辅因子S-腺苷-L-甲硫氨酸甲基类似物及其应用

甲基化在生物学过程中发挥着重要作用。S-腺苷-L-甲硫氨酸(S-adenosyl-L-methionine, SAM)作为一种广泛存在于生命体中的辅因子,是大多数生物甲基化反应的甲基供体。SAM依赖型甲基转移酶(methyltransferases, MTase)通过将甲基从SAM分子特异性转移到底物,从而改变底物分子的各种理化性质和生物活性。近年来,许多具有替代甲基取代基的SAM类似物被合成并应用于甲基转移酶,以将不同修饰的基团特异性地转移到甲基转移酶的底物上,从而引入标记官能团或者新的烷基修饰。本文主要综述了近年来该领域不同SAM甲基类似物在合成和应用方面取得的进展,并对这一领域未来的研究方向进行展望。     

Catalytic mechanism of glycine N-methyltransferase

Methyltransfer reactions are some of the most important reactions in biological systems. Glycine N-methyltransferase (GNMT) catalyzes the S-adenosyl-l-methionine- (SAM-) dependent methylation of glycine to form sarcosine. Unlike most SAM-dependent methyltransferases, GNMT has a relatively high value and is weakly inhibited by the product S-adenosyl-l-homocysteine (SAH). The major role of GNMT is believed to be the regulation of the cellular SAM/SAH ratio, which is thought to play a key role in SAM-dependent methyltransfer reactions. Crystal structures of GNMT complexed with SAM and acetate (a potent competitive inhibitor of Gly) and the R175K mutated enzyme complexed with SAM were determined at 2.8 and 3.0 A resolutions, respectively. With these crystal structures and the previously determined structures of substrate-free enzyme, a catalytic mechanism has been proposed. Structural changes occur in the transitions from the substrate-free to the binary complex and from the binary to the ternary complex. In the ternary complex stage, an alpha-helix in the N-terminus undergoes a major conformational change. As a result, the bound SAM is firmly connected to protein and a "Gly pocket" is created near the bound SAM. The second substrate Gly binds to Arg175 and is brought into the Gly pocket. Five hydrogen bonds connect the Gly in the proximity of the bound SAM and orient the lone pair orbital on the amino nitrogen (N) of Gly toward the donor methyl group (C(E)) of SAM. Thermal motion of the enzyme leads to a collision of the N and C(E) so that a S(N)2 methyltransfer reaction occurs. The proposed mechanism is supported by mutagenesis studies.     

Effects of L-dopa treatment on methylation in mouse brain: implications for the side effects of L-dopa

The effects of L-dopa on methylation process in the mouse brain were investigated. The study is based on recent findings that methylation may play an important role in Parkinson's disease (PD) and in the actions of L-dopa. The methyl donor, S-adenosylmethionine (SAM) and a product of SAM, methyl beta-carboline, were shown to cause PD-like symptoms, when injected into the brain of animals. Furthermore, large amounts of 3-O-methyl dopa, the methyl product of L-dopa, are produced in PD patients receiving L-dopa treatment, and L-dopa induces methionine adenosyl transferase, the enzyme that produces SAM. The results show that, at 0.5 hr, L-dopa (100 mg/kg) decreased the methyl donor, S-adenosylmethionine (SAM) by 36%, increased its metabolite S-adenosylhomocysteine (SAH) by 89% and increased methylation (SAH/SAM) by about 200%. All parameters returned to control values within 4 hr. But 2, 3 and 4 consecutive injections of L-dopa, given at 45 min intervals, depleted SAM by 60, 64 and 76% and increased SAM/SAH to 818, 896, and 1524%. L-dopa (50, 100 and 200 mg/kg) dose-dependently depleted SAM from 24.9 +/- 1.7 nmol/g to 13.0 +/- 0.8, 14.7 +/- 0.8 and 7.7 +/- 0.7 nmol/g, and increased SAH from 1.88 +/- 0.14 to 3.43 +/- 0.26, 4.22 +/- 0.32 and 6.21 +/- 0.40 nmol/g. Brain L-dopa was increased to 326, 335 and 779%, dopamine to 138, 116 and 217% and SAH/SAM to 354, 392 and 1101%. The data show that L-dopa depletes SAM, and increases methylation 4-5 times more than dopamine, therefore, methylation may play a role in the actions of L-dopa. This and other studies suggest that the high level of utilization of methyl group by L-dopa leads to the induction of enzymes to replenish SAM and to increase the methylation of L-dopa as well as DA. These changes may be involved in the side effects of L-dopa.     

