Involvement of chlorophyllase in chlorophyll metabolism in olive varieties with high and low chlorophyll content

Olive fruits of the Arbequina variety are differentiated from those of Hojiblanca and Picual by the differing presence of 13²-OH-chlorophyll a and of dephytylated chlorophyll derivatives during the life cycle of the fruit. During the fruit growth stage, which coincides with chlorophyll synthesis, chlorophyllase (EC: 3.1.1.14) is present in the three varieties but only yields chlorophyllides in Arbequina. The presence of oxidized catabolites of chlorophyll a in fruits of the Arbequina variety during this same period confirms the activity of oxidative enzyme systems. The low synthesis of chlorophylls in the fruits of the Arbequina variety is associated with the fact that, during the natural biosynthetic turnover, the catabolic pathway is more potentiated than the anabolic one. In the ripening phase, in the Hojiblanca and Picual fruits, chlorophyllase activity was measured but the absence of chlorophyllides showed that this enzyme remains latent and that oxidative enzymes are the ones taking part in the chlorophyll disappearance. In the Arbequina variety, both chlorophyllase and oxidative enzymes are responsible for the chlorophyll degradation.     

Localization of chlorophyllase in the chloroplast envelope

Chlorophyllase catalyzes the first step in the catabolic pathway of chlorophyll. It is a constitutive enzyme located in chloroplast membranes. In isolated plastids the hydrolysis of the endogenous chlorophyll does not take place unless the membranes are solubilized in the presence of detergent. The structural latency of chlorophyllase activity appears to be due to the differential locations of substrate and enzyme within the plastids. Envelope membranes prepared from both chloroplasts and gerontoplasts contain chlorophyllase activity. The isolation of envelopes is associated with a marked increase in chlorophyllase activity per unit of protein. Yields of chlorophyllase and of specific envelope markers in the final preparations are similar, suggesting that the enzyme may be located in the envelope. It is hypothesized that the breakdown of chlorophyll during leaf senescence requires a mechanism that mediates the transfer of chlorophyll from the thylakoidal pigment-protein complexes to the sites of catabolic reactions in the envelope.Abbreviations ACT acyl CoA thioesterase - Chl chlorophyll - Chlide chlorophyllide - PC phosphatidylcholine     

Mechanistic analysis of wheat chlorophyllase

Chlorophyllase catalyzes the initial step in the degradation of chlorophyll and plays a key role in leaf senescence and fruit ripening. Here, we report the cloning of chlorophyllase from Triticum aestivum (wheat) and provide a detailed mechanistic analysis of the enzyme. Purification of recombinant chlorophyllase from an Escherichia coli expression system indicates that the enzyme functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll (k(cat) = 566 min(-1); K(m) = 63 microM) and was active over a broad temperature range (10-75 degrees C). In addition, the enzyme displays carboxylesterase activity toward p-nitrophenyl (PNP)-butyrate, PNP-decanoate, and PNP-palmitate. The pH-dependence of the reaction showed the involvement of an active site residue with a pK(a) of approximately 6.5 for both k(cat) and k(cat)/K(m) with chlorophyll, PNP-butyrate, and PNP-decanoate. Using these substrates, solvent kinetic isotope effects ranging from 1.5 to 1.9 and from 1.4 to 1.9 on k(cat) and k(cat)/K(m), respectively, were observed. Proton inventory experiments suggest the transfer of a single proton in the rate-limiting step. Our analysis of wheat chlorophyllase indicates that the enzyme uses a charge-relay mechanism similar to other carboxylesterases for catalysis. Understanding the activity and mechanism of chlorophyllase provides insight on the biological and chemical control of senescence in plants and lays the groundwork for biotechnological improvement of this enzyme.     

