactivation of phosphorylase was stimulated by Mg²⁺ with a reduction in the apparent K_m for ATP. Casein-degrading activities with the same properties of ATP and/or Mg²⁺ stimulation, heat stability, and susceptibility to proteinase inhibitors were detected suggesting that phorphorylase inactivation was due to proteolysis. The activity was greatest at about the time of flowering and also appeared to depend on the light regime.     

Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

Elongation Pathway for alpha-Linolenic Acid Synthesis in Spinach Leaves: A Reexamination

Leaf slices from spinach exhibited considerable variation in their incorporation of [¹⁴C]bicarbonate and [1-¹⁴C]acetate into fatty acids. Reductive ozonolysis studies indicated that all the ¹⁴C-labeled fatty acids were synthesized de novo; no in vivo evidence was found for the previously proposed elongation of hexadecatrienoate to α-linolenate.     

Oxidation of Betaine Aldehyde by Betaine Dehydrogenase from Spinach Leaves

Betaine content in leaves of fifteen plant species was determined. The results showed higher betaine levels in those salt-, drought-, and chilling-resistant species. Betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8 ) was isolated and partially purified from spinach leaves. Some properties of this enzyme were studied. BADH was precipitated by 60% saturation of (NH4)2SO4. Its activity was not detected in 70% saturation of (NH4)2SO4. BADH has two isoenzymes. The activity of BADH was quite stable below –80℃. It was inhibited by 0.125–1.0 mol/L NaG1 or KC1 but not by Mn2+ and Mo6+, and slightly increased by Mg2+.     

Chloroplast DNA in Expanding Spinach Leaves

The proportion of chloroplast DNA in total DNA from spinachleaves has been measured using the second order reassociationkinetics of a ³H-labelled chloroplast DNA probe in total DNAextracts. There was no significant difference between the proportionof chloroplast DNA in the basal and distal halves of 2 cm leavesand in the distal halves of 5, 8, and 10 cm leaves. The meanof all the observations was 21.1 ± 0.7%. There was littlechange in the average total DNA content of cells from any ofthe leaves but cells from larger leaves contained 130–170chloroplasts while cells from the basal half of 2 cm leavescontained about 20 chloroplasts which were smaller than thosefrom the larger leaves. Consequently the average number of copiesof the plastome per chloroplast in large leaves was about 30(5 x 10^–15 g DNA) and in the smaller chloroplasts in thebase of 2 cm leaves was 200 (32 x 10^–15 g DNA). Stainingwith the DNA fluorochrome 4, 6-diamidino-2 phenyl indole (DAPI)showed 10–15 plastid nucleoid areas in chloroplasts oflarger leaves, suggesting there are 2–3 copies of theplastome per plastid nucleoid.     

Biochemical Characterization of Photosystem II Antenna Polypeptides in Grana and Stroma Membranes of Spinach

The photosystem (PS) II antenna system comprises several biochemically and spectroscopically distinct complexes, including light-harvesting complex II (LHCII), chlorophyll-protein complex (CP) 29, CP26, and CP24. LHCII, the most abundant of these, is both structurally and functionally diverse. The photosynthetic apparatus is laterally segregated within the thylakoid membrane into PSI-rich and PSII-rich domains, and the distribution of antenna complexes between these domains has implications for antenna function. We report a detailed analysis of the differences in the polypeptide composition of LHCII, CP29, and CP26 complexes associated with grana and stroma thylakoid fractions from spinach (Spinacia oleracea L.), making use of a very high-resolution denaturing gel system, coupled with immunoblots using monospecific antibodies to identify specific antenna components. We first show that the polypeptide composition of the PSII antenna system is more complex than previously thought. We resolved at least five type I LHCII apoproteins and two to three type II LHCII apoproteins. We also resolved at least two apoproteins each for CP29 and CP26. In state 1-adapted grana and stroma thylakoid membranes, the spectrum of LHCII apoproteins is surprisingly similar. However, in addition to overall quantitative differences, we saw subtle but reproducible qualitative differences in the spectrum of LHCII apoproteins in grana and stroma membrane domains, including two forms of the major type II apoprotein. The implications of these findings for models of PSII antenna function in spinach are discussed.     