The Role of 3-<Emphasis Type="Italic">O</Emphasis>-Methyldopa in the Side Effects of <Emphasis Type="SmallCaps">l</Emphasis>-dopa

Long-term treatment of l-dopa for Parkinson’s disease (PD) patients induces adverse effects, including dyskinesia, on–off and wearing-off symptoms. However, the cause of these side effects has not been established to date. In the present study, therefore, 3-O-methyldopa (3-OMD), which is a major metabolite of l-dopa, was tested to determine whether it plays a role in the aforementioned adverse effects. The effects of 3-OMD on the dopaminergic nervous system in the brain were investigated, by examining behavioral, biochemical, and cellular changes in male Sprague–Dawley rats and catecholamine-producing PC12 neuronal cells. The results revealed that the intracerebroventricular (icv) injection of 1 μmol of 3-OMD impaired locomotor activities by decreasing movement time (MT), total distance (TD), and the number of movement (NM) by 70, 74 and 61%, respectively. The biochemical analysis results showed that a single administration of 1 μmole of 3-OMD decreased the dopamine turnover rate (DOPAC/DA) by 40.0% in the rat striatum. 3-OMD inhibited dopamine transporter and uptake in rat brain striatal membranes and PC12 cells. The subacute administration of 3-OMD (5 days, icv) also significantly impaired the locomotor activities and catecholamine levels. 3-OMD induced cytotoxic effects via oxidative stress and decreased mitochondrial membrane potential in PC12 cells, indicating that 3-OMD can damage neuronal cells. Furthermore, 3-OMD potentiated l-dopa toxicity and these toxic effects induced by both 3-OMD and l-dopa were blocked by vitamin E (α-tocopherol) in PC12 cells, indicating that 3-OMD may increase the toxic effects of l-dopa to some extent by oxidative stress. Therefore, the present study reveals that 3-OMD accumulation from long-term l-dopa treatment may be involved in the adverse effects of l-dopa therapy. Moreover, l-dopa treatment might accelerate the progression of PD, at least in part, by 3-OMD.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Acetyl-l-carnitine influences the fluidity of brain microsomes and of liposomes made of rat brain microsomal lipid extracts

The fluorescence anisotropy (r) of diphenylhexatriene (DPH) was measured in different preparations (bovine spinal cord phosphatidylserine liposomes, rat brain microsomes, liposomes made with rat brain microsomal lipid having different phospholipid:cholesterol ratios) at temperatures ranging from 10° to 55°C. Phosphatidylserine liposomes exhibited an exponential relationship of rversus temperature, whereas the relationship shown by microsomes and liposomes prepared with microsomal lipid extracts was a linear one. The removal of protein and high phospholipid:cholesterol ratios decreased the slope of the lines (fluidity increased), although the intercept was unaffected. This means that differences were better appreciated at high temperatures and were well evident at 37°C. Acetyl-l-carnitine decreased r in rat brain microsomes and in liposomes made with microsomal lipids with different phospholipid:cholesterol ratios. The fluidifying effect of acetyl-l-carnitine was mild but statistically significant and could explain, at least in part, the data reported in the literature of acetyl-l-carnitine acting on some parameters affected by ageing. Besides, acetyl-l-carnitine seemed to oppose the changes of viscosity due to lipid peroxidation, which has been reported to increase in ageing and dementia.l-carnitine shares the properties of its acetyl ester, but only in part.Abbreviations DPH diphenylhexatriene - HEPES 4-(2-hydroxyethyl-l-piperazineethansulfonic) acid - r fluorescence anisotropy - SHB sucrose-HEPES-buffer (0.32 M sucrose, 2 mM HEPES, pH 7.0)     

Emodin O-methyltransferase from Aspergillus terreus

Emodin O-methyltransferase, an enzyme catalyzing methylation of the 8-hydroxy group of emodin, was identified in the mould Aspergillus terreus IMI 16043, a (+)-geodin producing strain. The enzyme catalyzed the formation of questin from emodin and S-adenosyl-l-methionine. By chromatography on DEAE-cellulose, Phenyl Sepharose, Q-Sepharose, Hydroxyapatite, and CM-cellulose, emodin O-methyltransferase was purified to apparent homogeneity. The purified protein had a molecular weight of 322 kDa as estimated by gel filtration and 53.6 kDa as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme was a homohexamer. The enzyme showed pI 4.4 and optimum pH 7–8. Magnesium ion or manganese ion was not an absolute requirement, nor increased the enzyme activity. The enzyme had strict substrate specificity and very low Km values for both emodin (3.4×10^-7 M) and S-adenosyl-l-methionine (4.1×10^-6 M).Abbreviations EOMT emodin O-methyltransferase from A. terreus - SAM S-adenosyl-l-methionine - PAGE polyacrylamide gel electrophoresis     

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.)

