Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

Inhibition of Aldehyde Dehydrogenases in Rat Brain and Liver by Disulfiram and Coprine

Abstract: Rats were treated with either coprine or disulfiram and the inhibition of aldehyde dehydrogenase (ALDH) in liver and brain mitochondria was measured with acetaldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL), and succinate semialdehyde at different concentrations. The inhibition pattern was similar for both inhibitors, but the degree of inhibition was lower with disulfiram. The ALDH activity both in the liver and the brain was inhibited at low concentrations of acetaldehyde and DOPAL, but not with succinate semialdehyde. The high- K _m enzyme activities with acetaldehyde were not inhibited in liver and brain. The activity at high concentration of DOPAL was inhibited in the liver, but only slightly affected in the brain, suggesting the presence of a brain enzyme with an intermediate K _m value for DOPAL. In contrast with the results observed in viva, it was found that the high- K _m activities with acetaldehyde and DOPAL in brain mitochondrial preparations were more sensitive to the inhibitors in vitro than the low- K _m activities. Kinetic studies on ALDH preparations from brain and liver mitochondria suggested that acetaldehyde and DOPAL are metabolized by the same low- K _m ALDH.     

Subcellular and Perisynaptic Distribution of Rat Brain Aldehyde Dehydrogenase Activity

Abstract: The nuclear mitochondrial and synaptosomal fractions of rat brain were each found to contain some 25–30% of the total aldehyde dehydrogenase activity. The cytoplasmic fraction had a very low total aldehyde dehydrogenase activity. There were differences in the distribution of the activity when different aldehydes were used as substrates, suggesting the presence of isoenzymes in the various subcellular compartments. When rats were treated intra-cisternally with 6-hydroxydopamine there was no change in brain aldehyde dehydrogenase activity, although the noradrenaline content and the activities of tyrosine hydroxylase and dopamine-β-hydroxylase were markedly decreased. Treatment with 6-hydroxydopamine also had no significant effect on the aldehyde dehydrogenase activity in retinal homogenates. The results suggest that the aldehyde dehydrogenase activity in rat brain is predominantly outside the catecholaminergic nerve terminals.     

Subcellular Distribution of Human Brain Aldehyde Dehydrogenase

Abstract: Two human brain surgery biopsies and one autopsy sample were subjected to subcellular fractionation. With either 0.12 or 6 mM-acetaldehyde as substrate, about half of the total aldehyde dehydrogenase activity was found in the mitochondrial (+ synaptosomal) fraction and less activity in the cytosolic, nuclear, and microsomal fractions. High-affinity activity was found only in the mitochondrial fraction. The enzyme in all fractions had a higher affinity for indole-3-acetaldehyde than for acetaldehyde. The kinetic data indicate the presence of several distinct aldehyde dehydrogenase isozymes that have ample capacity to oxidize both aliphatic and aromatic aldehydes in human brain.     

Biogenic Aldehyde Metabolism in Rat Brain: Subcellular Distribution of Aldose Reductase and Valproate-Sensitive Aldehyde Reductase

Reductase activity towards two aldose substrates has been examined in subcellular fractions prepared from rat brain. The reduction of glucuronate, which is sensitive to inhibition by the anticonvulsant drug sodium valproate, corresponds to the major high-Km aldehyde reductase in brain. Xylose reduction that is insensitive to valproate inhibition has characteristics consistent with the activity of aldose reductase (EC 1.1.1.21). Both enzymes are predominantly localized in the cytosolic fraction. The significance of the location of these two reductases is discussed in relation to the compartmentation of catecholamine metabolism in brain.     

Age-Related Changes in Aldehyde Dehydrogenase Activity of Rat Brain, Liver, and Heart

Abstract: Aldehyde dehydrogenase (ALDH) activity was measured in brains, livers, and hearts of 23–26-month-old and 3-month-old rats. A significant increase of ALDH activity was found in whole brain of old rats with both acetaldehyde (39%) and propionylaldehyde (15%) used as substrates. In different brain areas of old rats, with acetaldehyde used as substrate, a significant increase of ALDH activity was found in striatum (30–50%) and cerebral cortex (37%). However, no significant difference in ALDH activity was found in livers and hearts of young and old rats. Preliminary experiments showed a significant increase of aldehyde reductase activity (52%) with p -nitrobenzaldehyde used as substrate in whole brain of old rats compared with young rats. The present work indicates that an increase of ALDH activity in brain of old rats may be an adaptive phenomenon.     

