Cysteine proteinase forms in sprouting potato tuber

Transformation of plants with exogenous proteinase inhibitor genes represents an attractive strategy for the biological control of insect pests. However, such a strategy necessitates a thorough characterization of endogenous proteinases. which represent potential target enzymes for the exogenous inhibitors produced. In the present study. changes in general endoproteolytic activity were monitored during sprouting of potato ( Solanum tuberosum L. cv. Kennebec) tuber. Quantitative data obtained using standard procedures showed that an increase in cysteine proteinase (EC 3.4.22) activity occurs during sprouting. This increased activity results from the gradual appearance of new cysteine proteinase forms, as demonstrated by the use of class-specific proteinase activity gels. While only one cysteine proteinase form was present during early sprouting, at least six new active forms of the same class were shown to appear gradually after the mature tuber was sown, suggesting the involvement of a complex cysteine proteolytic system in the last stages of tuber protein breakdown. Interestingly, oryzacystatins I and II. two cysteine proleinase inhibitors potentially useful for insect control, had no effect on any tuber proteinase delected. Similar results were obtained with leaf, stem and stolon proteinases. This apparent absence of direct interference supports the potential of oryzacystatin genes for production of insect-tolerant transgenie potato plants.     

A channel that allows inwardly directed fluxes of anions in protoplasts derived from wheat roots

An anion channel that only allows outward current flow (anion influx) has been identified in protoplasts derived from wheat (Triticum aestivum L., Triticum turgidum L.) roots. The anion outward rectifier (anion OR) measured by patch-clamp of whole cells activated very quickly, usually reaching a steady-state level in less than 100 ms and was easily distinguished from the cation outward rectifier (cation OR) which activated more slowly during membrane depolarisation. The anion OR is permeable to NO ₃ ^– and Cl^–, moderately permeable to I^–, and relatively impermeable to H₂PO₄/^– and ClO₄/^–. An anomalous mole-fraction effect between ClO_4/ ^– and Cl^– was observed on the outward current, indicating that the channel is a multi-ion pore. The anion OR is gated by both voltage and external anion concentration such that it activates near to the equilibrium potential for the permeant anion. It activated at more negative membrane potentials when NO ₃ ^– was substituted for Cl^– in the external medium, indicating that the channel may function to allow NO ₃ ^– influx under luxuriant external NO ₃ ^– concentrations. For most experiments, K⁺ and Cl^– were the main cation and anion in solution, and under these conditions it appeared likely that the anion OR functioned in membrane-potential regulation by facilitating a Cl^– influx at membrane potentials more positive than the chloride reversal potential (E_Cl). If E_Cl was more negative than the K⁺ reversal potential (E_K) then the anion OR dominated but both the anion and cation ORs occurred together when the membrane potential difference (V_m) was positive of both E_Cl and E_K. The cation OR was inhibited by increasing external Cl^– concentrations, but the anion OR was not affected by external K⁺ or Na⁺ concentration. The anion-transport inhibitors, zinc and phenylglyoxal were ineffective in blocking the anion OR. 4,4-Di-isothiocyanostilbene-2, 2-disulfonic acid (DIDS) irreversibly blocked about 34% of the current when applied extracellularly at a concentration of 25 M, and about 69% at a concentration of 200 M. However, DIDS (200 M) also occasionally acted as an irreversible blocker of the cation OR. Perchlorate blocked irreversibly 75% of the current at an external concentration of 10 mM and did not block the cation OR. Whole-cell currents also indicated that the anion OR was insensitive to external pH (pH=5–7) and calcium concentration ([Ca²⁺]=0.1–10 mM). Increasing intracellular calcium concentration significantly increased the occurrence of the fast outward current in whole cells (P < 0.005, X² test). With approximately 10 nM calcium inside the cell the anion outward current was observed in 64% (n = 45) of cells and with 50 nM calcium inside the cell the anion current was observed in 88% (n = 69) of cells. Single-anion OR channels observed in outside-out patches had a conductance in 300 mM KCl (external) of about 4 pS. When voltage pulses were applied to outside-out patches the average currents were similar to those observed in whole cells. The significance of the anion OR as a likely route for Cl uptake in high salinities is discussed.Abbreviations Bath solution bathing the extracellular face of the membrane - DIDS (4,4-diisothiocyanostilbene-2,2-disulfonic acid) - E_x reversal potential for ion x - OR outward rectifier - Pip solution inside the pipette - TEACl (tetraethyl-ammonium chloride) - V_m membrane potential difference We thank the Australian Research Council for financial support, G.P. Findlay and A. Garrill for helpful discussions, and K. Morris and D. Mackenzie for expert technical assistance. M.S. was supported by an Australian Postgraduate Research Award.     

Similarity between pyridoxal/pyridoxamine phosphate-dependent enzymes involved in dideoxy and deoxyaminosugar biosynthesis and other pyridoxal phosphate enzymes.

A multiple sequence alignment among aspartate aminotransferase, dialkylglycine decarboxylase, and serine hydroxymethyltransferase (DAS) was used for profile databank search. The DAS profile could detect similarities to other pyridoxal or pyridoxamine phosphate-dependent enzymes, like several gene products involved in dideoxysugar and deoxyaminosugar synthesis. The alignment among DAS and such gene products shows the conservation of aspartate 222 and lysine 258, which, in aspartate aminotransferase, interacts with the N1 of the coenzyme pyridine ring and forms the internal Schiff base, respectively. The lysine is replaced by histidine in the pyridoxamine phosphate-dependent gene products. The alignment indicates also that the region encompassing the coenzyme binding site is the most conserved.     

