Intracerebroventricular injections of noradrenaline affect brain energy metabolism of rainbow trout

To assess the role of noradrenaline (NA) as a possible regulator of brain energy metabolism in teleost fish, the impact of increased noradrenaline levels within the brain on several parameters of energy metabolism was assessed in rainbow trout brain. Accordingly, two different doses of noradrenaline, producing increases in brain NA levels comparable to those occurring in several physiological processes in nature, were selected. In a subsequent set of three different experiments, fish were intracerebroventricularly injected with 1 microL 100 g(-1) body weight of Cortland saline alone (control) or containing NA (5 nmol NA and 10 nmol NA); after 30 min, brain and plasma samples were taken to assess changes in parameters of energy metabolism due to NA treatment. The results obtained clearly show dose-dependent changes in NA-treated fish in several parameters, including decreased glycogen and ATP levels, increased lactate and pyruvate levels, decreased fructose 1,6-bisphosphatase activity, and increased pyruvate kinase and lactate dehydrogenase activities. Altogether, the present experiments show for the first time in a teleost fish evidence supporting that increased noradrenaline levels in the brain elicit metabolic changes in the brain (enhanced glycogenolysis and glycolysis), resulting in an increased energy demand. These metabolic changes may be related to those occurring under several physiological conditions in nature such as hypoxia, in which increased energy demand and increased noradrenaline levels occur in the brain simultaneously.     

硬骨鱼类血红蛋白转换表达的新模式——以黄河裸裂尻鱼胚胎型血红蛋白研究为例

为了探讨高原硬骨鱼类胚胎型血红蛋白的转换表达模式,研究利用转录组数据和克隆方法研究了黄河裸裂尻鱼(Schizopygopsis pylzovi)胚胎型血红蛋白的组成和基因结构,并通过qRT-PCR和整胚原位杂交方法确定了胚胎发育过程中血红蛋白基因的表达情况。结果表明,黄河裸裂尻鱼胚胎型血红蛋白基因由hbae1、hbae4、hbbe1和hbbe3组成,均包含3个外显子和2个内含子,其中hbbe1/hbae1以"头对头"(3′-5′—5′-3′)转录方式串联在一起, hbbe3/hbae4则以"尾对尾"(5′-3′—3-5′)的转录方式串联在一起。进一步的研究初步判断hbae3、hbae5和hbbe2基因在裂腹鱼亚科鱼类进化过程中可能发生了丢失。qRT-PCR和整胚原位杂交结果均表明在早期胚胎发育过程中4个胚胎型血红蛋白基因的表达都是从受精后第5天(120 hpf)开始明显上升,但是hbae4和hbbe3基因的高表达只维持在受精后第5天(120 hpf)至破膜期(216 hpf),随后急剧下降,而hbae1和hbbe1基因表达从破膜(216 hpf)开始急剧上升。研究揭示了硬骨鱼类一种全新...     

Tissue specificity of chordin

Chordin is a tissue-specific protein antigen of notochord. Earlier this protein was discovered in the notochords of sturgeon (Acipenseridae) species; the notochord-specific antigenic determinants were detected in the notochord residues of teleost fish species and in notochord derivatives (nuclei pulposi) of mammals. Using the RIA technique, extracts from 35 samples of normal, fetal and tumour tissues of man were screened for chordin. Among other tissue samples tested, extracts from fetal brain and rectal adenocarcinoma exhibited marked cross-reactivity towards antibodies against chordin. Cross-reactivity towards chordin was observed in rabbit brain extract. This extract contained an antigen which was immunologically related (but not fully identical) to chordin. In total, in this and previous studies, 58 samples of fish and mammalian tissues were analyzed for chordin. However, antigenic determinants of chordin were identified only in extracts prepared from the notochords and nuclei pulposi as well as from brain and rectal adenocarcinoma. These findings suggest that chordin is an antigen with a restricted tissue specificity.     

