Formation of 12-oxo-trans-10-dodecenoic acid in chloroplasts from Thea sinensis leaves

Linolenic acid-[1-¹⁴C] was converted to 12-oxo-trans-10-dodecenoic acid, via 12-oxo-cis-9-dodecenoic acid by incubation with chloroplasts of Thea sinensis leaves. Thus, it was confirmed that linolenic acid is split into a C₁₂-oxo-acid, 12-oxo-trans-10-dodecenoic acid, and a C₆-aldehyde, trans-2-hexenal, leaf aldehyde, by an enzyme system in chloroplasts of tea leaves.     

Biosynthesis of trans-2-hexenal in chloroplasts from Thea sinensis

The biosynthetic pathway of trans-2-hexenal, leaf aldehyde, in isolated chloroplasts of Thea sinensis leaves. was examined using a tracer experiment. A high and specific incorporation of radioactivity into cis-3-hexenal and trans-2-hexenal, was observed when linolenic acid-[U-¹⁴C] was incubated with the isolated chloroplasts. Thus, trans-2-hexenal was biosynthesized via cis-3-hexenal from linolenic acid in the chloroplasts.     

The effects of soybean lipoxygenase-1 on chloroplasts from wheat

Chloroplasts were isolated from primary leaves of wheat 12 days after germination and incubated at 25° for 45 min in the dark with soybean lipoxygenase-1. The lipoxygenase action was evident from a weak oxygen uptake of ca 0.18, μmol/hr per mg chloroplast protein. The lipoxygenase treatment caused a marked decrease in the photochemical activity, as measured by the reduction rate of 2,6-dichlorophenolindophenol. However, both the content and composition of the lipids as well as those of total fatty acids remained largely unchanged except for a slight but significant decrease in the total linolenic acid content. It is proposed that soybean lipoxygenase-1 selectively attacks free linolenic acid present in chloroplasts, followed by a chlorophyll-catalysed reaction of hydroperoxylinolenic acid with components of the electron transfer system.     

Effects of inhibitors on the enzyme system producing C6-aldehydes from C18-unsaturated fatty acids in chloroplasts of Japanese silver (Farfugium japonicum) leaves

When chloroplasts isolated from Farfugium japonicum (Japanesesilver) leaves were used as an enzyme source, the activity ofthe enzyme system producing C₆-aldehydes (cis-3-hexenal andn-hexanal) from C₁₈-unsaturated fatty acids (linolenic and linoleicacids) decreased upon treatment with LAHase from potato. Thisenzyme system could not be separated from chlorophylls and lipidsby detergent treatment and was not affected by light illumination,CCCP or DCMU. The activity of the enzyme system was inhibitedby MB and NTB used as a redox reagent, SKF 525-A as an oxidaseinhibitor and DABCO as a quencher of singlet oxygen, but notby DCIP, PMS and SOD. These data suggest that; i) interactionof the enzyme system with lipids is required for maximal enzymeactivity, ii) this enzyme system may involve electron mediator(s),and iii) singlet oxygen takes part in the enzyme reaction. (Received October 28, 1977; )     

Substrate specificity of potato lipoxygenase

The substrate specificity of potato lipoxygenase was examined using a partially purified enzyme preparation from tubers of a potato variety with low lipolytic acyl hydrolase activity. Potato lipoxygenase is fully active only on free linoleic acid or linolenic acid, and only acts directly on more complex glyceride moieties in the absence of any significant endogenous lipolytic acyl hydrolase activity.     

Purification and properties of alcohol oxidase from Tanacetum vulgare

Alcohol oxidase (alcohol: O₂ oxidoreductase) from leaves of Tanacetum vulgare has been purified 5150-fold to homogeneity on disc electrophoresis and gel electrofocussing. The enzyme which is probably flavoprotein, has molecular weight 180 000 daltons and is comprised of two sub-units of 94 000 and 75 000 daltons. It is active over a broad range (pH 5–9) and best accepts primary aliphatic alcohols with 6 to 10 carbons, especially those with a 2-ene group. K_m values for hex-trans-2-ene-1-ol, geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) and n-octanol were 0.19, 1.56 and 0.49 mM respectively. The significance of the enzyme in the formation of leaf aldehyde (hex-trans-2-ene-1-al) and in terpene metabolism is discussed.     

Incorporation of shikimate and other precursors into aromatic amino acids and prenylquinones of isolated spinach chloroplasts

Under conditions of photosynthesis, shikimate-[1,6-¹⁴C] and D,L-tyrosine-[β-¹⁴C] were incorporated into the aromatic amino acids Phe, Tyr and Trp, and the prenylquinone and α-tocopherol by intact spinach chloroplasts. This might indicate the presence of enzymes of shikimate pathway in chloroplasts.     

