Isolation of a Ca2+-Dependent Actin-Fragmenting Protein from Brain, Spinal Cord, and Cultured Neurones

Extracts of ox spinal cord and chicken brain were fractionated by ion-exchange chromatography and assayed for their ability to reduce the viscosity of muscle F-actin solutions. Two distinct peaks of activity were obtained, one of which was further purified by affinity chromatography on a DNAase-actin Sepharose column. Following molecular exclusion chromatography, the actin component appeared as a complex of 1 molecule of a protein with molecular weight 90,000 and 2 molecules of actin (42,000). This tightly bound complex was resistant to most methods of protein separation, but was resolvable into its component proteins by sodium dodecyl sulphate acrylamide gel electrophoresis. The protein of molecular weight 90,000 could be eluted from such a gel in a fully active form. The activity of the protein from ox spinal cord was closely similar to that of gelsolin, an actin-fragmenting protein originally isolated from rabbit lung macrophages. Like gelsolin, the protein from ox spinal cord produced fragmentation of muscle F-actin filaments at Ca2+ concentrations greater than 10(-7) M, and had a nucleating effect on the polymerisation of muscle actin; the latter was measured most easily by the enhancement of fluorescence of muscle actin conjugated to N-(1-pyrenyl)iodoacetamide. Nucleation was more effective in the presence of Ca2+, but also occurred in its absence, and the same was true of complex formation between the 90,000 protein and muscle G-actin. On the basis of its actin-fragmenting activity, we estimate that the 90,000 molecular weight protein constitutes 0.2% of the protein initially extracted from ox spinal cord. A very similar protein, indistinguishable in its action on actin but containing variable amounts of a protein of molecular weight 85,000 as well as 90,000, was isolated from chicken brain. A similar protein was also detected in pure cultures of sympathetic neurones by enrichment on a DNAase-actin affinity column and by immune blotting and by immunofluorescence. We conclude that a protein similar, if not identical to macrophage gelsolin is present in neurones and that it probably plays a part in the actin-based movements of these cells.     

Mitochondrial creatine kinase activity alterations in skeletal muscle during long-distance running

In human gastrocnemius muscle obtained from long-distance runners, mitochondrial creatine kinase (CK) activities were significantly greater than nonrunning control skeletal muscle and significantly increased during training for and after a marathon race. Thus skeletal muscle tended to become similar to heart muscle in its mitochondrial CK composition. Total muscle CK activity was significantly different in males and females, was unaffected by marathon training and racing, and was similar to gastrocnemius muscle obtained from nonrunning controls. There was an inverse correlation between the maximum O2 uptake and the percentage increase in mitochondrial CK activity after training. These studies suggest that mitochondrial CK may play a key role in the intracellular transport of energy from mitochondrial to myofibrils in skeletal muscle during endurance exercise such as long-distance running.     

Structural characterization of myosin from bovine brain

Myosins isolated from bovine brain, rabbit skeletal muscle, and chicken gizzard smooth muscle and their heavy meromyosin and light meromyosin fractions were studied in the electron microscope by negative staining with uranyl acetate. Under similar conditions of preparation and polymerization, the three myosins formed paracrystals of different structures. The light meromyosin portion of the skeletal muscle myosin also assembled in a different fashion than the brain or smooth muscle light meromyosins; the latter two assembled similarly. The heavy meromyosin portion from each of the three myosins was shown to interact with the actins isolated from each of the three tissue sources by the formation of the characteristic arrowhead patterns with similar periodicities. The brain heavy meromyosin attachment to both skeletal and brain actins was dissociated by ATP. It is suggested that differences in the light meromyosin portions of the three myosins may account in part for their differences in assembly in vivo.     

Comparison of soft tissue calcium levels throughout the life spans of normal and myopathic hamsters

Comparisons of calcium levels in heart, skeletal muscle, brain, liver and plasma were made throughout the life spans of normal and genetically myopathic hamsters. A large calcium accumulation occurred in, and was restricted to, myopathic tissues (heart and skeletal muscle). Calcium elevation in myopathic tissues corresponded to documented lesion appearance, and persisted throughout the remainder of the life span. Calcium buildup in heart and skeletal muscle, as a central feature of the myopathic process, does not reflect a general failure of calcium homeostasis; nonmyopathic tissues are unaffected. Calcium increased moderately in some normal tissues late in life.     