A Comparative Perspective on Brain Regeneration in Amphibians and Teleost Fish

Regeneration of lost cells in the central nervous system, especially the brain, is present to varying degrees in different species. In mammals, neuronal cell death often leads to glial cell hypertrophy, restricted proliferation, and formation of a gliotic scar, which prevents neuronal regeneration. Conversely, amphibians such as frogs and salamanders and teleost fish possess the astonishing capacity to regenerate lost cells in several regions of their brains. While frogs lose their regenerative abilities after metamorphosis, teleost fish and salamanders are known to possess regenerative competence even throughout adulthood. In the last decades, substantial progress has been made in our understanding of the cellular and molecular mechanisms of brain regeneration in amphibians and fish. But how similar are the means of brain regeneration in these different species? In this review, we provide an overview of common and distinct aspects of brain regeneration in frog, salamander, and teleost fish species: from the origin of regenerated cells to the functional recovery of behaviors.     

ON THE MECHANISM OF STIMULATION OF AMP-AMINOHYDROLASE ACTIVITY BY HEXOKINASE IN BRAIN TISSUE

—A hexokinase has been isolated from brain tissue on Sephadex G-100 and DEAE cellulose which is similar to yeast enzyme in stimulating the AMP-aminohydrolase activity of rat brain soluble fractions. This effect of hexokinase is influenced neither by N-acetyl-glucosamine nor noradrenaline. An isoenzyme of hexokinase isolated from brain tissue on DEAE cellulose, having properties similar to that of the muscle enzyme, has no effect on AMP-aminohydrolase activity. The activating effect of yeast hexokinase is not due to its oligomeric structure. Enzyme subunits obtained by the treatment of native yeast enzyme by urea also activate AMP-aminohydrolase of rat brain soluble fractions.     

A NEW LARVAL FISH BIOASSAY FOR TESTING THE PATHOGENICITY OF PFIESTERIA SPP. (DINOPHYCEAE)1

Water quality, microbial contamination, prior fish health, and variable results have been major impediments to identifying the cause and mechanism of fish mortality in standard aquarium‐format Pfiesteria bioassays. Therefore, we developed a sensitive 96‐h larval fish bioassay for assessing Pfiesteria spp. pathogenicity using six‐well tissue culture plates and 7‐day‐old larval cyprinodontid fish. We used the assay to test pathogenicity of several clonal lines of Pfiesteria piscicida Steidinger and Burkholder and P. shumwayae Glasgow and Burkholder that had been cultured with algal prey for 2 to 36 months. The P. shumwayae cultures exhibited 80%–100% cumulative mortality in less than 96 h at initial zoospore densities of approximately 1000 cells·mL^?1. No fish mortalities occurred with P. piscicida at identical densities or in controls. In a dose‐response assay, we demonstrated a strong positive correlation between dinospore density and fish mortality in a highly pathogenic culture of P. shumwayae, generating a 96‐h LD₅₀ of 108 zoospores·mL^?1. Additionally, we applied the assay to evaluate a 38‐L P. shumwayae bioassay that was actively killing fish and compared results with those from exposures of juvenile tilapia (Oreochromis niloticus) in a 500‐mL assay system. Water from the fish‐killing 38‐L assay was filtered and centrifuged to produce fractions dominated by dinoflagellates, bacteria, or presumed ichthyotoxin (cell‐free fraction). After 96 h, the larval fish assay exhibited 50%–100% cumulative mortality only in fractions containing dinoflagellates, with no mortalities occurring in the other fractions. The 500‐mL bioassay with tilapia produced inconsistent results and demonstrated no clear correlation between mortality and treatment. The new larval fish bioassay was demonstrated as a highly effective method to verify and evaluate dinoflagellate pathogenicity.     

翘嘴鳜miR-222的表达特征

为了解翘嘴鳜miR-222的时空表达规律, 研究利用实时荧光定量PCR的方法检测miR-222在翘嘴鳜不同组织、胚胎发育及胚后发育中的相对表达丰度。研究结果显示, miR-222在肌肉相关的组织中表达较高, 特别是在成年翘嘴鳜的白肌中表达最高; 胚胎发育阶段结果显示, miR-222在胚胎发育的2细胞期就有表达, 而表达量在心动期达到最高。不同组织及不同发育阶段的差异性表达结果表明, miR-222很可能参与调控鳜鱼肌肉的生长发育。为研究合成代谢过程中miR-222在肌肉生长调控中的表达规律, 通过对翘嘴鳜幼鱼在饥饿一周后饱食一餐的实验处理下, 利用实时荧光定量的方法测定miR-222在骨骼肌中的相对表达变化。结果显示, miR-222的表达量在恢复喂食后的1h显著上升(P0.05), 表明miR-222很可能是调节鱼类骨骼肌生长过程中, 参与快速应答信号系统的一类miRNA。研究为miR-222在鱼类发育中的调控作用提供一些理论依据。       

