Subcellular distribution of cysteine oxidase activity in ox retina.

Cysteine oxidase activity has been determined in the primary and secondary subfractions of ox retina. About 30% of enzyme activity is found in the soluble fraction while about 70% is associated with particulate components.In the secondary subcellular fractions about 36% of enzyme activity, recovered from crude mitochondria, is present in the synaptosomal fraction.Enzymic activity is stimulated by Fe⁺⁺ and NAD⁺. The reason and significance of the cysteine oxidase activity in synaptosomal fraction are briefly discussed in relation with taurine function in retina.     

Cysteine oxidase activity in rat retina during development.

Cysteine oxidase activity has been determined in developing rat retina. Enzymic activity is present in 55, 000 × $\text{g}$ supernate and in the crude mitochondria.Activity is rather low at birth; but increases with age; in mitochondria it reaches its maximum value at the 25th day while in supernate it increases more rapidly, reaching its maximum value 20 days after birth; unlike in the mitochondria, the activity of supernate considerably decreases during further development.The reason and significance of the postnatal changes in the mitochondrial cysteine oxidase activity are briefly discussed in relation with taurine formation.     

Subcellular localization of aldehyde reductase activities in ox brain.

The distribution of the two principal isoenzymes of aldehyde reductase (EC 1.1.1.2) has been studied in ox brain. The more active of these, which has been termed the high-Km enzyme, has been shown to be located in the cytosol and the less abundant low-Km form has a similar localization. p-Nitrobenzaldehyde, which has been used as a substrate in previous studies, caused the reduction of NADH in the presence of the mitochondrial fraction, but mixed substrate experiments with 1,3-dinitrobenzene and the effects of pH on the activity indicate that this is due to the presence of a nitro reductase activity which has been recently described (Köchli, Wermuth & von Wartburg (1980) Biochim. Biophys. Acta 616, 133-142] rather than to the low-Km aldehyde reductase activity. Fractionation of the mitochondria indicated this activity to be largely confined to the mitochondrial inner membrane.     

Subcellular distribution of zinc and the presence of a metallothionein-like protein in bovine retina

The mammalian retinas contain the highest concentrations of Zn in any known living tissues. Metallothionein is a low-molecular-weight, cysteine-rich metal-binding protein which occurs ubiquitously in nature and which has been recently identified in mammalian brains with a high affinity to bind Zn. In order to study the metabolism of Zn further, we have measured its subcellular distribution in the bovine retina and have found the distribution to be nonuniform, with the outer rod segments and the pellet 1 fraction, known to be highly enriched in photoreceptor cell synaptosomes, containing the highest amounts of 0.230 ± 0.040 and 0.119 ± 0.04 μ Zn/mg protein, respectively. In addition, the bovine retina contains a low-molecular-weight metallothionein-like protein which exhibits an elution volume (V_c/V₀) of 1.9 on gel permeation chromatography and which produces only one isoform on reverse-phase high performance liquid chromatography with a retention time of 16.67 min. The precise function of this metallothioneinlike protein, which may be related to the transport and compartmentation of Zn in the retina, remains to be elucidated.     

Subcellular localization of tubulin in chick retina

Tubulin was measured through [3H]colchicine-binding in membrane and soluble components of chick retinal subcellular fractions. Total tubulin content was concentrated in the synaptosomal and rod outer segment fractions. Although in total retinal homogenate only 20% of total tubulin was associated to the membrane, in synaptosomes and photoreceptor outer segments, up to 50% of tubulin was bound to the membrane fraction. Results raise the possibility of tubulin participation in transmembrane phenomena which are common to transmitter release and photoexcitation.     

Subcellular distribution of OM cytochrome b-mediated NADH-semidehydroascorbate reductase activity in rat liver

Tissue, cellular, and subcellular distributions of OM cytochrome b-mediated NADH-semidehydroascorbate (SDA) reductase activity were investigated in rat. NADH-SDA reductase activity was found in the post-nuclear particulate fractions of liver, kidney, adrenal gland, heart, brain, lung, and spleen of rat. Liver, kidney, and adrenal gland had higher NADH-SDA reductase activity than other tissues, and OM cytochrome b-dependent activity was 60-70% of the total activity. On the other hand, almost all of the reductase activity of heart and brain cells was mediated by OM cytochrome b. The ratio of the OM cytochrome b-mediated activities of NADH-SDA reductase to rotenone-insensitive NADH-cytochrome c reductase varied among these tissues. OM cytochrome b-mediated NADH-SDA reductase and rotenone-insensitive NADH-cytochrome c reductase activities were mainly present in the parenchymal cells of rat liver. The localization of the cytochrome-mediated reductase activities in the outer mitochondrial membrane was confirmed by subfractionation of liver mitochondria. Among the submicrosomal fractions, OM cytochrome b-mediated NADH-SDA reductase activity was highest in the cis-Golgi membrane fraction, in which monoamine oxidase activity was also highest. On the other hand, OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase activity showed a slightly different distribution pattern from the NADH-SDA reductase activity. Thenoyltrifluoroacetone (TTFA), a metal chelator, effectively inhibited the NADH-SDA reductase activity, though other metal chelators did not affect the activity. TTFA failed to inhibit rotenone-insensitive NADH-cytochrome c reductase activity at the concentration which gave complete inhibition of NADH-SDA reductase activity.     