Aerobic metabolism of human quadriceps muscle: in vivo data parallel measurements on isolated mitochondria

The aim of the present study was to examine whether parameters of isolated mitochondria could account for the in vivo maximum oxygen uptake (VO2max) of human skeletal muscle. VO2max and work performance of the quadriceps muscle of six volunteers were measured in the knee extensor model (range 10-18 mmol O2 x min(-1) x kg(-1) at work rates of 22-32 W/kg). Mitochondria were isolated from the same muscle at rest. Strong correlations were obtained between VO2max and a number of mitochondrial parameters (mitochondrial protein, cytochrome aa3, citrate synthase, and respiratory activities). The activities of citrate synthase, succinate dehydrogenase, and pyruvate dehydrogenase, measured in isolated mitochondria, corresponded to, respectively, 15, 3, and 1.1 times the rates calculated from VO2max. The respiratory chain activity also appeared sufficient. Fully coupled in vitro respiration, which is limited by the rate of ATP synthesis, could account for, at most, 60% of the VO2max. This might be due to systematic errors or to loose coupling of the mitochondrial respiration under intense exercise.     

A monoclonal antibody against cytochrome c oxidase distinguishes cardiac and skeletal muscle mitochondria

The mitochondrial enzyme cytochrome c oxidase (COX) in eukaryotes consists of at least seven subunits, three of which (I-III) are encoded by mitochondrial DNA (mitDNA) and the others (IV-VII) by the nuclear genome. There is increasing evidence that COX in mammals exists in multiple tissue-specific forms, presumably specified by nuclearly encoded subunits. We performed immunologic studies in human cardiac and skeletal muscle, using a monoclonal antibody raised against subunit IV of COX purified from human cardiac muscle. In immunotitration studies, the antibody bound with high affinity to mitochondria from cardiac muscle, but reacted only weakly with mitochondria from skeletal muscle. Similarly, immunocytochemical studies showed prominent mitochondrial staining in frozen sections of heart, but no staining in sections of mature skeletal muscle. Although this antibody did not stain mitochondria in mature skeletal muscle, it clearly stained mitochondria in myoblasts and immature myotubes of human muscle cultures, suggesting that mitochondria in immature muscle cells are different from those in mature muscle, and similar to heart mitochondria. Immunotitration data using either native or denatured COX protein from heart or skeletal muscle showed similar immunoreactivity. These studies indicate that the epitope for recognition by this antibody is exposed in mitochondria from heart and immature muscle cells, but masked in mitochondria from mature skeletal muscle.     

Chronic systemic complex I inhibition induces a hypokinetic multisystem degeneration in rats

In Parkinson's disease, nigral dopaminergic neurones degenerate, whereas post-synaptic striatal target neurones are spared. In some atypical parkinsonian syndromes, both nigral and striatal neurones degenerate. Reduced activity of complex I of the mitochondrial respiratory chain has been implicated in both conditions, but it remains unclear if this affects the whole organism or only the degenerating brain structures. We therefore investigated the differential vulnerability of various brain structures to generalized complex I inhibition. Male Lewis rats infused with rotenone, a lipophilic complex I inhibitor [2.5 mg/kg/day intraveneously (i.v.) for 28 days], were compared with vehicle-infused controls. They showed reduced locomotor activity and loss of striatal dopaminergic fibres (54%), nigral dopaminergic neurones (28.5%), striatal serotoninergic fibres (34%), striatal DARPP-32-positive projection neurones (26.5%), striatal cholinergic interneurones (22.1%), cholinergic neurones in the pedunculopontine tegmental nucleus (23.7%) and noradrenergic neurones in the locus ceruleus (26.4%). Silver impregnation revealed pronounced degeneration in basal ganglia and brain stem nuclei, whereas the hippocampus, cerebellum and cerebral cortex were less affected. These data suggest that a generalized mitochondrial failure may be implicated in atypical parkinsonian syndromes but do not support the hypothesis that a generalized complex I inhibition results in the rather selective nigral lesion observed in Parkinson's disease.     