Enzymatic chlorophyll degradation in wheat near-isogenic lines elicited by cereal aphid (Homoptera: Aphididae) feeding

Chlorophyll degradation enzyme (i.e., chlorophyllase, Mg-dechelatase, and chlorophyll oxidase) activities of aphid-infested and uninfested 'Tugela' and Tugela near-isogenic wheat lines (i.e., Tugela-Dn1, Tugela-Dn2, and Tugela-Dn5) were assayed. Chlorophyllase activity was higher in bird cherry-oat aphid, Rhopalosiphum padi (L.) (Homoptera: Aphididae),-infested wheat lines compared with Russian wheat aphid, Diuraphis noxia (Mordvilko) (Homoptera: Aphididae)]-infested and uninfested plants. Mg-dechelatase activity was higher in D. noxia-infested wheat lines than in R. padi-infested and uninfested plants. Also, Mg-dechelatase activity was lower in Tugela wheat infested with D. noxia than in Tugela near-isogenic lines with Dn genes. Based on the in vitro assays of chlorophyll degradation enzyme (i.e., chlorophyllase and Mg-dechelatase) activities, we proposed that the chlorotic symptoms observed on D. noxia-infested Tugela wheat were most likely to be elicited by unbalanced chlorophyll biosynthesis and degradation.     

Studies on chlorophyllase in tea leaves III. Properties of soluble and insoluble chlorophyllases

In contrast to previous knowledge of chlorophyllase activityin higher plants, significant enzyme activity was isolated fromtea leaves in a soluble state. Soluble chlorophyllase was partially purified by proceduresincluding ammonium sulfate fractionation (Preparation I). Theinsoluble fraction was extracted, by solubilizing it with SDC,from the methanol-acetone powder of sediments of the leaf homogenate,from which the water-soluble enzyme had been completely removedby repeated extraction. This initially insoluble enzyme wasalso partially purified (Preparation II). Specific activities(mg chlorophyll a hydrolyzed per hr per mg protein, 7.2 forPreparation I, and 12.4 for Preparation II), were much higherthan those reported for other plant material. The soluble enzyme was more resistant to PCMB, lipase and heattreatment. The two enzymes differed in optimum temperature andoptimum acetone concentration needed for the reaction, but showedthe same optimum pH, and same Km value. The Km value was thesame (7 µM) for reactions with 30% and 50% acetone. These results suggest that, in spite of differences in locationand extractability, activities of the soluble and insoluble(solubilized) chlorophyllase in tea leaves are attributableto the same enzyme. (Received March 8, 1972; )     

铁胁迫诱导的赤潮异弯藻细胞生化组成变化

研究了铁胁迫对赤潮异弯藻细胞生化组成的影响．结果表明。铁胁迫下的细胞内所有色素浓度均降低,同富铁条件相比。铁胁迫下的细胞内叶绿素a浓度降低2倍多。叶绿素c也有相当程度的降低,因此,铁胁迫下的胞内叶绿素c与叶绿素a的比率变化不大．铁胁迫下的细胞内类胡萝卜素的含量降低了1．5倍,因此在铁胁迫下的细胞内类胡萝卜素相对于叶绿素a的比例升高。碳水化合物含量随培养基内铁浓度降低而下降,与铁丰富条件(10μmol·L-1)相比,受铁胁迫的细胞可溶蛋白电泳图谱中17kDa和55kDa附近的带明显增加,而在20kDa和35kDa附近的蛋白带降低     

Chlorophyllase versus pheophytinase as candidates for chlorophyll dephytilation during senescence of broccoli

Degradation of chlorophylls during senescence is a highly regulated process which requires the concerted action of several enzymes. Traditionally, it has been stated that the dismantling process of the chlorophyll molecule begins with a dephytilation step, followed by Mg²⁺ removal and other breakdown reactions. Recently, new evidence suggests the possibility of a rearrangement in the first two steps of this process, occurring Mg²⁺ removal prior to the loss of the phytol side chain. With the purpose of approximating to the real sequential order of these reactions and to assess if dephytilation occurs on intact (catalyzed by chlorophyllase) or Mg-free (catalyzed by pheophytinase) chlorophyll, expression of both genes was analyzed in broccoli tissue during senescence. Samples of broccoli florets treated with plant hormones, such as cytokinin and ethylene were utilized, as to assess the effect of such compounds on the expression of these genes. Results showed that chlorophyllase expression did not correlate to typical expression patterns for genes related to senescence, since a decrease in expression during senescence was found for one of the two chlorophyllase genes analyzed, and the hormonal-treatment effects on gene expression did not match those observed on chlorophyll content for both chlorophyllase genes. Pheophytinase expression patterns, on the other hand, displayed an increase in the first 3 days of induced senescence, followed by lower expression values towards the end of the experiment. Samples subjected to postharvest treatments mostly showed an inhibition of pheophytinase expression, especially in samples in which degradation of chlorophylls had been delayed. These results suggest that pheophytinase expression correlates to the visual manifestation of postharvest treatments, supporting the possibility that this enzyme is responsible for the dephytilation step in chlorophyll breakdown.     

