过氧化物酶体膜对各种光呼吸代谢物的透性和Percoll对其完整性的影响

利用氧极谱法测定依赖乙醇酸和乙醛酸的氧吸收,利用分光光度计测定转氨酶活性的方法,检查了光呼吸碳代谢中各种中间产物对过氧化物酶体膜的透性,结果表明,O2,乙醇酸,乙醛酸,丝氨酸,天冬氨酸,草酰乙酸,α－酮戊二酸能透过过氧体膜,过氧体膜可能有谷氨酸和NADH进入的屏障,过氧体悬浮液Percoll存在,很快使过氧体膜破坏,Percoll也使羟基丙酮酸还原酶和苹果酸脱氢酶的活性很快降低。     

过氧化物酶体膜对各种光呼吸代谢物的透性和Percoll对其完整性的影响

利用氧极谱法测定依赖乙醇酸和乙醛酸的氧吸收,利用分光光度计测定转氨酶活性的方法,检查了光呼吸碳代谢中各种中间产物对过氧化物酶体膜的透性,结果表明,O2,乙醇酸,乙醛酸,丝氨酸,天冬氨酸,草酰乙酸,α－酮戊二酸能透过过氧体膜,过氧体膜可能有谷氨酸和NADH进入的屏障,过氧体悬浮液Percoll存在,很快使过氧体膜破坏,Percoll也使羟基丙酮酸还原酶和苹果酸脱氢酶的活性很快降低。     

菠菜叶过氧化物酶体转变甘油酸为丝氨酸的NAD供应

完整的菠菜(Spinacia oleracea L.)叶过氧化物酶体在NAD和丙氨酸存在时,转变甘油酸成为丝氨酶。完整过氧化物酶体的转变速率低于破碎的过氧化物酶体转变速率。反应对NAD是绝对依赖的,对草酰乙酸只是部分依赖。草酰乙酸可以用α-酮戊二酸和天冬氨酸代替。过氧化物酶体膜能缓慢透过NAD/NADH。提出了膜运载体可能参与叶过氧体NAD/NADH的再产生的穿梭系统。     

类胡萝卜素生物合成途径及其控制与遗传操作

类胡萝卜素在真菌和植物细胞胞液／内质网上是由乙酰CoA经甲羟戊酸途径合成的,在细菌与植物质体中由磷酸甘油醛与丙酮酸经1-脱氧木酮糖-5-磷酸途径合成。形成的异戊烯基焦磷酸经多次缩合生成第一个类胡萝卜素八氢番茄红素,再经脱氢、环化、羟基化、环氧化等转变为其它类胡萝卜素。类胡萝卜素生物合成中涉及的酶都是膜结合的或整合入膜中的。类胡萝卜素合成是通过底物可利用性与环化分支方式进行控制的。白色体到叶绿体的转变以及花与果实成熟时类胡萝卜素合成增加是在基因转录水平调节的。进行类胡萝卜素合成酶基因的转化,可增加转化体类胡萝卜素的积累。     

过氧物酶体和乙醛酸循环体结构与功能研究新进展

本文叙述了利用完整的过氧物酶体和乙醛酸循环体,研究其结构与功能取得的新进展。     

过氧物酶体和乙醛酸循环体结构与功能研究新进展

本文叙述了利用完整的过氧物酶体和乙醛酸循环体,研究其结构与功能取得的新进展。     

血清谷丙转氨酶测定的生物医学意义

谷丙转氨酶(GPT)全称谷氨酸-丙酮酸氨基移换酶,其辅酶是磷酸吡哆醛或磷酸吡哆胺。此酶在人体内催化谷氨酸和丙酮酸之间进行转氨基反应,是最重要的转氨酶之一。GPT广泛分布于人体各组织器官,但以肝脏活性最高,血清中此酶活性很低。以每克湿组织所含的GPT加以比较,肝脏的GPT是44000卡门氏单位,而血清的GPT只是15卡门氏单位。但是,当人体某些组织器官尤其是肝脏有病变时,由于细胞膜通透性增高,或组织坏死,细胞破坏,细胞内的GPT就会逸入血液,引起血清GPT活性明显升高(临床测定常采用赖氏法,     

几种有机酸对芸苔光呼吸代谢的影响（简报）

芸苔叶圆片在乙醇酸氧化酶受抑对照光,其乙醇酸的积累受而酮酸促进,而被柠檬酸、琥珀酸及苹果酸抑制、离体条件下．添加苹果酸、柠檬酸和琥珀酸时,乙醇酸氧化酶活性增高：添加丙酮酸、柠檬酸、苹果酸和琥珀酸时．甘氨酸氧化酶活性也增大。     

蛇足石杉中两个新的过氧羟基取代的石松生物碱

从我国民间中药蛇足石杉（Huperzia serrata(Thunb.)Trev.)的全草的总碱部位分得3个石松生物碱。经光谱分析鉴定为11α-过氧羟基马尾杉碱乙（1）,7-过氧羟基马尾杉碱乙（2）和马尾杉碱乙（3）,1和2为首次发现的过氧羟基取代的石松生物碱。     

蛇足石杉中两个新的过氧羟基取代的石松生物碱

从我国民间中药蛇足石杉(Huperzia serrata(Thunb.)Trey.)的全草的总碱部位分得3个石松生物碱,经光谱分析鉴定为11α-过氧羟基马尾杉碱乙(1)、7-过氧羟基马尾杉碱乙(2)和马尾杉碱乙(3).1和2为首次发现的过氧羟基取代的石松生物碱.     