Studies on Analogues of Succinic Semialdehyde as Substrates for Succinate Semialdehyde Dehydrogenase from Rat Brain

The methyl ester of succinic semialdehyde (SSA) was examined as a substrate for succinate semialdehyde dehydrogenase (SSADH) from rat brain. It was found that the ester can be oxidized by the enzyme. Values of Km for SSA-Me were higher than for those for SSA, and for this substrate the enzyme showed a substrate-dependent inhibition. This finding suggests that the carboxylate group of SSA is not essential in the process of inhibition of SSADH by the substrate. Cyclopropyl analogues of SSA, cis- and trans-1-formyl-cyclopropan-2-carboxylic acids, were also individually tested as substrates of SSADH. Only the trans isomer was found to be oxidized to the corresponding dicarboxylic acid; it inhibited the enzyme in the same range of concentrations as SSA. The above data suggest that, as for gamma-aminobutyric acid, SSA is present in an unfolded, transoid conformation at the active site of SSADH.     

Regional and Subcellular Localization in Rat Brain of the Enzymes That Can Synthesize γ-Hydroxybutyric Acid

Abstract: Rat brain contains two major NADPH-linked aldehyde reductases that can reduce succinate semialdehyde to 4-hydroxybutyrate. One of these enzymes appears to be fairly specific for succinate semialdehyde and is not significantly inhibited by classic aldehyde reductase inhibitors such as barbiturates. The other enzyme can reduce several aromatic aldehydes and is strongly inhibited by barbiturates and branched-chain fatty acids. Using one such inhibitor, it was possible to distinguish between and measure the two enzyme activities separately in various rat brain regions and in subcellular fractions. Both enzymes are mainly cytoplasmic but there is some activity in the synaptosomal fraction. The activity of the specific succinic semialdehyde reductase is highest in the cerebellum, where it represents 21% of the total activity, and lowest in the cortex, where it represents about 11% of the total activity.     

Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.     

Histochemical Study of Aldehyde Dehydrogenase in the Rat CNS

A quantitative histochemical method was developed to determine aldehyde dehydrogenase (EC 1.2.1.3; ALDH) activity in the CNS. The distribution of ALDH activity in all rat brain and spinal cord regions is described. Among the CNS neuron structures, high enzyme activity was found in receptor and effector neurons, whereas low activity was noted in perikarya of the majority of intermediate neurons, including all aminergic neurons. A positive correlation was demonstrated between the distribution of ALDH activity among rat CNS microregions (our own data) and the density of dopaminergic terminals, dopamine content, and monoamine oxidase activity (literature data) among the same microregions. They may reflect a spatial linkage between ALDH and the predicted sites of natural aldehyde production. Lower enzyme activity was found in phylogenetically younger brain structures. It may explain the differential resistance of CNS structures to ethanol (acetaldehyde). Among the barrier CNS structures, moderate ALDH activity was found in capillaries and surrounding astrocytes and high activity was noted in ependimocytes covering the brain cavities and those of the vascular plexus. This provides realization of the function of ALDH as a brain metabolic barrier for aldehydes.     

Biogenic Aldehydes in Brain: On Their Preparation and Reactions with Rat Brain Tissue

When 1 mM serotonin, dopamine, or norepinephrine was incubated with a monoamine oxidase preparation (mitochondrial membranes) in the presence of 4 mM sodium bisulfite, 85-95% of the amines were oxidized to the corresponding aldehydes. In the absence of bisulfite, the recoveries were only approximately 30%, and dark colored products were formed during the incubations. The aldehydes derived from tyramine, octopamine, methoxytyramine, and normetanephrine were also prepared by the use of this method. The bisulfite-aldehyde compounds were stable during storage at -20 degrees C. Bisulfite-free aldehyde solutions were made by diethylether extraction. When the aldehydes derived from dopamine or serotonin were incubated with rat brain homogenates, they were found to disappear in an aldehyde dehydrogenase- and aldehyde reductase-independent manner. The disappearance of the latter aldehyde was more pronounced, and the results indicated that this aldehyde may react with both proteins and phospholipids.     