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-l-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20–56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.     

Alterations in <Emphasis Type="SmallCaps">D</Emphasis>-amino acid levels in the brains of mice and rats after the administration of <Emphasis Type="SmallCaps">D</Emphasis>-amino acids

Summary. To mutant ddY/DAO⁻ mice lacking D-amino-acid oxidase activity and normal ddY/DAO⁺ mice, five D-amino acids (D-Asp, D-Ser, D-Ala, D-Leu and D-Pro) were orally administered for two weeks, and the D-amino acid levels were examined in seven brain regions. The levels of D-Asp markedly increased in the pituitary and pineal glands in both strains. In the ddY/DAO⁺ mice, the levels of the other D-amino acids did not significantly change in most of the brain regions. While in the ddY/DAO⁻ mice the levels of D-Ser significantly increased in most of the brain regions except for the cerebrum and hippocampus. The levels of D-Ala and D-Leu increased in all regions but the levels of D-Pro did not significantly change. The same five D-amino acids were intravenously injected into Wistar rats and the D-amino acid levels in their brains were examined for 60 min after the administration. The levels of D-Asp markedly increased in the pineal gland 3 min after the administration, while the levels of D-Ser, D-Ala, and D-Pro increased both in the pineal and pituitary glands, the levels of D-Leu increased in all brain regions. These results are useful for the elucidation of the origins and regulation of D-amino acids in the mammalian body.     

Alpha‐Synuclein as a Pathological Link Between Chronic Traumatic Brain Injury and Parkinson's Disease

The long‐term consequences of traumatic brain injury (TBI) are closely associated with the development of histopathological deficits. Notably, TBI may predispose long‐term survivors to age‐related neurodegenerative diseases, such as Parkinson's disease (PD), which is characterized by a gradual degeneration of the nigrostriatal dopaminergic neurons. However, preclinical studies on the pathophysiological changes in substantia nigra (SN) after chronic TBI are lacking. In the present in vivo study, we examined the pathological link between PD‐associated dopaminergic neuronal loss and chronic TBI. Sixty days post‐TBI, rats were euthanized and brain tissues harvested. Immunostaining was performed using tyrosine hydroxylase (TH), an enzyme required for the synthesis of dopamine in neurons, α‐synuclein, a presynaptic protein that plays a role in synaptic vesicle recycling, and major histocompatibility complex II (MHCII), a protein found in antigen presenting cells such as inflammatory microglia cells, all key players in PD pathology. Unbiased stereology analyses revealed significant decrease of TH‐positive expression in the surviving dopaminergic neurons of the SN pars compacta (SNpc) relative to sham control. In parallel, increased α‐synuclein accumulation was detected in the ipsilateral SN compared to the contralateral SN in TBI animals or sham control. In addition, exacerbation of MHCII+ cells was recognized in the SN and cerebral peduncle ipsilateral to injury relative to contralateral side and sham control. These results suggest α‐synuclein as a pathological link between chronic effects of TBI and PD symptoms as evidenced by significant overexpression and abnormal accumulation of α‐synuclein in inflammation‐infiltrated SN of rats exposed to chronic TBI. J. Cell. Physiol. 230: 1024–1032, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

N-acetyl-l-glutamate in brain: Assay,levels, and regional and subcellular distribution

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200–300 times lower than the levels of N-acetyl-l-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 g/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-l-aspartyl-l-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.Special issue dedicated to Dr. Santiago Grisolía     

Intra-mitochondrial Methylation Deficiency Due to Mutations in SLC25A26

S-adenosylmethionine (SAM) is the predominant methyl group donor and has a large spectrum of target substrates. As such, it is essential for nearly all biological methylation reactions. SAM is synthesized by methionine adenosyltransferase from methionine and ATP in the cytoplasm and subsequently distributed throughout the different cellular compartments, including mitochondria, where methylation is mostly required for nucleic-acid modifications and respiratory-chain function. We report a syndrome in three families affected by reduced intra-mitochondrial methylation caused by recessive mutations in the gene encoding the only known mitochondrial SAM transporter, SLC25A26. Clinical findings ranged from neonatal mortality resulting from respiratory insufficiency and hydrops to childhood acute episodes of cardiopulmonary failure and slowly progressive muscle weakness. We show that SLC25A26 mutations cause various mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation, and the biosynthesis of CoQ10 and lipoic acid.     