Improved rates of CO2-fixation by intact chloroplasts isolated in media with KCl as the osmoticum

Intact chloroplasts were isolated from spinach leaves using media with either 330 mM sorbitol or 200 mM KCl as the osmoticum. Chloroplasts isolated in KCl exhibited higher rates of CO₂-dependent oxygen evolution in nine out of ten experiments, the average increase being 43%. Chloroplasts isolated in KCl routinely achieved rates of CO₂-dependent oxygen evolution of 200–300 mol·mg chlorophyll^-1·hour^-1 at 20°C. Intact chloroplasts were also isolated in media with 200 mM NaCl or choline chloride but the rates of CO₂ fixation were not superior to those isolated in sorbitol media. The K⁺ content of chloroplasts isolated in KCl media was higher than for chloroplasts isolated in sorbitol. It is suggested that the use of KCl as an osmoticum prevents the loss of chloroplast K⁺ which can occur during isolation in sorbitol media. Chloroplasts isolated in KCl lost, on average, 36% of the initial CO₂ fixation activity after storage for four hours on ice, compared to 24% loss of activity for chloroplasts isolated in sorbitol. This increased loss of activity was not observed if KCl was used in the grinding medium and sorbitol or glycinebetaine in the resuspension media. For measurement of the maximum photosynthetic capacity in vitro, the use of KCl in the grinding medium may be better than sorbitol.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - EDTA ethlenediamine tetraacetic acid     

Determination of plastoquinone-9 in chloroplasts by high pressure liquid chromatography

A method for the specific determination of nmol quantities of plastoquinone-9 in chloroplasts is described. HPLC of a heptane extract of chloroplasts separated the plastoquinone-9 from other material permitted a final spectrophotometric assay. Levels of plastoquinone-9 plastoquinol-9 were determined in dark adapted and illuminated chloroplasts.     

脂肪酶的底物特异性及其应用潜力

不同来源的Lipase对底物油脂中脂肪酸的链长、不饱和度及不饱和脂肪酸的双键位置表现出不同的脂肪酸特异性;对甘油酯中Sn-1(3)和Sn-2位酯键具有不同的位置特异性;对甘油酯中立体对映结构的1位和3位酯键呈现不同的立体特异性,脂肪酶能够催化酯水解和酯合成(或酯交换)反应,用于制备甘油单酯、多不饱和脂肪酸及其酯和具有光学活性的有机化合物,因此它在油酯加工和有机合成中具有很大的应用潜力.     

Interspecific Association and Substrate Specificity in Tardigrades from Florida, Southeastern United States

The distribution of terrestrial tardigrades in the Gulf Coast states of the United States is poorly known. Only one species has been reported in Florida. In this study moss, liverwort, lichen and fern samples (47 identified species) from trees and shrubs were collected from all 67 Florida counties. These samples contained 20 species of tardigrade. All possible pairs of tardigrade species and tardigrade and substrate were tested for interspecific association. Only three significant negative and one positive interspecific association between tardigrades were detected. Evidence for substrate specificity was weak. Although some tardigrade species were significantly associated with mosses or foliose lichens in general, no significant association between a tardigrade species and a substrate species was detected.     

Substrate Specificity of a Novel Alcohol Resistant Metalloproteinase,Vimelysin, from Vibrio sp. T1800

Vimelysin is a novel alcohol resistant metalloproteinase from Vibrio sp. T1800. The substrate specificity of vimelysin was studied by using natural and furylacryloyl dipeptide substrates. Vimelysin cleaved mainly Pro⁷-Phe⁸ bond and slightly Tyr⁴-Ile⁵ bond in human angiotensin I. Vimelysin also cleaved mainly Phe²⁴-Phe²⁵ and Tyr¹⁶-Leu¹⁷ bonds, and slightly His⁵-Leu⁶, His¹⁰-Leu¹¹, Ala¹⁴-Leu¹⁵, and Gly²³-Phe²⁴ bonds in oxidized insulin B-chain. The substrate specificity of vimelysin, by using furylacryloyl (Fua) dipeptides were also studied. The ratio of k_cat/K_m for Fua-Gly-Phe-NH₂/Fua-Gly-Leu-NH₂, Fua-Phe-Leu-NH₂/Fua-Gly-Leu-NH₂, and Fua-Phe-Phe-NH₂/Fua-Gly-Leu-NH₂ were 15.9, 27.8, and 59.0, respectively. These results indicate that vimelysin easily recognizes phenylalanine in P1′ positions, which is different from thermolysin.     

The distribution of flavonoids in chloroplasts of twenty five species of vascular plants

A survey was made of the major flavonoids in whole leaf extracts and in chloroplast preparations from twenty five species of vascular plants including Anthophyta (20), Coniferophyta (1), Ginkophyta (1), Pterophyta (2), and Arthrophyta (1). The chloroplasts variously contained derivatives of flavones, C-glycosylflavones, flavonols, flavanones, isoflavones, 3-deoxyanthocyanidins, and anthocyanins. Twenty three species contain one or more flavonoids in isolated chloroplast, usually in a pattern quite similar to that found in whole-leaf extracts but occasionally showing enrichment of one or more flavonoids in the chloroplasts. Flavonoids are apparently absent from chloroplasts of Phaseolus aureus and Morus alba although whole-leaf extracts of these species are rich in quercetin derivatives.     