Metabolism of glycolate and glyoxylate in intact spinach leaf peroxisomes

Intact and broken (osmotically disrupted) spinach (Spinacia oleracea) leaf peroxisomes were compared for their enzymic activities on various metabolites in 0.25 molar sucrose solution. Both intact and broken peroxisomes had similar glycolate-dependent o₂ uptake activity. In the conversion of glycolate to glycine in the presence of serine, intact peroxisomes had twice the activity of broken peroxisomes at low glycolate concentrations, and this difference was largely eliminated at saturating glycolate concentrations. However, when glutamate was used instead of serine as the amino group donor, broken peroxisomes had slightly higher activity than intact peroxisomes. In the conversion of glyoxylate to glycine in the presence of serine, intact peroxisomes had only about 50% of the activity of broken peroxisomes at low glyoxylate concentrations, and this difference was largely overcome at saturating glyoxylate concentrations. In the transamination between alanine and hydroxypyruvate, intact peroxisomes had an activity only slightly lower than that of broken peroxisomes. In the oxidation of NADH in the presence of hydroxypyruvate, intact peroxisomes were largely devoid of activity. These results suggest that the peroxisomal membrane does not impose an entry barrier to glycolate, serine, and O₂ for matrix enzyme activity; such a barrier does exist to glutamate, alanine, hydroxypyruvate, glyoxylate, and NADH. Furthermore, in intact peroxisomes, glyoxylate generated by glycolate oxidase is channeled directly to glyoxylate aminotransferase for a more efficient glycolate-glycine conversion. In related studies, application of in vitro osmotic stress to intact or broken peroxisomes had little effect on their ability to metabolize glycolate to glycine.     

Cell organelles from crassulacean-acid-metabolism (CAM) plants

Cell organelles were isolated from the CAM plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen by isopycnic centrifugation in sucrose gradients. The inclusion of 2.5% Ficoll in the grinding medium proved to be essential for a satisfactory separation of cell organelles during the subsequent centrifugation. Peroxisomes, mitochondria, and whole and broken chloroplasts were at least partially resolved as judged by marker-enzyme-activity profiles. The isolated peroxisomes contained activities of glycollate oxidase, catalase, hydroxypyruvate reductase, glycine aminotransferase, serine-glyoxylate aminotransferase, and aspartate aminotransferase, comparable to activities found in spinach (Spinacia oleracea L.) leaf peroxisomes. In contrast to spinach, however, only little, if any, particulate malate dehydrogenase activity could be attributed to isolated peroxisomes of the three CAM plants.     

Conversion of serine to glycerate in intact spinach leaf peroxisomes: role of malate dehydrogenase

In photorespiration, leaf peroxisomes convert serine to glycerate via serine-glyoxylate aminotransferase and NADH-hydroxypyruvate reductase. We isolated intact spinach leaf peroxisomes in 0.25 M sucrose, and characterized their enzymatic conversion of serine to glycerate using physiological concentrations of substrates and coenzymes. In the presence of glycolate (glyoxylate), and NADH and NAD alone or together in physiological proportions, the rate of serine-to-glycerate conversion was enhanced and sustained by the addition of malate. The rate was similar at 1 and 5 mM serine, but was two to three times higher in 50 mM than 5 mM malate. In the presence of NAD and malate, there was 1:1 stoichiometric formation of glycerate and oxaloacetate. Addition of 1 or 5 mM glutamate resulted in a negligible enhancement of the conversion of hydroxypyruvate to glycerate. Intact peroxisomes produced glycerate from either serine or hydroxypyruvate at a rate two times higher than osmotically lysed peroxisomes. These results suggest that under physiological conditions, the peroxisomal malate dehydrogenase operates independent of aspartate-alpha-ketoglutarate aminotransferase in supplying NADH for hydroxypyruvate reduction. This supply of NADH is the rate-limiting step in the conversion of serine to glycerate. The compartmentation of hydroxypyruvate reductase and malate dehydrogenase in the peroxisomes confers a higher efficiency in the supply of NADH for hydroxypyruvate reduction under a normal, high NAD/NADH ratio in the cytosol.     

Reconstitution of amino acid synthesis by combining spinach chloroplasts with other leaf organelles

By adding leaf peroxisomes to purified intact chloroplasts, glycine synthesis was reconstituted. On adding leaf mitochondria, serine synthesis was also reconstituted. However, aromatic amino acid synthesis which was effected by purified chloroplasts was not enhanced on adding peroxisomes or mitochondria although the rate in whole leaves was considerably higher.     

Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.     