Expression of <Emphasis Type="SmallCaps">l</Emphasis>-Serine Biosynthetic Enzyme 3-Phosphoglycerate Dehydrogenase (Phgdh) and Neutral Amino Acid Transporter ASCT1 Following an Excitotoxic Lesion in the Mouse Hippocampus

The nonessential amino acid l-serine functions as a glia-derived trophic factor and strongly promotes the survival and differentiation of cultured neurons. The l-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase (Phgdh) and the small neutral amino acid transporter ASCT1 are preferentially expressed in specific glial cells in the brain. However, their roles in pathological progression remain unclear. We examined the expression of Phgdh and ASCT1 in kainic acid (KA)-induced neurodegeneration of the mouse hippocampus using immunohistochemistry and Western blots. Our quantitative analysis revealed that Phgdh and ASCT1 were constitutively expressed in the normal brain and transiently upregulated by KA-treatment. At the cellular level, Phgdh was expressed in astrocytes in control and in KA-treated mice while ASCT1 that was expressed primarily in the neurons of the normal brain appeared also in activated astrocytes in KA treated mouse brain. The preferential glial expression of ASCT1 was consistent with that of Phgdh. These results demonstrate injury-induced changes in Phgdh and ASCT1 expression. It is hypothesized that the secretion of l-serine is regulated by astrocytes in response to toxic molecules such as glutamate and free radicals that promote neurodegeneration, and may correspond to the level of l-serine needed for neuronal survival and glial activation during brain insults. G. S. Jeon and D. H. Choi contributed equally to this work.     

Evidence for altered methionine methyl-group utilization in the diabetic rat's brain

The methionine (MET) derivative, S-adenosylmethionine (SAM), provides methyl-groups for methylation reactions in many neural processes. In rats made diabetic with streptozotocin (SZ), brain SAM levels were generally lower (10–20%) than in controls, with a constant decrease being observed five weeks after onset of diabetes. This decrease in SAM levels may be due to reduced precursor (MET) availability because greatly elevating plasma MET concentrations in SZ diabetic rats by dietary manipulation increased their neural SAM concentrations to be approximately or even greater than (5–20%) those of controls. In contrast, neural levels of SAM's demethylated product, S-adenosylhomocysteine (SAH), were reduced to a greater extent (17–44%) than SAM levels in all groups of SZ diabetic rats independent of their plasma MET concentrations or brain SAM levels. This indicates that the decrease in SAH levels is not simply due to substrate (SAM) restriction. These changes in MET metabolites appear to be a general effect of diabetes rather than a non-pancreatic side-effect of SZ, because genetically diabetic BB Wistar rats also exhibited reduced brain SAM (25%) and brain SAH (46%) levels. These results indicate that methyl-groups from MET are handled differently in the brain of the diabetic rat, which considering the variety and importance of neural methylation reactions, could have important consequences for the diabetic.Abbreviations MET methionine - SAM S-adenosylmethionine - SAH S-adenosylhomocysteine - SZ streptozotocin - BBW BB Wistar - LNAA large neutral amino acids - BCAA branchedchain amino acids - MET:BCAA methionine to branched-chain amino acid ratio - MET:LNAA methionine to large neutral amino acid ratio     

Studies of aluminum in rat brain

The effects of high aluminum concentrations in rat brain were studied using¹⁴C autoradiography to measure the uptake of [¹⁴C]2deoxy-d-glucose ([¹⁴C]2dG) and microbeam proteon-induced X-ray emission (microPIXE) with a 20-μm resolution to measure concentrations of magnesium, aluminum, potassium, and calcium. The aluminum was introduced intracisternally in the form of aluminum tartrate (Al-T), and control animals were given sodium tartrate (Na-T). The¹⁴C was administered intravenously. The animals receiving Al-T developed seizure disorders and had pathological changes, which included cerebral cortical atrophy. The results showed that there was a decreased uptake of [¹⁴C]2dG in cortical regions in which increased aluminum levels were measured, i.e., there was a correlation between the aluminum in the rat brain and decreased brain glucose metabolism. A minimum detection limit of about 16 ppm (mass fraction) or 3×10⁹ Al atoms was obtained for Al under the conditions employed.     