Sources of Carbon for Export from Spinach Leaves throughout the Day

Rates of net carbon exchange, export, starch, and sucrose synthesis were measured in leaves of spinach (Spinacia oleracea L.) throughout a 14-hour period of sinusoidal light to determine the sources of carbon contributing to export. Net carbon exchange rate closely followed light level, but export remained relatively constant throughout the day. In the morning when photosynthesis was low, starch degradation provided most of the carbon for export, while accumulated sucrose was exported during the evening. At high photosynthesis rate, the regulatory metabolite fructose 2,6-bisphosphate was low, allowing more of the newly fixed carbon to flow to sucrose through cytosolic fructose bisphosphatase. When the rate of sucrose synthesis exceeded the rate of export from the leaf, sucrose accumulated and soon thereafter sucrose synthesis declined. A decreasing sucrose synthesis rate resulted in additional carbon moving to the synthesis of starch, which was maintained throughout the remainder of the day. The declining sucrose synthesis rate coincided with decreasing activity of sucrose phosphate synthase present in gel-filtered leaf extracts. A rise in the leaf levels of uridine diphosphoglucose and fructose 6-phosphate throughout the day was consistent with this declining activity.     

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.     

Purification and Characterization of Leu-Proteinase, the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves

The leucine specific serine proteinase present in the soluble fraction of leaves from Spinacia oleracea L. (called Leu-proteinase) has been purified by acetone precipitation and a combination of gel-filtration, ion exchange, and adsorption chromatography. This enzyme shows a molecular weight of 60,000 ± 3,000 daltons, an isoelectric point of 4.8 ± 0.1, and a relative electrophoretic mobility of 0.58 ± 0.03. The Leu-proteinase catalyzed hydrolysis of p-nitroanilides of N-α-substituted(-l-)amino acids as well as of chromogenic macromolecular substrates has been investigated between pH 5 and 10 at 23 ± 0.5°C and I = 0.1 molar. The enzyme activity is characterized by a bell-shaped profile with an optimum pH value around 7.5, reflecting the acid-base equilibrium of groups with pK_a values of 6.8 ± 0.1 and 8.2 ± 0.1 (possibly the histidyl residue present at the active site of the enzyme and the N-terminus group). Among the substrates considered, N-α-benzoyl-l-leucine p-nitroanilide shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 1 × 10⁻⁹ molar. In agreement with the enzyme specificity, only N-α-tosyl-l-leucine chloromethyl ketone, di-isopropyl fluorophosphate and phenylmethylsulfonyl fluoride, among compounds considered specific for serine enzymes, strongly inhibit the Leu-proteinase. Accordingly, the enzyme activity is insensitive to cations, chelating agents, sulfydryl group reagents, and activators.     

Activation of 20S Proteasomes from Spinach Leaves by Fatty Acids

In order to clarify the mechanism of activation of plant 20Sproteasomes by fatty acids, we examined the effects of oleic,linoleic and linolenic acids on the three peptidase activitiesof purified 20S proteasomes from spinach leaves and comparedthem with the effects of SDS, a previously characterized activatorof 20S proteasomes. The three fatty acids all activated thehydrolysis of succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide(Suc-LLVYMCA) and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide(Cbz-LLE-2NA) at low concentrations (one-third to one-sixthof that required for activation by SDS). The range of concentrationsof linolenic acid for the activation of Suc-LLVY-MCA hydrolysiswas very narrow. All the fatty acids inhibited the hydrolysisof tert-butoxycarbonyl-Leu-Arg-Arg-4-methylcoumaryl-7-amide(Boc-LRR-MCA)at extremely low concentrations (one-fifth to one-fifteenthof that required for the activation of the hydrolysis of Suc-LLVY-MCAand Cbz-LLE-2NA). In the case of hydrolysis of Suc-LLVY-MCA,SDS and the three fatty acids increased the V_max value and decreasedthe apparent K_m value to similar relative extents. In the caseof hydrolysis of Boc-LLE-MCA, SDS and the three fatty acidsalso decreased the K_m and increased the V_max. However, SDS markedlyincreased V_max. The curves representing the SDS-dependent activationwere shifted to a lower range by the addition of linoleic acid,but the maximum activity at the optimum concentration of SDSwas essentially unchanged. These results suggest that the activationby SDS and that by the fatty acids has an additive effect. Theresults imply that fatty acids, such as linolenic acid, mightact as physiological regulators in plant cells. (Received April 10, 1995; Accepted December 22, 1995)     