CEPHALOPODS AND FISH: THE LIMITS OF CONVERGENCE

Resemblances between cephalopods and fish 1. Modern cephalopods (coleoids < 1000 species) resemble modern fish (30,000 species) more closely than any of their ancestors did. They have not been replaced by the more diversified group in geological time. 2. The main body of the article (pp. 245–283) reviews these resemblances. They are to be found at all levels of analysis. 3. Basic physiological mechanisms of molluscs (pp. 262–5) have been incorporated into systems with performances comparable to those of vertebrate systems. For instance the cephalopod locomotory system (pp. 249–56) and hydrostatic control system (pp. 256–60), structurally very different from their fish counterparts, have similar adaptive radiation. 4. Behaviour (p. 278) and growth of the brain (p. 265) are characteristically vertebrate-like. 5. Cephalopods and fish are considered as occupying the same broad adaptive zone though modal differences (pp. 283–5)-in reproductive habits, growth rate and light-dependent behaviour where extraocular photoreceptors appear to be important - mean that they occupy different areas within that zone. (ii) Evolution of convergence 6. Evidence is presented (pp. 287–293) for considering the convergence as due not merely to similar physical demands of the marine environment, but to dynamic interactions between cephalopods and vertebrates from the late Palaeozoic onwards. 7. The convergence was set on its way when the two groups, independently of each other, acquired locomotory methods that allowed them to increase in size. 8. It is argued that reduction and eventually complete loss of the chambered shell (in all but sepiids) was an evolutionary response to the needs of increased mobility and to the need to go deeper as vertebrate predators pushed out into oceanic waters. 9. The ammonites (pp. 291–2) present a partial model of the course that coleoid ancestors may have taken. 10. Coleoids subsequently reinvaded surface and coastal waters, competing successfully in a teleost habitat. Their most striking adaptations are ones that they share with teleosts. 11. Behavioural interactions in a vertebrate-dominated environment have probably been responsible for the vertebrate-type eye of cephalopods (p. 293). 12. The conclusion that the common adaptive zone shared by cephalopods and fish has been achieved by mutual interactions between the two groups evokes no special evolutionary principles. It assumes that all cephalopod species have (at some period of their evolutionary history) been in competition for food with some vertebrate species and that vertebrates are a source of selection pressures - largely operating through visual behaviour - that maintain and promote convergence upon the ‘fish’ modal type. 13. Selection pressures also operate within behaviour space to maintain and promote the special differences that separate any competitively successful species from all others. Coleoids as a group appear to have retained adaptations associated with such molluscan features as high growth rate and rapid turnover of the population. They are still characteristically crepuscular in habits and have extensive vertical mobility.     

Microstructure,mineral and mechanical properties of teleost intermuscular bones

There is an increasing interest in understanding teleost bone biomechanics in several scientific communities, for instance as interesting biomaterials with specific structure-function relationships. Intermuscular bones of teleost fish have previously been described to play a role in the mechanical force transmission between muscle and bone, but their biomechanical properties are not yet fully described. Here, we have investigated intermuscular bones (IBs) of the North Atlantic Herring with regard to their structure and micro-architecture, mineral-related properties, and micro-mechanical tensile properties. A total of 115 IBs from 18 fish were investigated. One cohort of IBs, containing 20 bones from 2 smaller fish and 23 bones of 3 larger fish, was used for mechanical testing, wide-angle X-ray scattering, and scanning electron microscopy. Another cohort, containing 36 bones from 7 smaller fish and 36 bones from 6 larger fish, was used for microCT. Results show some astonishing properties of the IBs: (i) IBs present higher ductility, lower Young's modulus but similar strength and TMD (Tissue Mineral Density) compared to mammalian bone, and (ii) IBs from small fish were 49% higher in Young’s modulus than fish bones from larger fish while their TMD was not statistically different and crystal length was 8% higher in large fish bones. Our results revealed that teleost IB presents a hybrid nature of soft and hard tissue that differs from other bone types, which might be associated with their evolution from mineralized tendons. This study provides new data regarding teleost fish bone biomechanical and micro-structural properties.     