质体醌重组的D1／D2／Cytb559复合物的荧光衰减动力学和光破坏作用

光系统Ⅱ反应中心Ｄ１／Ｄ２／Ｃｙｔｂ５５９ 在分离纯化过程中失去了电子受体ＱＡ 和ＱＢ,人工合成的质体醌可以与Ｄ１／Ｄ２／Ｃｙｔｂ５５９ 复合物发生重组。癸基质体醌（ＤＰＱ）与Ｄ１／Ｄ２／Ｃｙｔｂ５９９ 复合物的重组导致该复合物的荧光强度下降及发射光谱蓝移,同时两个与光化学活性相关的长寿命（２４ ｎｓ和７３ ｎｓ）荧光衰减组分占整个荧光的百分数下降,这些结果表明ＤＰＱ作为Ｐｈｅｏ－ 的电子受体,限制了Ｐ６８０＋ ·Ｐｈｅｏ－ 的电荷重组。ＤＰＱ 的加入对Ｄ１／Ｄ２／Ｃｙｔｂ５５９复合物中Ｃｈｌａ 分子的光破坏敏感性影响不大,但β－胡萝卜素在加入ＤＰＱ 之后可以被光照破坏,这个过程可能与β－胡萝卜素的生理功能相关。     

油菜素内酯促进绿豆上胚轴伸长与蛋白质和核酸合成的关系

用饲喂蛋白质和核酸合成的放射性前体［３ Ｈ］－Ｐｈｅ、［３ Ｈ］－尿嘧啶和［３ Ｈ］－胸腺嘧啶证实了油菜素内酯（ＢＲ）能促进绿豆上胚轴的生长和蛋白质、ＲＮＡ 及ＤＮＡ 的合成。用蛋白质和核酸合成抑制剂（ＣＨ、Ａｃｔ．Ｄ、５－Ｆｕ）进一步探讨它们对上胚轴伸长的抑制作用与蛋白质、ＲＮＡ、ＤＮＡ 和ｍ ＲＮＡ 合成之间的关系。证明了上胚轴的伸长依赖于蛋白质和核酸的合成,尤其是依赖于ｍ ＲＮＡ 的合成。说明ＢＲ是在转录水平上调节基因的表达,进而促进上胚轴的伸长     

Hydrogen peroxide-mediated inactivation of microsomal cytochrome P450 during monooxygenase reactions

Cytochrome P450 can undergo inactivation following monooxygenase reactions in liver microsomes of untreated, phenobarbital and 3-methylcholanthrene-treated rats and rabbits. The acceleration of cytochrome P450 loss in the presence of catalase inhibitors (sodium azide, hydroxylamine) indicates that hydrogen peroxide is involved in hemoprotein degradation. It was revealed that cytochrome P450 is inactivated mainly by H₂O₂ formed through peroxy complex breakdown, whereas H₂O₂ formed via the dismutation of superoxide anions produces a slight inactivating effect. The hydrogen peroxide added outside or formed by a glucose-glucose oxidase system has less of an inactivating effect than H₂O₂ produced within the cytochrome P450 active center. Self-inactivation of cytochrome P450 during oxygenase reactions is highly specific. Other components of the monooxygenase system, such as cytochrome b₅, NADH- and NADPH-specific flavorproteins, undergo no inactivation. The alterations in phospholipid content and in the rate of lipid peroxidation were not observed as well. The inactivation of cytochrome P450 by H₂O₂ is the result of heme loss or destruction without cytochrome P420 formation. Such. a mechanism operates with different substrates and cytochrome P450 species catalyzing the partially coupled monooxygenase reactions.     

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg²⁺-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase -dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergenta availability and cation sensitivity on the apparent distribution of PAPH in neutrophil sub-cellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzyme was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.     

Bayesian signal extraction from noisy FT NMR spectra

Summary The statistical interpretation of the histogram representation of NMR spectra is described, leading to an estimation of the probability density function of the noise. The white-noise and Gaussian hypotheses are discussed, and a new estimator of the noise standard deviation is derived from the histogram strategy. The Bayesian approach to NMR signal detection is presented. This approach homogeneously combines prior knowledge, obtained from the histogram strategy, together with the posterior information resulting from the test of presence of a set of reference shapes in the neighbourhood of each data point. This scheme leads to a new strategy in the local detection of NMR signals in 2D and 3D spectra, which is illustrated by a complete peak-picking algorithm.     

Latency of cathepsin B secreted by human colon carcinoma cells is not linked to secretion of cystacin C and is relieved by neutrophil elastase

The lysosomal cystein proteinase cathepsin B is shown to be secreted by ten human colon carcinoma cell lines and to accumulate in culture media as a latent enzyme. The cell lines also secrete a physiological inhibitor of cathepsin B, cystatin C. A significant correlation was found between secretion of the latent enzyme and the inhibitor (r = 0.755, P < 0.01). The aim of the present study was to modulate the respective secretion of the two antagonists to test whether or not latency of cathepsin B was due to the concomitant secretion of the inhibitor. SW480 colon carcinoma cells were treated with the acidotropic agent ammonium chloride, phorbol 12-myristate 13-acetate, and the inflammatory cytokines TGF-β, TNF-α, and IL-1β. Ammonium chloride significantly increased latent cathepsin B levels without affecting the constitutive secretion of cystatin C. Phorbol 12-myristate 13-acetate induced a 4- to 5-fold increase in secreted latent cathepsin B, but did not alter significantly the accumulation of cystatin C in media. The cytokines, TGF-β, TNF-α, and IL-1β, had no major effect on the expression of these two antagonists. Latent cathepsin B released from human carcinoma cells could be efficiently activated by neutrophil elastate at neutral pH. It is concluded that latent cathepsin B is a true proenzyme rather than an enzyme-inhibitor comples. In addition, our data from neutrophil elastate activation experiments indicate that a proteolytic system for activation of the tumor cell-secreted latent enzyme may exist in vivo.