Effect of aluminum on the blood-brain barrier permeability during nitric oxide-blockade-induced chronic hypertension in rats

We examined the effect of aluminum on the permeability of the blood-brain barrier (BBB) during nitric oxide-blockade-induced chronic hypertension in rats. Animals were given the inhibitor of nitric oxide synthase, l-NAME (N ^ω-nitro-l-arginine methyl ester), for 4 wk to induce chronic hypertension. Two groups of rats were given an intraperitoneal injection of aluminum chloride. The integrity of the BBB was assessed by a quantitative measurement for Evans blue (EB) dye. The arterial blood pressure in l-NAME- and l-NAME plus aluminum-treated animals was significantly elevated from 115±2.8 and 110±1.7 mm Hg to 174±5.2 and 175±4.8 mm Hg, respectively (p<0.01). The EB dye content in the brain regions of the rats in the l-NAME group was increased, but there was no statistical significance compared to the saline group. The extravasation of EB dye was significantly increased in the brain regions of the animals treated with aluminum compared to the rats treated with saline (p<0.05). A significantly higher EB dye content in the brain regions was observed in the l-NAME plus aluminium group compared to l-NAME, aluminum, and saline groups (p<0.01). These findings indicate that exposure to a high level of aluminum leads to an additional increase in BBB permeability where nitric oxide-blockade-induced chronic hypertension potentiates the effect of aluminum to enhance BBB permeability to EB dye.     

Activity of Lactate Dehydrogenase in Serum and Cerebral Cortex of Immature and Mature Rats after Hypobaric Hypoxia

In our previous studies we have found both an increase of lipid peroxidation damage (expressed as levels of thiobarbituric acid-reactive substances) in brain and plasma lactate concentration in 21-day-old rats after a 30-min exposure to hypobaric hypoxia. Pretreatment of rats with l-carnitine decreased both parameters. The aim of our present study was to determine if the l-carnitine-dependent decrease of plasma lactate could be due to a modification of lactate dehydrogenase (LDH) activity. We followed brain and blood serum LDH activity of 14-, 21- and 90-day-old Wistar rats. We found an increase of brain LDH activity with age. However, we did not observe any significant differences in LDH activity after exposure to hypobaric hypoxia or l-carnitine pretreatment. In contrast to brain, serum LDH activity did not show any clear age-dependence. The hypoxia exposure increased LDH activity of 21-day-old rats only. Pretreatment of rats with l-carnitine decreased serum LDH activity of 21- and 90-day-old rats probably due to membrane stabilizing role of l-carnitine. In conclusions, acute hypobaric hypoxia and/or l-carnitine pretreatment modified serum but not brain LDH activity.     

Overexpression of yeast <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase <Emphasis Type="Italic">metK</Emphasis> in <Emphasis Type="Italic">Streptomyces actuosus</Emphasis> leads to increased production of nosiheptide

S-Adenosylmethionine (SAM) is synthesized via the metabolic reaction involving adenosine triphosphate and l-methionine that is catalyzed by the enzyme S-adenosyl-l-methionine synthetase (SAM-s) and encoded by the gene metK. In the present study, metK with the absence of introns from Saccharomyces cerevisiae was introduced into Streptomyces actuosus, a nosiheptide (Nsh) producer. Intracellular SAM levels were determined by high-pressure liquid chromatography. Through optimizing the nutrient content of the medium, it was shown that increased SAM production induced by the overexpression of SAM-s leads to an increase in the intracellular cysteine pool and overproduction of Nsh in S. actuosus. This investigation shows that increased SAM promotes the elevated production of the non-ribosomal thiopeptide Nsh in Streptomyces sp.     

Action ofl-acetylcarnitine on age-dependent modifications of mitochondrial membrane proteins from rat cerebellum

Protein patterns of mitochondrial outer membrane, inner membrane, and matrix from nonsynaptic (free) mitochondria from rat cerebellum at different ages (4, 8, 12, 16, 20, and 24 months) were analyzed by gel electrophoresis. Acutel-acetylcarnitine treatment was per-formed by a single i.p. injection (100 mg/kg body weight) of the substance 60 min before the sacrifice of the animals. Different age-dependent changes were obtained for the proteins of the three fractions. The amount of some protein subunits increased and/or decreased after drug treatment. In particular, protein composition of the inner mitochondrial membrane showed significant age-related modifications. This result probably indicates differences in protein synthesis and/or turnover rates in the various mitochondrial compartments during aging. Acutel-acetylcarnitine treatment caused: a high increase in the amount of one inner membrane protein with Mw 16 kDa, at all the ages studied; a decrease in the amount of many other inner membrane proteins; modifications of some matrix proteins. Our results show that in vivo administration ofl-acetylcarnitine affects mainly the inner membrane protein composition of cerebellar mitochondria.