Inactivation of S-adenosylhomocysteine hydrolase by 5'-deoxy-5'-methylthioadenosine

In the coupling of ATP pyrophosphorolysis to Ca²⁺ transport in beef heart mitochondria, for each molecule of ATP cleaved, one proton is released and one Ca²⁺ is transported into the interior space. With the use of tritium labelled ATP, it could be shown that ATP is pyrophosphorylyzed into a species equivalent to Pi that moves inward, and a species equivalent to ADP that is extruded into the aqueous space on the exterior of the mitochondrion. The species equivalent to Pi has been identified as a negatively charged form of Pi (PO^?) and the species equivalent to ADP as a positively charged form (ADP⁺). The inward flow of PO^? is coupled to the inward flow of Ca²⁺ in 1:1 stoichiometry—a token that Ca²⁺ must enter in the form of Ca²⁺A^?, where A^? is a monovalent anion. During ATP pyrophosphorolysis protons are released on the I side and taken up on the M side—one proton for each molecule of ATP cleaved. The alkalinization of the matrix space leads to the deposition of Ca₃(PO₄)₂ and to the extrusion of the two species released by this deposition (Pi, K⁺). Two thirds of the PO^? is trapped as Ca₃(PO₄)₂ in the matrix space and one third is extruded into the external space. The extrusion of K⁺ provides a mechanism by which protons can be continuously brought into the matrix space to sustain a high rate of coupled pyrophosphorolysis of ATP. The coupling pattern for Ca²⁺ transport driven by ATP pyrophosphorolysis is identical with the corresponding pattern for Ca²⁺ transport driven by electron transfer. This identity is suggestive that coupling mediated by the Fo-F₁ system and coupling mediated by electron transfer complexes are mechanistically indistinguishable.     

Antifungal antibiotics and Siba inhibit 1-aminocyclopropane-1-carboxylic acid synthase activity

The antifungal antibiotics Sinefungin and A9145C isolated from Streptomyces griseolus and the synthetic nucleoside Siba, which are analogs of S-adenosylmethionine, inhibit the activity of 1-aminocyclopropane 1-carboxylic acid synthase from tomato fruits. Sinefungin and Siba were shown to be more potent inhibitors than A9145C. In extracts of green fruits, the enzyme activity was inhibited by Sinefungin with an I50 of 1 microM, which was similar to that caused by aminoethoxyvinylglycine, and by Siba with an I50 of 100 microM; in extracts from red tomatoes, the I50's were 25 microM and 100 microM, respectively. The inhibition of ACC synthase by these analogs could be reversed by gel filtration chromatography.     

Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes

Spermidine synthase (EC 2.5.1.16) was purified to homogeneity for the cytosol of soybean (Glycine max) axes using ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, ω-aminooctyl-Sepharose and ATPA-Sepharose. The molecular mass of the enzyme estimated by gel filtration and SDS–PAGE is 74 kDa. Cadaverin and 1,6-diaminohexane could not replace putrescine as the aminopropyl acceptor. Kinetic behaviors of the substrate are consistent with a ping pong mechanism. The kinetic mechanism is further supported by direct evidence confirming the presence of an aminopropylated enzyme and identification of product, 5′-deoxy-5′-methylthioadenosine, prior to adding putrescine. The K_m values for decarboxylated S-adenosylmethionine and putrescine are 0.43 μM and 32.45 μM, respectively. Optimum pH and temperature for the enzyme reaction are 8.5 and 37°C, respectively. The enzyme activity is inhibited by N-ethylmaleimide and DTNB, but stimulated by Co²⁺, Cu²⁺ and Ca²⁺ significantly, suggesting that these metal ions could be the cellular regulators in polyamine biosynthesis.     

Synthesis and Degradation of 1-Aminocyclopropane-1-carboxylic Acid by Penicillium citrinum

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M _r 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the K _m for S-adenosyl-L-methionine of 1.74 mM and k _cat of 0.56 s^-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M _r 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a K _m for ACC of 4.8 mM and k _cat of 3.52 s^-1. The presence of 7 mM Cu²⁺ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.     

Effects of Cold Acclimation and Salicylic Acid on Changes in ACC and MACC Contents in Maize during Chilling

The effect of 0.5 mM salicylic acid (SA) pretreatment and of growing at hardening temperatures on chilling-induced changes in 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl 1-aminocyclopropane-1-carboxylic acid (MACC) was investigated in young maize (Zea mays L.) plants grown in hydroponic solution at 22/20 °C. Chilling at 5 °C caused an increase in ACC content;however, this increase was less pronounced in plants cold acclimated at 13/11 °C 4 d before the chilling treatment, and in those which were pretreated with SA for 1 d before the cold stress. Changes in MACC at low temperature showed no correlation with chilling tolerance in maize.     