Fractionation of Nitrogen Isotopes by Glutamine Synthetase Isolated from Spinach Leaves

The isotopic fractionation of nitrogen in the reaction in vitroof glutamine synthetase isolated from spinach (Spinacia oleraceaL.) leaves was calculated from the changes in natural ¹⁵N abundance(     

热预处理菠菜叶绿体的能量转换特征

经热预处理（湿度为２５－４５℃,部分实验为２０－３６℃,时间为５－１０min)的菠菜（Ｓpinacia oleracea L.)叶绿体。严重影响其能量转换的各步反应。（１）循环光合磷酸化速率随处理温度而下降。（２）类囊体膜上腺三磷酶失活。（３）光照诱导叶绿体的质子吸收减小;叶绿体的９－氨基吖啶（９－ＡＡ）荧光猝灭减弱,但加二环己基磷二亚胺（ＤＣ－ＣＤ）可以部分恢复９－ＡＡ荧光猝死。（４）叶绿体的延迟发光和△Ａ５１５nm电色效应均出现明显变化。（５）免疫印迹反应结果表明,经４５℃预处理叶绿体的膜上腺三磷酶出现解离,其α亚单位的蛋白量明显减少。（６）预处理温度超过３３℃,叶绿体光系统Ⅰ介导的氧吸收速率也下降,在反应介质中加芥子碱可以部分恢复其氧吸收能力,就这些结果与膜透性变化、耦联因子复合物解离和自由基积累的关系进行了讨论。     

Characterization of Energy-Transduction in Thermal Pretreated Chloroplast from Spinach

Characterization of energy-transduction on the chloroplast thylakoid membranes from spinach (Spinacia oleracea L.) after thermal pretreatment was investigated. The related reactions of energy-transduction in chloroplasts were seriously affected by thermal pretreatment. The results were obtained as following: (1) The rate of cyclic photophosphorylation declined when the pretreatment temperature increased in the range of 25 to 45 ℃. (2) The thermal pretreatment led to a decrease of the activity of thylakoid membrane-bounded ATPase. (3) Proton uptake of chloroplasts and the fluorescence quenching of 9-aminoacridine (9-AA) in thylakoid membrane decreased after the thermal pretreatment, but addition of dicyclohexylcarbodiimide (DCCD) could partially restore the fluorescence quenching of 9-AA. (4) Both the rates of fast phase in electrochroism absorption change at 515 nm and the millisecond delayed light emission (ms-DLE) of chloroplast showed a progressive decrease upon raising the temperature of pretreatment. (5) Immunbloting analysis showed that the thermal pretreatment caused the changes of protein content and the electrophoresis mobility of thylakoid membrane-bound ATPase and its α-subunit. (6) If the temperature of pretreatment were higher than 33 ℃, oxygen uptake of PSⅠ -mediated in the samples was rapidly inhibited, but addition of sinapine into the reaction medium could partially restore the ability of oxygen uptake in the samples. These results are briefly discussed in relation to the change of permeability of thylakoid membranes, the dissociation of coupling factor complex as well as accumulation of the radicals in the thylakoid membranes after thermal pretreatment.