Aldehyde Dehydrogenases and Their Role in Carcinogenesis

Abstract

Aldehydes are highly reactive molecules that may have a variety of effects on biological systems. They can be generated from a virtually limitless number of endogenous and exogenous sources. Although some aldehyde-mediated effects such as vision are beneficial, many effects are deleterious, including cytotoxicity, mutagenicity, and carcinogenicity. A variety of enzymes have evolved to metabolize aldehydes to less reactive forms. Among the most effective pathways for aldehyde metabolism is their oxidation to carboxylic acids by aldehyde dehydrogenases (ALDHs).

ALDHs are a family of NADP-dependent enzymes with common structural and functional features that catalyze the oxidation of a broad spectrum of aliphatic and aromatic aldehydes. Based on primary sequence analysis, three major classes of mammalian ALDHs — 1, 2, and 3 — have been identified. Classes 1 and 3 contain both constitutively expressed and inducible cytosolic forms. Class 2 consists of constitutive mitochondrial enzymes. Each class appears to oxidize a variety of substrates that may be derived either from endogenous sources such as amino acid, biogenic amine, or lipid metabolism or from exogenous sources, including aldehydes derived from xenobiotic metabolism.

Changes in ALDH activity have been observed during experimental liver and urinary bladder carcinogenesis and in a number of human tumors, including some liver, colon, and mammary cancers. Changes in ALDH define at least one population of preneoplastic cells having a high probability of progressing to overt neoplasms. The most common change is the appearance of class 3 ALDH dehydrogenase activity in tumors arising in tissues that normally do not express this form. The changes in enzyme activity occur early in tumorigenesis and are the result of permanent changes in ALDH gene expression.

This review discusses several aspects of ALDH expression during carcinogenesis. A brief introduction examines the variety of sources of aldehydes. This is followed by a discussion of the mammalian ALDHs. Because the ALDHs are a relatively understudied family of enzymes, this section presents what is currently known about the general structural and functional properties of the enzymes and the interrelationships of the various forms.

The remainder of the review discusses various aspects of the ALDHs in relation to tumorigenesis. The expression of ALDH during experimental carcinogenesis and what is known about the molecular mechanisms underlying those changes are discussed. This is followed by an extended discussion of the potential roles for ALDH in tumorigenesis. The role of ALDH in the metabolism of cyclophosphamidelike chemotherapeutic agents is described. This work suggests that modulation of ALDH activity may be an important determinant of the effectiveness of certain chemotherapeutic agents. The evidence that changes in ALDH are part of an adaptive response of preneoplastic and neoplastic cells to altered cell physiology or stress is then considered. Roles in the metabolism of aldehydes generated from lipid peroxidation and as part of the Ah gene-mediated response to xenobiotic exposure are both discussed. The data are consistent with a role for certain ALDHs in lipid aldehyde metabolism. Biochemical and genetic data also imply that changes in ALDH may be linked, in part, to cellular adaptation to oxidative stress.

Finally, a model of inducible ALDH gene regulation is proposed. The model incorporates current information about ALDH gene expression with the regulation of other genes known to be part of the adaptive responses occurring in neoplastic cells. The model suggests that regulation of class 1 and 3 ALDH gene activity may be complex, involving the tissue-specific ability to respond to a variety of physiological cues. The model also suggests several avenues for future research that should provide a clearer understanding of the regulation of this important gene family in response to a variety of factors.     

Rat Brain 3-Hydroxy-3-Methylglutaryl-CoA Reductase: Subcellular Distribution and Cold Lability

Abstract: Data are provided indicating that the rat brain 3-hydroxy-3-methyl-glutaryl-CoA reductase is similar to the enzyme from other tissues as far as diurnal rythmicity, cold lability and half-life measurements at 0°C are concerned. The enzyme activity in the brain decreased with age of the animals. Subcellular fractionation studies demonstrate that while 77% of the activity was associated with the microsomal fraction, 19% of the enzyme activity was recovered in the mitochondrial fraction. The possible function of such a mitochondrially located 3-hydroxy-3-methylglutaryl-CoA reductase in rat brain is discussed.     