Development of a high-throughput purification method and a continuous assay system for chlorophyllase

In the degradation of chlorophyll, chlorophyllase catalyzes the initial hydrolysis of the phytol moiety from the pigment. Since chlorophyll degradation is a defining feature of plant senescence, compounds inhibiting chlorophyllase activity may delay senescence, thereby improving shelf life and appearance of plant products. Here we describe the development of a 96-well plate-based purification and assay system for measuring chlorophyllase activity. Integrated lysis and immobilized metal affinity chromatography plates were used for purifying recombinant hexahistidine-tagged Triticum aestivum (wheat) chlorophyllase from Escherichia coli. Chlorophyllase assays using chlorophyll as a substrate showed that the immobilized fusion protein displayed kinetic parameters similar to those of recombinant enzyme purified by affinity chromatography; however, the need to extract reaction products from a multiwell plate limits the value of this assay for high-throughput screening applications. Replacing chlorophyll with p-nitrophenyl-ester substrates eliminates the extraction step and allows for continuous measurement of chlorophyllase activity in a multiwell plate format. Determination of steady state kinetic constants, pH rate profile, the inhibitory effects of metal ions and esterase inhibitors, and the effect of functional group-modifying reagents validated the utility of the plate-based system. The combined purification and assay system provides a convenient and rapid method for the assessment of chlorophyllase activity.     

Properties of chlorophyllase from Capsicum annuum L. fruits

The in vitro properties of semi-purified chlorophyllase (chlorophyll-chlorophyllido hydrolase, EC 3.1.1.14) from Capsicum annuum fruits have been studied. The enzyme showed an optimum of activity at pH 8.5 and 50 degrees C. Substrate specificity was studied for chlorophyll (Chl) a, Chl b, pheophytin (Phe) a and Phe b, with Km values of 10.70, 4.04, 2.67 and 6.37 microM respectively. Substrate inhibition was found for Phe b at concentrations higher than 5 microM. Chlorophyllase action on Chl a' and Chl b' was also studied but no hydrolysis was observed, suggesting that the mechanism of action depends on the configuration at C-13(2) in the chlorophyll molecule, with the enzyme acting only on compounds with R132 stereochemistry. The effect of various metals (Mg2+, Hg2+, Cu2+, Zn2+ Co2+, Fe2+ and Fe3+) was also investigated, and a general inhibitory effect was found, this being more marked for Hg2+ and Fe2+. Functional groups such as -SH and -S-S- seemed to participate in the formation of the enzyme-substrate complex. Chelating ion and the carbonyl group at C3 appeared to be important in substrate recognition by the enzyme. The method for measuring Chlase activity, including HPLC separation of substrate and product, has been optimized.     

Ribonuclease and Chlorophyllase Activities in Senescing Leaves

The activities of two enzymes, ribonuclease and chlorophyllase were investigated during the senescence of leaves. Ribonuclease activities were measured in primary leaves of Phaseolus vulgaris, and related to the levels of nucleic acid, protein and chlorophyll. Similarly, changes in chlorophyllase activity during senescence of leaves of Raphanus sativus were measured and related to chlorophyll. During senescence the levels of each enzyme as well as its respective substrate declined. Retardation of senescence, by excision of young tissue from intact plants or by treatment of detached leaves with cytokinins resulted in a maintainace of both the substrate and enzyme levels. It was concluded that high levels of ribonuclease and chlorophyllase activity are not linked directly with the degradation of RNA and chlorophyll during leaf senescence.     

Degradation pathway(s) of chlorophyll: what has gene cloning revealed?