Participation of Mitochondrial Metabolism in Photorespiration : Reconstituted System of Peroxisomes and Mitochondria from Spinach Leaves

In this study the interplay of mitochondria and peroxisomes in photorespiration was simulated in a reconstituted system of isolated mitochondria and peroxisomes from spinach (Spinacia oleracea L.) leaves. The mitochondria oxidizing glycine produced serine, which was reduced in the peroxisomes to glycerate. The required reducing equivalents were provided by the mitochondria via the malate-oxaloacetate (OAA) shuttle, in which OAA was reduced in the mitochondrial matrix by NADH generated during glycine oxidation. The rate of peroxisomal glycerate formation, as compared with peroxisomal protein, resembled the corresponding rate required during leaf photosynthesis under ambient conditions. When the reconstituted system produced glycerate at this rate, the malate-to-OAA ratio was in equilibrium with a ratio of NADH/NAD of 8.8 × 10⁻³. This low ratio is in the same range as the ratio of NADH/NAD in the cytosol of mesophyll cells of intact illuminated spinach leaves, as we had estimated earlier. This result demonstrates that in the photorespiratory cycle a transfer of redox equivalents from the mitochondria to peroxisomes, as postulated from separate experiments with isolated mitochondria and peroxisomes, can indeed operate under conditions of the very low reductive state of the NADH/NAD system prevailing in the cytosol of mesophyll cells in a leaf during photosynthesis.     

Compartmentation studies on spinach leaf peroxisomes : evidence for channeling of photorespiratory metabolites in peroxisomes devoid of intact boundary membrane

In concurrence with earlier results, the following enzymes showed latency in intact spinach (Spinacia oleracea L.) leaf peroxisomes: malate dehydrogenase (89%), hydroxypyruvate reductase (85%), serine glyoxylate aminotransferase (75%), glutamate glyoxylate aminotransferase (41%), and catalase (70%). In contrast, glycolate oxidase was not latent. Aging of peroxisomes for several hours resulted in a reduction in latency accompanied by a partial solubilization of the above mentioned enzymes. The extent of enzyme solubilization was different, being highest with glutamate glyoxylate aminotransferase and lowest with malate dehydrogenase. Osmotic shock resulted in only a partial reduction of enzyme latency. Electron microscopy revealed that the osmotically shocked peroxisomes remained compact, with smaller particle size and pleomorphic morphology but without a continuous boundary membrane. Neither in intact nor in osmotically shocked peroxisomes was a lag phase observed in the formation of glycerate upon the addition of glycolate, serine, malate, and NAD. Apparently, the intermediates, glyoxylate, hydroxypyruvate, and NADH, were confined within the peroxisomal matrix in such a way that they did not readily leak out into the surrounding medium. We conclude that the observed compartmentation of peroxisomal metabolism is not due to the peroxisomal boundary membrane as a permeability barrier, but is a function of the structural arrangement of enzymes in the peroxisomal matrix allowing metabolite channeling.     

Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts

Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues.     

Compartmentation studies on spinach leaf peroxisomes

The synthesis of glycerate by isolated intact spinach (Spinacia oleracea L.) leaf peroxisomes upon the addition of glycolate, serine, and glutamate, with either NADH or malate as reductant, has been measured. Measurement of the concentration dependence of NADH-and malate-dependent glycerate synthesis, and the exclusion of various artefacts, clearly demonstrate that under in vivo conditions the transfer of reducing equivalents into the peroxisomes required for the reduction of hydroxypyruvate to glycerate, occurs exclusively via a malate shuttle. The results indicate that a direct uptake of NADH into the peroxisomes does not occur under invivo conditions to any appreciable extent. As these results have been observed with intact as well as with osmotically shocked peroxisomes, it is concluded that the specificity of redox transfer into the peroxisomes is not due to a selectivity of the peroxisomal boundary membrane, but to a multi-enzyme structure of the peroxisomal matrix.Abbreviations GDH glycerophosphate dehydrogenase - GOT glutamate oxaloacetate transaminase - HPR hydroxy-pyruvate reductase - MDH malate dehydrogenase The authors are indebted to Mr. Bernd Raufeisen for the art work. This work was supported by the Deutsche Forschungsgemeinschaft.     

The simultaneous determination of carbon dioxide release and oxygen uptake in suspensions of plant leaf mitochondria oxidizing glycine

The construction and operation of a device for continuous measurement of CO₂ release by suspensions of respiring mitochondria is described. A combination of this device with a Clark-type O₂ electrode was used for simultaneous measurement of respiration and of CO₂ release by spinach and pea leaf mitochondria with glycine as substrate. Both mitochondrial preparations showed high rates of respiration and high respiratory control ratios. The addition of oxaloacetate not only inhibited O₂ uptake substantially, but also greatly stimulated glycine oxidation as monitored by CO₂ release. In spinach leaf mitochondria, the maximal rates of glycine oxidation thus obtained, were two times higher than the rate of glycine oxidation required at average rates of photorespiration. It is concluded from these results that under saturating conditions the capacity of glycine oxidation by intact mitochondria exceeds the capacity of glycine-dependent respiration.