Regional and Subcellular Distribution of ATP-Citrate Lyase and Other Enzymes of Acetyl-CoA Metabolism in Rat Brain

The activities of ATP-citrate lyase in frog, guinea pig, mouse, rat, and human brain vary from 18 to 30 μmol/h/g of tissue, being several times higher than choline acetyltransferase activity. Activities of pyruvate dehydrogenase and acetyl coenzyme A synthetase in rat brain are 206 and 18.4 μmol/h/g of tissue, respectively. Over 70% of the activities of both choline acetyltransferase and ATP-citrate lyase in secondary fractions are found in synaptosomes. Their preferential localization in synaptosomes and synaptoplasm is supported by RSA values above 2. Acetyl CoA synthetase activity is located mainly in whole brain mitochondria (RSA, 2.33) and its activity in synaptoplasm is low (RSA, 0.25). The activities of pyruvate dehydrogenase, citrate synthase, and carnitine acetyltransferase are present mainly in fractions C and B_p. No pyruvate dehydrogenase activity is found in synaptoplasm. Striatum, cerebral cortex, and cerebellum contain similar activities of pyruvate dehydrogenase, citrate synthase, carnitine acetyltransferase, fatty acid synthetase, and acetyl-CoA hydrolase. Activities of acetyl CoA synthetase, choline acetyltransferase and ATP-citrate lyase in cerebellum are about 10 and 4 times lower, respectively, than in other parts of the brain. These data indicate preferential localization of ATP-citrate lyase in cholinergic nerve endings, and indicate that this enzyme is not a rate limiting step in the synthesis of the acetyl moiety of ACh in brain.     

Biogenic Aldehydes in Brain: Characteristics of a Reaction Between Rat Brain Tissue and Indole-3-Acetaldehyde

When indole-3-acetaldehyde was incubated with rat brain tissue, an aldehyde dehydrogenase-independent irreversible disappearance of the aldehyde was found. This was accompanied by an increase in absorbance at 240-400 nm, with a peak at 310 nm. The results suggested that this change in absorbance was caused by a membrane-bound nonenzymatic reaction between indole-3-acetaldehyde and phospholipids. A similar reaction occurred between indole-3-acetaldehyde and pure preparations of phosphatidylethanolamine and phosphatidylserine, but not phosphatidylcholine. Indole-3-acetaldehyde levels also decreased slightly when incubated with albumin but absorbance at 310 nm was unaltered. It is suggested that nonenzymatic reactions between indole-3-acetaldehyde (or other biogenic aldehydes) and membrane components might occur in vivo, and could be involved in the effects of drugs such as ethanol and barbiturates.     

Subcellular Distribution of UDP-Galactose:Ceramide Galactosyltransferase in Rat Brain Oligodendroglia

Oligodendrocytes isolated from 18-19-day-old rat brain were homogenized in 0.32 M sucrose. The homogenate was centrifuged at 100,000 g for 50 min in a gradient containing 0.8, 1.05, and 1.3 M sucrose. Three discrete bands were obtained at the interfaces 0.32-0.8 (F1), 0.8-1.05 (F2), and 1.05-1.3 M (F3). The distribution of UDP-galactose:ceramide galactosyltransferase (CgalT) activity in each fraction was measured using liposomes containing normal fatty acid-containing ceramides (NFA-CgalT activity) or 2-hydroxy fatty acid-containing ceramides (HFA-CgalT activity). Although detection of both CgalT activities was possible in all fractions, HFA-CgalT activity was enriched in F1 and F2 fractions, which also showed an enrichment of Golgi and endoplasmic reticulum markers, respectively. It is interesting that NFA-CgalT activity was significantly enriched in the F2 fraction. These results suggest that hydroxylated and nonhydroxylated galactocerebrosides may be synthesized at different intracellular locations.     

Ontogeny and Subcellular Distribution of Rat Brain Tele-Methylhistamine

Abstract: The whole brain content and subcellular distribution of histamine and its metabolite, tele-methylhistamine, were studied during postnatal development of the rat. Brain methylhistamine levels were similar to or greater than histamine levels, indicating that histamine methylation is a major metabolic pathway in neonatal brain, as it is in adults. When calculated per brain, histamine, methylhistamine, and histamine methyltransferase were all maximal 10 days after birth. In neonates, brain histamine was found almost entirely in nuclear fractions, whereas methylhistamine was found almost exclusively in supernatant fractions. By day 20, however, a greater proportion of both amines was localized in subcellular fractions containing synaptosomes, a finding consistent with histamine's suggested transmitter role. The ontogenic pattern of brain methylhistamine questions the mast cell origin of neonatal histamine, but may be consistent with a role for histamine in brain development.     