Energy metabolism of fish brain

This review focuses on recent research on the metabolic function of fish brain. Fish brain is isolated from the systemic circulation by a blood-brain barrier that allows the transport of glucose, monocarboxylates and amino acids. The limited information available in fishes suggests that oxidation of exogenous glucose and oxidative phosphorylation provide most of the ATP required for brain function in teleosts, whereas oxidation of ketones and amino acids occurs preferentially in elasmobranchs. In several agnathans and benthic teleosts brain glycogen levels rather than exogenous glucose may be the proximate glucose source for oxidation. In situations when glucose is in limited supply, teleost brains utilize other fuels such as lactate or ketones. Information on use of lipids and amino acids as fuels in fish brain is scarce. The main pathways of brain energy metabolism are changed by several effectors. Thus, several parameters of brain energy metabolism have been demonstrated to change post-prandially in teleostean fishes. The absence of food in teleosts elicits profound changes in brain energy metabolism (increased glycogenolysis and use of ketones) in a way similar to that demonstrated in mammals though delayed in time. Environmental factors induce changes in brain energy parameters in teleosts such as the enhancement of glycogenolysis elicited by pollutants, increased capacity for anaerobic glycolysis under hypoxia/anoxia or changes in substrate utilization elicited by adaptation to cold. Furthermore, several studies demonstrate effects of melatonin, insulin, glucagon, GLP-1, cortisol or catecholamines on energy parameters of teleost brain, although in most cases the results are quite preliminary being difficult to relate the effects of those hormones to physiological situations. The few studies performed with the different cell types available in the nervous system of fish allow us to hypothesize few functional relationships among those cells. Future research perspectives are also outlined.     

Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.)

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus labrax (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88 +/- 10% for gut-associated lymphoid tissue and 79 +/- 18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRV beta 3. By 3'-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes.     

Ouabain-insensitive Na-ATPase activity in homogenates from different animal tissues.

1. Two Na(+)-stimulated ATPase activities were determined in gill homogenates from squid, shrimp and teleost fish; in kidney slice homogenates from teleost fish, bullfrog, toad, iguana, chicken, duck, rat, pig and cow, as well as in homogenates from rat small intestinal cells, brain cortex and liver slices. The two Na(+)-stimulated ATPase activities, the Na- and the Na,K-ATPase, showed a different behavior toward K+ and ouabain. 2. The ouabain-insensitive, K(+)-independent, Na-ATPase activity for all the studied homogenates was completely inhibited by 2 mM furosemide. 3. An increase in cell volume of the kidney, brain cortex and liver slice preparations, as well as of the rat small intestinal cells, produced a concomitant increase of the ouabain-insensitive Na-ATPase.     

Characterization of microtubule-associated proteins in teleosts.

Although microtubules are known to play an important role in many cellular processes, they have been virtually neglected in fish. In this report, microtubule-associated proteins (MAPs) in fish (teleost) were characterized using antibodies (Abs) directed against the mammalian MAPs tau, MAP1A and B, and MAP 2. Two different populations of tau-like proteins (TLPs) were found in fish brain using the anti-tau Abs Tau-1, Tau-2, tau5', and tau3'. The TLPs that were recognized by Tau-1, Tau-2, and tau5' were (1) heat-stable; (2) the same molecular weight as mammalian TLPs: 59-62 kDa; (3) not enriched in microtubules prepared from catfish brain; and (4) localized to the cell body of neurons in fish brains. While the TLPs recognized by tau3' Abs were (1) heat-stable; (2) lower molecular weight than mammalian TLPs: 32-55 vs. 50-65 kDa; (3) enriched in microtubule fractions prepared from catfish brain, and (4) localized to the axons of neurons. These results are consistent with two different populations of TLPs being present in fish brains. While MAP2 was found to be approximately the same molecular weight, 250 kDa, in zebrafish and goldfish as in mammals and to be distributed to dendrites in the fish brain, both MAP1A and MAP1B were found to be about 25% the mass of their mammalian homologs. These results suggest that MAPS in fish have some characteristics similar to their mammalian counterparts, but also possess some unique properties that require further study to elucidate their function.     