Iron, neuromelanin and ferritin content in the substantia nigra of normal subjects at different ages: consequences for iron storage and neurodegenerative processes

Information on the molecular distribution and ageing trend of brain iron in post‐mortem material from normal subjects is scarce. Because it is known that neuromelanin and ferritin form stable complexes with iron(III), in this study we measured the concentration of iron, ferritin and neuromelanin in substantia nigra from normal subjects, aged between 1 and 90 years, dissected post mortem. Iron levels in substantia nigra were 20 ng/mg in the first year of life, had increased to 200 ng/mg by the fourth decade and remained stable until 90 years of age. The H‐ferritin concentration was also very low (29 ng/mg) during the first year of life but increased rapidly to values of ≈ 200 ng/mg at 20 years of age, which then remained constant until the eighth decade of life. L ‐Ferritin also showed an increasing trend during life although the concentrations were ≈ 50% less than that of H‐ferritin at each age point. Neuromelanin was not detectable during the first year, increased to ≈ 1000 ng/mg in the second decade and then increased continuously to 3500 ng/mg in the 80th year. A Mössbauer study revealed that the high‐spin trivalent iron is probably arranged in a ferritin‐like iron?oxyhydroxide cluster form in the substantia nigra. Based on this data and on the low H‐ and L‐ferritin content in neurones it is concluded that neuromelanin is the major iron storage in substantia nigra neurones in normal individuals.     

Oxidative Myocytes of Heart and Skeletal Muscle Express Abundant Sarcomeric Mitochondrial Creatine Kinase

Sarcomeric mitochondrial creatine kinase catalyzes the reversible transfer of a high energy phosphate between ATP and creatine. To study cellular distribution of the kinase, we performed immunocytochemical studies using a peptide antiserum specific for the kinase protein. Our results demonstrated that the sarcomeric mitochondrial creatine kinase gene is abundantly expressed in heart and skeletal muscle, with no protein detected in other tissues examined, including brain, lung, liver, spleen, kidney, bladder, testis, stomach, intestine, and colon. RNA blot study showed that there is no detectable expression of the kinase mRNA in the thymus gland. In heart and skeletal muscle, the kinase protein is expressed in atrial and ventricular cardiomyocytes and a subpopulation of skeletal myofibres. In skeletal muscle, fast myosin heavy chain co-localization studies demonstrated that the sarcomeric mitochondrial creatine kinase is highly expressed in type 1, slow-oxidative and type 2A, fast-oxidative-glycolytic myofibres. We conclude that the kinase gene is abundantly expressed in oxidative myocytes of heart and skeletal muscle and may contribute to oxidative capacity of these cells.     

The isolation and characterization of actin from porcine brain

Highly purified porcine brain actin has been prepared by a procedure involving anion-exchange chromatography, polymerization-depolymerization, and gel filtration. Electrophoresis of purified brain actin on polyacrylamide gels in the presence of sodium dodecyl sulfate shows a single protein band corresponding to more than 95% of the applied protein and migrating with the relative mobility of skeletal muscle actin. The amino acid composition of brain actin is similar but not identical to that of rabbit skeletal muscle actin. Brain actin activates the low ionic strength Mg²⁺-ATPase of skeletal muscle myosin to the same extent that skeletal muscle actin potentiates the muscle ATPase. Although similar to its skeletal muscle counterpart, brain actin is distinctly different. Isoelectric focusing experiments indicate that brain actin consists of at least two species, each of which is more basic than the α-species of skeletal muscle actin. The polymerization of brain actin was followed by viscometry and sedimentation techniques as a function of protein concentration, temperature, and ionic conditions. The critical actin concentrations of both brain and skeletal muscle actins polymerized at low ionic strength in the presence of 2.0 mm MgCl₂ are similar and show little dependence upon temperature. When polymerized in the presence of 0. 1 m KCl, brain actin has a critical actin concentration that is higher and more dependent upon temperature than the corresponding critical concentration of skeletal muscle actin.     

Improved brain and muscle mitochondrial respiration with CoQ. An in vivo study by 31P-MR spectroscopy in patients with mitochondrial cytopathies.