Subcellular Distribution of Two Spin Trapping Agents in Rat Heart: Possible Explanation for Their Different Protective Effects Against Doxorubicin-Induced Cardiotoxicity

Previous investigations, performed on isolated rat atria, showed that the lipophylic spin-trapping agent N-tert-butyl-alpha-phenylnitrone (PBN) is able to prevent the acute cardiotoxic effects produced by doxorubicin (DXR), whereas the hydrophylic compound 5,5-dimethyl-pyrroline-N-oxide (DMPO) is inactive. The present study was designed to ascertain whether differences in the pharmacological effects of the two spin traps are related to their different subcellular distribution. Langendorff rat hearts were perfused for 60 minutes with [^I4C]-DXR and either PBN or DMPO. The subcellular mapping of the three compounds was performed by measuring DXR by liquid scintillation counting, PBN by GC/MS, and DMPO by HPLC in the following isolated fractions: nuclei, mitochondria, sarcoplasmic reticulum, sarcolemma, cytosol. DMPO was shown to accumulate in the cytosolic compartment; both PBM and DXR are taken up by nuclei and mitochondria, while only trace amounts of DXR were detected in the sarcoplasmic reticulum. These results suggest that mitochondrial (and not sarcoplasmic) enzymes are mainly involved in DXR-induced free radical production, which is thought to cause the acute cardiotoxic effects of DXR. An involvement of DXR-induced free radical generation in the nuclear compartment seems unlikely in the short-term “in vitro” effects observed with the experimental model adopted for these studies, although it may play a role in the delayed pathology.     

镉在东南景天中的亚细胞分配

采用差速离心技术,比较研究了Cd在矿山生态型东南景天(Sedum alfredii Hance)根、茎、叶中的亚细胞分布.结果表明:矿山生态型东南景天对Cd具有很好的忍受耐和累积能力,非矿山生态型东南景天则不具有这种能力.Cd在矿山生态型东南景天根、茎、叶各部分的亚细胞分配满足F1(细胞壁部分)>F3(可溶部分)>F2(细胞器及膜)规律,且Cd在细胞壁部分的分配占绝对优势;同时矿山生态型东南景天地上部有很好的Cd累积能力.与众多超累积植物相似的Cd亚细胞分配规律和地上部的良好的Cd累积能力.对此植物的Cd亚细胞分布完全不同于非矿山生态型东南景天的情况作了比较分析,结果显示矿山生态型东南景天很可能是一种新的镉超累积种质资源.     

Developmental Pattern for Phosphatidylserine Decarboxylase in Rat Brain

In adult rats, a significant portion of brain ethanolamine glycerophospholipids are synthesized by a pathway involving phosphatidylserine decarboxylase, a mitochondrial enzyme. We have now examined whether this enzyme plays a particularly prominent role during development. Activities for both phosphatidylserine decarboxylase and succinate dehydrogenase (another mitochondrial enzyme) were determined in brain homogenates from rats 5 days of age to adulthood. Succinate dehydrogenase activity, expressed on a per unit brain protein basis, increased markedly during development. This pattern has been reported previously and is as expected from the postnatal increase in oxidative metabolism. In contrast, phosphatidylserine decarboxylase activity decreased 40% from 5 to 30 days of age. The apparent Km for brain phosphatidylserine decarboxylase was 85 microM in both young (8- and 20-day-old) and adult animals. Parallel studies in vivo were carried out to determine the contribution of the phosphatidylserine decarboxylase pathway, relative to pathways utilizing ethanolamine directly, to the synthesis of brain ethanolamine glycerophospholipids. Animals were injected intracranially with a mixture of L-[G-3H]serine and [2-14C]ethanolamine and incorporation into the base moieties of the phospholipids determined. The 3H/14C ratio of ethanolamine glycerophospholipids decreased about 50% during development. Our studies in vitro and in vivo both suggest that phosphatidylserine decarboxylase plays a significant role in the synthesis of brain ethanolamine glycerophospholipids at all ages, although it is relatively more prominent early in development.