Subcellular distribution of enkephalins and endogenous opioid activity in rat brain.

The subcellular distribution of leucine- and methionine-enkephalin in rat brain was studied using a highly selective and sensitive radioimmunoassay. About 85% of the total recoverable activity of each peptide was present in crude synaptosomal and microsomal fractions which contained about 60% and 25% respectively. Total opioid activity in brain subcellular extracts was measured by competition for opiat receptor binding. It is concluded that enkephalin accounts for the majority of the opioid activity in the brain extracts. It seems unlikely that the enkephalin in microsomal fractions are exclusively associated with opiate receptors present in these fractions.     

Subcellular distribution and activity in different rat tissues of a deoxyinosine-activated nucleotidase.

A nucleotidase (EC 3.1.3.31) isolated previously from rat liver cytosol was specifically measured in 14 different rat tissues and in subcellular fractions of liver and spleen, taking advantage of the stimulation exerted on it by deoxyinosine. The intracellular distribution studies showed that the enzyme is located almost entirely in the soluble cytoplasm except for the possible presence of 1-2% of the enzyme in the nucleus. The enzyme was present in various amounts in all the tissues studied. Spleen, thymus, and intestinal mucosa showed higher specific activities than any other tissue. On a per cell basis spleen, liver and intestinal mucosa had the highest enzyme activity, whereas bone marrow, brain, thymus, heart and skeletal muscle had activities in the lower range. The results may suggest that the enzyme plays a role in the recovery of endogenous nuclear material for nucleic acid synthesis.     

Subcellular distribution of ascorbate in plants

The compartment specific distribution of ascorbate in plants is of great importance for plant development, growth and defense as this multifunctional metabolite plays important roles in the detoxification of reactive oxygen species (ROS), redox signaling, modulation of gene expression and is important for the regulation of enzymatic activities. Even though changes in ascorbate contents during plant growth and various stress conditions are well documented and the roles of ascorbate in plant defense during abiotic stress conditions are well established, still too little is known about its compartment specific roles during plant development and defense. This mini-review focuses on the subcellular distribution of ascorbate in plants and describes different methods that are currently used to study its compartment specific distribution. Finally, it will also briefly discuss data available on compartment specific changes of ascorbate during some abiotic stress conditions such as high light conditions and exposure to ozone.Key words: ascorbate, mitochondria, chloroplasts, electron microscopy, ozone, high light stress, reactive oxygen speciesAscorbate is one of the most important antioxidants in plants and animals. It detoxifies reactive oxygen species (ROS) either directly or through the glutathione-ascorbate cycle (Fig. 1) and is involved in redox signaling, modulation of gene expression and the regulation of enzymatic activities (extensively reviewed in ref. ¹ and ²). Ascorbate occurs in a reduced form (ascorbic acid) and two oxidized forms (mono- and dehydroascorbic acid). The ratio between reduced and oxidized ascorbate is essential for the ability of the plant to fight oxidative stress. During environmental stress situations when ROS are formed inside the cell, large amounts of dehydroascorbic acid can be formed by oxidation of ascorbic acid which shifts the ascorbate pool more towards the oxidative state and diminishes the antioxidative capacity of the plant. Additionally, environmental stress situations can change total ascorbate contents in plants which makes ascorbate an important stress marker during abiotic and biotic stress situations.^3–11 Ascorbate contents are typically measured biochemically in individual plant organs or tissues and the obtained values represent a combination of the ascorbate status of all individual organelles. As many environmental stress conditions induce highly compartment specific stress responses changes of ascorbate contents in individual organelles might not be detected when ascorbate is measured in whole organs or tissues. This is crucial as data obtained about the antioxidative status from individual organs are often used to interpret the stress response of the whole plant to the exposed stress conditions. Thus, in order to gain a deeper insight into the defense response of plants it is essential to measure changes in the subcellular distribution of these components during environmental stress situations.Open in a separate windowFigure 1Ascorbate-glutathione cycle in plants. Hydrogen peroxide (H₂O₂) within the plant cell can be detoxified by ascorbate peroxidase (APX). In this reaction the reduced form of ascorbate (Asc) is oxidized to monodehydroascorbate (MDHA). MDHA is then either reduced by monodehydroascorbate reductase (MDHAR) to Asc or, since very unstable, reacts to dehydroascorbate (DHA). DHA is reduced by dehydroascorbate reductase (DHAR) to Asc. In this reaction the reduced form of glutathione (GSH) is oxidized to glutathione disulfide (GSSG). GSSG is then reduced by glutathione reductase (GR) to GSH. The electron acceptor NADP is regenerated during the reduction of MDHA and GSSG by the respective enzymes. Asc and GSH are additional able to detoxify reactive oxygen species by direct chemical interaction. Thus, besides the total ascorbate level their redox state (reduced vs. oxidized state) which depends on the activity of the described enzymes (grey boxes) is also very important for successful plant protection.