Simultaneous measurement of changes in red and blue fluorescence in illuminated isolated chloroplasts and leaf pieces: The contribution of NADPH to the blue fluorescence signal

A newly developed nitrogen laser fluorimeter insensitive to actinic illumination was used to follow simultaneously the light induced changes in red and blue fluorescence of intact isolated spinach chloroplasts and leaf pieces. The recorded variable blue fluorescence was linked to a water soluble component of intact isolated chloroplasts, depended on Photosystem I, and was related to changes in carbon metabolism. From the comparison of changes in intact and broken chloroplasts and from fluorescence spectra under different conditions, it was concluded that the variation in NADPH was the major cause for the changes in blue fluorescence. This study opens a path towards continuous and non-destructive monitoring of NADPH redox state in chloroplasts and leaves.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - DLGA DL-glyceraldehyde - FNR ferredoxin-NADP reductase - FWHM full width at half maximum - LED light emitting diodes - OAA oxaloacetate - q_N non-photochemical quenching - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - q_P photochemical quenching - PPFD photosynthetic photon flux density - Q_A primary quinone acceptor of Photosystem II Preliminary results of this work were presented at the First Conference on the Physiology and Biochemistry of high Mountain Plants, 2–3 July 1992, Villar d'Arene, France.     

Association by 2,3-dihydroxybenzaldehyde of monomeric phenolase in spinach chloroplasts

In the presence of 5 mM 2,3-dihydroxybenzaldehyde, the monomeric phenolase (MW 36000) of spinach chloroplasts is completely converted to its dimer within 6 hr without significant change in activity. The aldehyde at concentrations higher than 0.25 mM could bring about this conversion after 18 hr treatment. The association of the two monomers becomes tighter with increasing concentration of the aldehyde. The dimer gave rise to a higher MW protein after freezing briefly. Several mono- and dihydroxybenzaldehydes, 2,3-dihydroxybenzoic acid, and o-vanillin did not produce the dimer.     

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.     

The cyclic nucleotide phosphodiesterases of spinach chloroplasts and microsomes

Cyclic nucleotide phosphodiesterase was extracted from intact chloroplasts and partially purified. Peak 1_c activity from Sephadex G-200 was resolved by electrophoresis into two major bands (MWs 1.87 × 10⁵ and 3.7 × 10⁵). Both also possessed acid phosphatase, ribonuclease, nucleotidase and ATPase. The chloroplast peak 1_c cyclic nueleotide phosphodiesterase was located in the envelope. Peak 1_m cyclic nucleotide phosphodiesterase obtained from the microsomal fraction had a MW of 2.63 × 10⁵. Electrophoresis separated 1_m into two bands of cyclic nucleotide phosphodiesterase activity (MWs 2.63 × 10⁵ and 1.28 × 10⁵). Both contain ATPase, ribonuclease, nucleotidase, but not acid phosphatase. Peak 1_c has high activity towards 3′:5′-cyclic AMP and 3′:5′-cyclic GMP but little towards 2′:3′-cyclic nucleotides. Peak 1_m showed most activity towards 2′:3′-cyclic AMP, 2′:3′-cyclic GMP and 2′:3′-cyclic CMP with little activity towards 3′:5′-cyclic nucleotides. With 1_c, 3′:5′-cyclic AMP and 3′:5′-cyclic GMP exhibit mixed-type inhibition towards one another. The 2′:3′-cyclic AMP phosphodiesterase 1_m was competitively inhibited by 2′:3′-cyclic GMP. p-Chloromercuribenzoate inhibits 1_c but not 1_m. Electrophoresis after dissociation indicates that 1_c and 1_m are both enzyme complexes. After dissociation, the 1_c complex but not that of 1_m could be reassociated. The ribonuclease of the 1_m complex hydrolyses RNA to yield 2′:3′-cyclic nucleotides as the main products. These results are compatible with the 1_c cyclic nucleotide phosphodiesterase complex being involved in the metabolism of 3′:5′-cyclic AMP, and the 1_m complex being concerned with RNA catabolism.     

Occurrence and biosynthesis of cyclopentenyl fatty acids in leaves and chloroplasts of flacourtiaceae

Cyclopentenyl fatty acids, the unusual fatty acids occurring naturally in certain Flacourtiaceae, have been detected for the first time in leaves of various plants belonging to the tribes Pangieae, Oncobeae and Flacourtieae. In leaves and chloroplasts of Caloncoba echinata (Oncobeae) cyclopentenyl fatty acids are synthesized from aspartate plus pyruvate or glutamate plus acetate. The biogenesis of the cyclopentene ring occurs from a C₇, compound, that may be formed by either pair of substrates.     

Purification and Substrate Specificity of Honeybee,Apis mellifera L., α-Glucosidase III

α-Glucosidase III, which was different in substrate specificity from honeybee α-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of α-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in α-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl α-glucoside, maltose, and maltotriose, and by extremely high K_m for substrates, compared with those of α-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k₀ values for maltose, sucrose, p-nitrophenyl α-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the K_m values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (A_i’s) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the A_i value at subsite 3 was larger than that of subsite 1.