Developmental changes in the brain-stem serotonergic nuclei of teleost fish and neural plasticity

Summary 1. During early ontogeny, the serotonergic neurons in the brain stem of the three-spined stickleback shows a temporal and spatial developmental pattern that closely resembles that of amniotes.2. However, in the adult fish, only the midline nuclei of the rostral group (dorsal and median raphe nuclei) and the dorsal lateral tegmental nucleus are consistently serotonin-immunoreactive (5-HTir), whereas the groups of the upper and lower rhombencephalon (raphe pontis, raphe magnus, and raphe pallidus/obscurus nuclei) are variable and, when present, contain relatively small numbers of 5-HTir neurons.3. Using specific antisera against tryptophan 5-hydroxylase and aromaticl-amino acid decarboxylase, we have shown that the lateral B9 group and the groups of the upper and lower rhombencephalon are consistently present in adult sticklebacks. The results are discussed in relation to other known instances of neurotransmitter plasticity or transient neurotransmitter expression in teleost fish.4. While there are several instances of transient expression of neurotransmitter markers by discrete neuronal populations, there is so far no evidence of changes from one neurotransmitter phenotype to another in the brain of teleost fish. However, there are indications of plasticity of expression of catecholamines and indoleamines, and their respective synthesizing enzymes, as reflected in age-dependent changes and variation between individuals of different physiological status.5. As the brain grows continuously in teleost fish, and new neurons are added from proliferative regions, synaptic connections may be expected to undergo remodeling in all brain regions throughout life. Thus, the teleostean brain may be considered a suitable model for experimental studies of different aspects of neural plasticity.     

Macroglial cells of the teleost central nervous system: a survey of the main types

Following our previous review of teleost microglia, we focus here on the morphological and histochemical features of the three principal macroglia types in the teleost central nervous system (ependymal cells, astrocyte-like cells/radial glia and oligodendrocytes). This review is concerned with recent literature and not only provides insights into the various individual aspects of the different types of macroglial cells plus a comparison with mammalian glia, but also indicates the several potentials that the neural tissue of teleosts exhibits in neurobiological research. Indeed, some areas of the teleost brain are particularly suitable in terms of the establishment of a “simple” but complete research model (i.e. the visual pathway complex and the supramedullary neuron cluster in puffer fish). The relationships between neurons and glial cells are considered in fish, with the aim of providing an integrated picture of the complex ways in which neurons and glia communicate and collaborate in normal and injured neural tissues. The recent setting up of successful protocols for fish glia and mixed neuron-glia cultures, together with the molecular facilities offered by the knowledge of some teleost genomes, should allow consistent input towards the achievement of this aim.     

FRACTIONATION OF OLFACTORY TISSUE HOMOGENATES. ISOLATION OF A CONCENTRATED PLASMA MEMBRANE FRACTION

Abstract— Differential and sucrose-density-gradient centrifugation techniques were used for studies on the separation of subcellular particles from rabbit brain and olfactory tissue. Comparisons were made among various fractions from the two types of tissue. These comparisons included protein concentration and enzyme activities of the individual fractions as well as their distribution in subfractions from density gradient separations. In tissue whole homogenates, the percentage of total ATPase activity as ouabain sensitive Na⁺-K⁺ ATPase activity was about 4 times greater in brain cortex (63 per cent) than in olfactory tissue (17 per cent). Cytochrome oxidase and Na⁺-K⁺ ATPase activities were used to indicate the presence and the concentration of mitochondria and of the plasma membranes. A fraction with properties similar to the mitochondria plus nerve ending fraction from brain homogenates (fraction B) was obtained from olfactory tissue. Nerve ending concentration subfractions (B₂) were prepared from the B primary fractions. Plasma membrane subfractions were obtained by osmotic shock treatment of B₂, In the fraction of plasma membrane from olfactory tissue (E₂), 56 per cent of the total ATPase activity was Na⁺-K⁺ ATPase activity. In E₂ from brain 71 per cent was Na⁺-K⁺ ATPase activity. Deoxycholate (DOC)-treated fractions containing nerve endings from brain preparations showed much greater increase in cytochrome oxidase activity than did similar fractions from olfactory tissue. DOC treatment increased the NADH cytochrome c reductase activity of all fractions and subfractions from brain, while it decreased activity in all but one fraction from olfactory tissue. DOC treatment decreased both the Mg²⁺ and Na⁺-K⁺ ATPase activities in both types of tissue. Electron photomicrographs of olfactory B₂, B₃, E₂ and E₃ show clear morphological differences among these subfractions. The presence of possible cilia and basal bodies on vesicles in B₂ gives morphological evidence for the presence of terminal swellings in this subtraction in agreement with enzyme marker activity results.     