Dynamic interaction between functional groups in the active site of glycogen phosphorylase b

The quenching of coenzyme fluorescence in glycogen phosphorylase b is reinvestigated. Data with anionic quenchers show deviations from the original Stern-Volmer kinetics. A kinetic analysis based on measured lifetime data indicates a collisional quenching process, which is, however, not diffusion-controlled. It is proposed, that the quenching takes place primarily by enzyme-bound quencher species. The observed inhibition of the enzyme reaction by I- and IO-3 is consistent with this hypothesis. The inhibition pattern and spectral investigation refer to a true competition with the substrate, glucose-1-phosphate. So, this dynamic quenching can be regarded as an indicator of rapid conformational fluctuations which bring the two important active-site groups in contact. Effect of ligand binding on the quenching of coenzyme fluorescence should also be revaluated according to these results.     

Chemiluminescent assay of lipid peroxide in plasma using cytochrome c heme peptide

A convenient assay specific to lipid hydroperoxide in plasma is presented. Cytochrome c heme peptide obtained from Saccharomyces was found to emit a strong chemiluminescence with any hydroperoxide, but not with TBA-reactive substances. The benefit of measuring this luminescence using photon counting is discussed with respect to in vivo lipid peroxidation.     

An ethylene-forming enzyme in Citrus unshiu fruits

An ethylene-forming enzyme from Citrus unshiu fruits was purified some 630-fold. The enzyme catalysed ethylene formation from 1-aminocyclopropane-1-carboxylic acid in the presence of pyridoxal phosphate, β-indoleacetic acid, Mn²⁺ and 2,4-dichlorophenol. It behaved as a protein of MW 40 000 on Sephacryl S-200 gel filtration, and gave one band corresponding to a MW of 25 000 on SDS-PAGE. It had a specific activity of 0.025 μmol/min·mg protein. It exhibited IAA oxidase activity and had no guaiacol peroxidase or NADH oxidase activity. Its K_m for ACC was 2.8 mM, and its pH optimum was 5.7. It was inhibited by potassium cyanide n-propyl gallate and Tiron. d-Mannose, histidine, iodoacetate, PCMB, dimethylfuran and superoxide dismutase showed no inhibition. β-Indoleacrylic acid against IAA competitively inhibited ethylene formation. Other IAA analogues, such as β-indolepropionic acid, β-indolecarboxylic acid and β-indolebutylic acid, slightly stimulated ethylene formation. β-Indoleacrylic acid against 1-aminocyclopropane-1-carboxylic acid non-competitively inhibited ethylene formation. Ascorbate was a potent inhibitor. The inhibitory effects, however, were not always reproduced in vivo. It is difficult to identify this enzyme system as a natural in vivo system from the above observations. Nevertheless, the possible in vivo participation of this in vitro enzyme system is discussed.     

乙烯与水浮莲(Pistia stratiotes)种子需光性休眠的关系

水浮莲种子是一种奇特的需光种子。在黑暗中,GA_2或BA均不能代替光照诱导萌发,可是0.1μl/l乙烯却能引起部分种子萌发,在1000μ1/1乙烯的作用下,发芽率可达80％,接近全光照处理的萌发水平(91％发芽率)。ACC也能诱导水浮莲种子的萌发,0.1 mM浓度可获30％发芽率。在较短光照下,ACC对种子萌发有增效作用。在光照前应用ACC,其诱导效应大于两者同时施用。在照光萌发中,种子的内源ACC含量及乙烯释放量均显著增加。CoCl_2和AOA均能抑制光的诱导萌发。推论光打破休眠诱导萌发的作用是与乙烯的生成密切相关。     

Identification and characterization of three putative genes for 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments

The polymerase chain reaction (PCR) was used to produce 3 putative clones for ACC synthase from etiolated mung bean (Vigna radiata Rwilcz cv. Berken) hypocotyls. This was accomplished by utilizing genomic DNA from mung bean and degenerate primers made from information derived from highly conserved regions of ACC synthase from different plant tissues. The total length of pMAC-1, pMAC-2 and pMAC-3 are 308, 321, and 326 bp, respectively, all of which code for 68 amino acids. The introns for pMAC-1, pMAC-2 and pMAC-3 are 92, 105, and 110 bp, respectively. The degrees of homology at the DNA level for each of these clones is ca. 80% in their coding region and ca. 50% in their respective introns. This is the first report providing evidence that there are at least 3 genes for ACC synthase in etiolated mung bean.     

Effects of 1-aminocyclopropane-1-carboxylic acid and oxygen concentrations on in vivo and in vitro activity of ACC oxidase of sunflower hypocotyl segments

In vivo ethylene production by hypocotyl segments of sunflower seedlings and in vitro activity of 1-aminocyclopropane-1-carboxylic acid oxidase (formerly ethylene-forming enzyme) extacted from the same tissues increase with increasing concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and oxygen. ACC oxidase activity follows Michaelis-Menten kinetics. The apparent Km values of the enzyme towards ACC, estimated in vivo and in vitro, are respectively 219 M and 20.6 M. Both Km values towards O₂ are similar, ca 10.6–11.4%. A decrease in concentration in one of the substrates (ACC or O₂) results in an increase in in vivo apparent Km of ACC oxidase for the other substrate. On the contrary, Km values of the enzyme towards ACC or O₂ estimated in vitro are not dependent upon the concentration of the other substrate (ACC or O₂).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - MACC malonylate 1-aminocyclopropane-1-carboxylic acid - SD standard deviation