The mechanism responsible for the degreening of plants and the degradation of chlorophyll was unclear for many years. However, recent studies have identified the colorless intermediates and helped to construct a basic pathway for degradation. After the successive removal of phytol and Mg21 from the chlorophyll molecule by chlorophyllase and 'Mg dechelatase', pheophorbide a is cleaved and reduced to yield a colorless, open tetrapyrrole intermediate. After further modifications, this is finally transported to the vacuole. Cloning the genes for chlorophyllase isozymes and the reductase should help to elucidate the physiological roles of each enzyme at a molecular level.     

Purification and Characterization of Chlorophyllase from Alga (Phaeodactylum tricornutum) by Preparative Isoelectric Focusing

Chlorophyllase from a diatom alga (Phaeodactylum tricornutum) was obtained and the partially purified extract has been further purified using preparative isoelectric focusing on a Rotofor cell. Three fractions, FI, FII, and FIII, were separated from the Rotofor cell and salt and ampholytes were removed to give fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′ showed specific activities of 15.2 × 10^?4, 226.7 ×10^?4 and 33.8 × 10^?4 µmol/mg protein/min, respectively. Most of the enzyme activity (84%) was in fraction FII′. The optimum pH for chlorophyllase activity was 8.0 for FI′ and 8.5 for both FII′ and FIII′. Apparent K_m values for enzyme fractions FI′, FII′, and FIII′ were 2.1nM, 2.3nM, and 2.0 nM, respectively. Enzyme fractions FII′ and FIII′ showed higher chlorophyllase activity towards the partially purified chlorophyll when it was compared to that with the crude chlorophyll as well as with both chlorophylls a and b. However, the enzyme fraction FI′ had higher activity towards the crude chlorophyll when it was compared to that with both chlorophylls a and b, but with a preference for chlorophyll a over chlorophyll b. The inhibitory effect of diisopropyl flurophosphate (DIFP) on chlorophyllase activity demonstrates a noncompetitive inhibitor kinetics with K_i values of 1.29mM, 2.14mM, and 0.71mM for FI′. FII′, and FIII′, respectively.     

Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either 'mitochondrial' or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk.     

Effect of indoleacetic Acid and hydroxyproline on isoenzymes of peroxidase in wheat coleoptiles

Indoleacetic acid at 0.017 millimolar inhibited the formation of three peroxidase isoenzymes in both soluble and wall-bound enzyme fractions of wheat coleoptile (Triticum vulgare) tissue. Hydroxyproline at 1 millimolar prevented the indoleacetic acid-induced inhibition. Indoleacetic acid oxidase activity in the soluble fraction was decreased by indoleacetic acid and was restored by hydroxyproline. Most of the indoleacetic acid oxidase activity was located in the electrophoretic zones occupied by two of the peroxidase isoenzymes influenced by indoleacetic acid and hydroxyproline. At least part of the effect of hydroxyproline on auxin-induced elongation of coleoptile tissue may be through control of auxin levels by indoleacetic acid oxidase.     

Succinyl-CoA synthetase in greening maize leaves

In extracts of greening maize leaves succinyl-CoA synthetase was present in both a particulate and a soluble fraction. Aqueous and non-aqueous fractionation together with determination of chlorophyll content and cytochrome oxidase activity indicated that the enzyme was neither located, nor originated in plastids. Pre-illumination of leaves caused only small increases in the activity of either the particulate or the soluble enzyme. The soluble enzyme was ATP specific and had a low affinity for succinate (Km = 63 mM).     

Fasciola hepatica: the presence of particle-associated and soluble nonspecific acid phosphatases.

The kinetic and physical properties of acid phosphatases in the lysosomal and microsomal fractions of F. hepatica were found to be similar, indicating that they are one and the same enzyme. In contrast, the biochemical properties of the soluble acid phosphatase (EC 3.1.3.2) were quite different from those of the lysosomal and microsomal fractions. This indicated the presence of two distinct forms of the enzyme one particle associated and the other soluble. Electrophoretic heterogeneity of these two types of acid phosphomonoesterase was seen. Two bands of activity were observed in both lysosomal and microsomal fractions and three bands in the soluble fraction.