THE EFFECT OF HYPOCHOLESTEREMIC AGENTS ON MYELINOGENESIS

Abstract— Three drugs known to inhibit biosynthesis of cholesterol, Clofibrate, 20, 25-diazacholesterol and AY-9944 were administered by stomach intubation to suckling rats. At weaning the rats were killed and subcellular fractions, including myelin, were prepared from the brains and spinal cords and analysed for sterol content. Central nervous tissue fractions from Clofibrate-treated rats showed some decrease in total sterols, but the sterol species were qualitatively normal. AY-9944 given to rats caused high amounts of 7-dehydro-cholesterol to accumulate in all brain and spinal cord fractions with the highest amounts (32–38 percent of total sterols) in myelin. In diazasterol-treated rats desmosterol reached 48 per cent of the sterols of myelin. A group of rats was allowed to survive after the final drug intake (21 days) and their brain and spinal cord sterol content followed up to 60 days. At 30 days the proportion of dehydrocholesterol or desmosterol comprised over half the total myelin sterol. By 60 days of age the 7-dehydrocholesterol had almost completely disappeared from all fractions while substantial amounts of desmosterol were retained in myelin. Myelination was retarded by treatment with AY-9944 and 20, 25-diazasterol, possibly by the limited amount of sterols available. The metabolism of the abnormal myelin constituents in drug-treated animals is discussed in relation to the molecular structure of the myelin membrane.     

The endocrinology of 1α-hydroxycorticosterone in elasmobranch fish: a review

The endocrine underpinnings of the stress response in fish have been the subject of intense research for well over 50years. Much of the research has focussed on teleost fish and so the endocrine mechanisms for cortisol production, transport and action at the target site have received significant attention. However, corticosteroidogenesis in elasmobranchs is exceptional on a number of levels. Unlike teleost fish the interrenal tissue is anatomically distinct from both renal and chromaffin (catecholamine producing) tissue; further the final product, 1α-hydroxycorticosterone (1α-OH-B), is unique to chondrichthyans where the carbon atom at position 1 of corticosterone has a hydroxyl group attached in the α orientation. The homologous nature of interrenal tissue in elasmobranchs presents an obvious advantage in the study of corticosteroidogenesis, however, the unique chemical nature of 1α-OH-B has presented distinct disadvantages as it has proven to be difficult to synthesise, and therefore studies examining the mineralocorticoid and glucocorticoid actions of this steroid are limited. Over the last decade molecular techniques have provided significant insight in the involvement of corticosteroiogenic enzymes in the elasmobranch interrenal in addition to the evolution of corticosteroid receptors. Given the number of excellent reviews focussing on the role of cortisol in the stress response of teleost fish, this short review aims to synthesise the endocrine basis for the synthesis, release, and action, of the enigmatic 1α-OH-B in elasmobranch fish.     

THE SUBCELLULAR DISTRIBUTION OF CARBONIC ANHYDRASE IN HOMOGENATES OF PERFUSED RAT BRAIN

Abstract— The distribution of carbonic anhydrase was examined in subcellular fractions of perfused rat brain and compared with those of markers for cytosol (lactic dehydrogenase), mitochondrial matrix (glutamic dehydrogenase), and mitochondrial membranes (succinic dehydrogenase). About half of the total carbonic anhydrase was found in particulate fractions, with the greatest part of this in the crude mitochondrial fraction. This fraction was separated into its components on a discontinuous sucrose gradient either as such or after isotonic mechanical disruption with a French pressure cell, and the resultant fractions were characterized by electron microscopy and by assay of marker enzymes.
Carbonic anhydrase was solubilized by mechanical disruption, but not to the same extent as lactic dehydrogenase. The highest specific activity for carbonic anhydrase was found in the myelin fraction of the gradient. A mitochondrial locus for carbonic anhydrase is unlikely, but the presence of the enzyme in synaptosomes remains in question.
Addition of soluble carbonic anhydrase did not significantly increase the activity of particulate fractions. Treatment of particulate fractions with detergent was necessary to reveal latent activity; this procedure resulted in a more than ten-fold increase in the measurable carbonic anhydrase activity of myelin fragments.