We used in vivo phosphorus magnetic resonance spectroscopy (31P-MRS) to study the effect of CoQ10 on the efficiency of brain and skeletal muscle mitochondrial respiration in ten patients with mitochondrial cytopathies. Before CoQ, brain [PCr] was remarkably lower in patients than in controls, while [Pi] and [ADP] were higher. Brain cytosolic free [Mg2+] and delta G of ATP hydrolysis were also abnormal in all patients. MRS also revealed abnormal mitochondrial function in the skeletal muscles of all patients, as shown by a decreased rate of PCr recovery from exercise. After six-months of treatment with CoQ (150 mg/day), all brain MRS-measurable variables as well as the rate of muscle mitochondrial respiration were remarkably improved in all patients. These in vivo findings show that treatment with CoQ in patients with mitochondrial cytopathies improves mitochondrial respiration in both brain and skeletal muscles, and are consistent with Lenaz's view that increased CoQ concentration in the mitochondrial membrane increases the efficiency of oxidative phosphorylation independently of enzyme deficit.     

Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acid binding protein

The rat contains at least three homologous cytosolic proteins that bind long chain fatty acids, termed liver (L-), intestinal (I-), and heart (H-) fatty acid binding protein (FABP). I-FABP mRNA is confined to the gastrointestinal tract while L-FABP mRNA is abundantly represented in hepatocytes as well as enterocytes. We have isolated a rat heart FABP cDNA clone and determined the pattern of H-FABP mRNA accumulation in a wide variety of tissues harvested from late fetal, suckling, weaning, and adult rats. RNA blot hybridizations and primer extension analysis disclosed that the distribution of H-FABP mRNA in adult rat tissues is different from that of I- or L-FABP mRNA. H-FABP mRNA is most abundant in adult heart. This mRNA was also present in an adult slow twitch (type I) skeletal muscle (soleus, 63% of the concentration in heart), testes (28%), a fast twitch skeletal muscle (psoas, 17%), brain (10%), kidney (5%), and adrenal gland (5%). H-FABP mRNA was not detected in adult small intestine, colon, spleen, lung, or liver RNA. Distinct patterns of developmental change in H-FABP mRNA accumulation were documented in heart, placenta, brain, kidney, and testes. Myocardial H-FABP mRNA levels rise rapidly during the 48 h prior to and after birth, reaching peak levels by the early weaning period. The postnatal increase in myocardial H-FABP mRNA concentration and its relative distribution in adult fast and slow twitch skeletal muscle are consistent with its previously proposed function in facilitating mitochondrial beta-oxidation of fatty acids. However, the presence of H-FABP mRNA in brain, a tissue which does not normally significantly oxidize fatty acids in late postnatal life, suggests that H-FABP may play a wider role in fatty acid metabolism than previously realized. Mouse-hamster somatic cell hybrids were utilized to map H-FABP. Using stringencies which did not produce cross-hybridization between L-, I-, and H-FABP DNA sequences, we found at least three loci in the mouse genome, each located on different chromosomes, which reacted with our cloned H-FABP cDNA. None of these H-FABP-related loci were linked to the gene which specifies a highly homologous adipocyte-specific protein termed aP2 or to genes encoding two other members of this protein family, cellular retinol binding protein and cellular retinol binding protein II.(ABSTRACT TRUNCATED AT 400 WORDS)     

The protein phosphatases involved in cellular regulation. Antibody to protein phosphatase-2A as a probe of phosphatase structure and function

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.     

Deleted mitochondrial DNA in the skeletal muscle of aged individuals.

Human mitochondrial DNA deletions occur mainly in the major region between the origins of replication of the heavy and light strands both in mitochondrial myopathy and in the ageing process. To determine whether deletions in the minor region also contribute to the ageing process, we analyzed a 3,610-basepair deletion (nucleotide position 1,837-5,447, from the 16S rRNA gene to the ND2 gene) in the skeletal muscle from individuals of various ages. The direct repeated sequence at each boundary of the deletion was identified as 5'-CCCC-3'. This minor-region deletion was detected in one of five individuals of the sixth decade, two of five in the seventh decade, and all of five in the eighth decade, but not in individuals below age 60. These results indicate that age-related accumulation of mtDNA deletions occurs not only in the major region but also in the minor region.     

High‐fat diet affects skeletal muscle mitochondria comparable to pressure overload‐induced heart failure

In heart failure, high‐fat diet (HFD) may exert beneficial effects on cardiac mitochondria and contractility. Skeletal muscle mitochondrial dysfunction in heart failure is associated with myopathy. However, it is not clear if HFD affects skeletal muscle mitochondria in heart failure as well. To induce heart failure, we used pressure overload (PO) in rats fed normal chow or HFD. Interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) from gastrocnemius were isolated and functionally characterized. With PO heart failure, maximal respiratory capacity was impaired in IFM but increased in SSM of gastrocnemius. Unexpectedly, HFD affected mitochondria comparably to PO. In combination, PO and HFD showed additive effects on mitochondrial subpopulations which were reflected by isolated complex activities. While PO impaired diastolic as well as systolic cardiac function and increased glucose tolerance, HFD did not affect cardiac function but decreased glucose tolerance. We conclude that HFD and PO heart failure have comparable effects leading to more severe impairment of IFM. Glucose tolerance seems not causally related to skeletal muscle mitochondrial dysfunction. The additive effects of HFD and PO may suggest accelerated skeletal muscle mitochondrial dysfunction when heart failure is accompanied with a diet containing high fat.     

一次性力竭运动对大鼠PHB1表达及线粒体功能的影响

目的：观察一次性力竭运动后大鼠脑、心、骨骼肌组织和线粒体中PHB1含量的变化及对大鼠线粒体功能的影响,探寻PHB1与线粒体功能和能量代谢的关系。方法：健康雄性SD大鼠40只,随机分为2组（n=20）：对照组和一次性力竭运动组,大鼠进行一次性急性跑台运动建立力竭运动模型。收集各组大鼠的心、脑和骨骼肌组织样品并提取线粒体,检测其呼吸功能和ROS的变化。用Western blot方法检测组织和线粒体中PHB1蛋白表达水平;用分光光度计检测各器官中ATP含量以及线粒体中复合体V活性（ATP合酶活性）。结果：①一次性力竭运动后脑、心肌、骨骼肌中ATP含量显著性降低;②一次性力竭运动后脑、心肌、骨骼肌线粒体中复合体V活性、RCR、ROS显著性降低,ST4均显著性升高,ST3无显著性差异。③一次性力竭运动后心、脑、骨骼肌线粒体中PHB1的表达显著性减少。④通过相关性分析得出：一次性力竭运动后心、脑、骨骼肌中ATP含量与心、脑、骨骼肌中复合体V活性呈正相关;心、脑、骨骼肌中ATP含量和心、脑骨骼肌中PHB1的表达呈正相关。结论：一次性力竭运动后,降低线粒体氧化磷酸化功能,使大鼠脑、骨骼肌线粒体内ROS生成增加,PHB1的表达、ATP含量和复合体V活性均下降。一次性力竭运动使得大鼠线粒体内PHB1表达降低,线粒体功能减弱,机体能量代谢降低。     

Skeletal muscle mitochondrial FAT/CD36 content and palmitate oxidation are not decreased in obese women

A reduction in fatty acid oxidation has been associated with lipid accumulation and insulin resistance in the skeletal muscle of obese individuals. We examined whether this decrease in fatty acid oxidation was attributable to a reduction in muscle mitochondrial content and/or a dysfunction in fatty acid oxidation within mitochondria obtained from skeletal muscle of age-matched, lean [body mass index (BMI) = 23.3 +/- 0.7 kg/m2] and obese women (BMI = 37.6 +/- 2.2 kg/m2). The mitochondrial marker enzymes citrate synthase (-34%), beta-hydroxyacyl-CoA dehydrogenase (-17%), and cytochrome c oxidase (-32%) were reduced (P < 0.05) in obese participants, indicating that mitochondrial content was diminished. Obesity did not alter the ability of isolated mitochondria to oxidize palmitate; however, fatty acid oxidation was reduced at the whole muscle level by 28% (P < 0.05) in the obese. Mitochondrial fatty acid translocase (FAT/CD36) did not differ in lean and obese individuals, but mitochondrial FAT/CD36 was correlated with mitochondrial fatty acid oxidation (r = 0.67, P < 0.05). We conclude that the reduction in fatty acid oxidation in obese individuals is attributable to a decrease in mitochondrial content, not to an intrinsic defect in the mitochondria obtained from skeletal muscle of obese individuals. In addition, it appears that mitochondrial FAT/CD36 may be involved in regulating fatty acid oxidation in human skeletal muscle.