Subcellular distribution of taurine and cysteinesulphinate decarboxylase in developing rat brain

The concentration of taurine and the activities of cysteinesulphinate decarboxylase and glutamate decarboxylase have been measured in rat brain. During development, taurine exhibited a decrease in concentration unrelated to the activity of cysteinesulphinate decarboxylase which increased during the same period. The distribution of taurine in subcellular fractions of adult and 7-day-old rat brain was typical of most amino acids, whereas half of the cysteinesulphinate decarboxylase activity was found in the nerve-ending cytoplasm. In anatomical distribution, taurine displayed great regional heterogeneity but both cysteinesulphinate decarboxylase and glutamate decarboxylase were more evenly distributed. Hypertaurinaemia was shown to have no effect on the entry of glycine into the brain or on its utilization in protein synthesis.     

Sulfur Amino Acid Metabolism in the Developing Rhesus Monkey Brain: Subcellular Studies of Taurine, Cysteinesulfinic Acid Decarboxylase, γ-Aminobutyric Acid, and Glutamic Acid Decarboxylase

Abstract: Taurine, cysteinesulfinic acid decarboxylase (CSAD), glutamate, γ-aminobutyric acid (GABA), and glutamic acid decarboxylase (GAD) were measured in subcellular fractions prepared from occipital lobe of fetal and neonatal rhesus monkeys. In addition, the distribution of [³⁵S]taurine in subcellular fractions was determined after administration to the fetus via the mother, to the neonate via administration to the mother prior to birth, and directly to the neonate at various times after birth. CSAD, glutamate, GABA, and GAD all were found to be low or unmeasurable in early fetal life and to increase during late fetal and early neonatal life to reach values found in the mother. Taurine was present in large amounts in early fetal life and decreased slowly during neonatal life, arriving at amounts found in the mother not until after 150 days of age. Significant amounts of taurine, CSAD, GABA, and GAD were associated with nerve ending components with some indication that the proportion of brain taurine found in these organelles increases during development. All subcellular pools of taurine were rapidly labeled by exogenously administered [³⁵S]taurine. The subcellular distribution of all the components measured was compatible with the neurotransmitter or putative neuro-transmitter functions of glutamate, GABA, and taurine. The large amount of these three amino acids exceeds that required for such function. The excess of glutamate and GABA may be used as a source of energy. The function of the excess of taurine is still not clear, although circumstantial evidence favors an important role in the development and maturation of the CNS.     

Taurine deficiency in the kitten subcellular distribution of taurine and [35S]taurine in brain

Taurine concentration decreases rapidly in the tissues and physiological fluids of kittens fed a diet of partially purified casein which lacks taurine. We have studied the subcellular distribution in cerebrum of taurine and [³⁵S]taurine administered intravenously to these animals. The taurine concentration of all the fractions isolated from the cerebrum of taurine-deficient kittens was approximately sevenfold less than that observed in the fractions of cerebrum isolated from control kittens. The [³⁵S]taurine was approximately twofold greater in all the brain fractions isolated from the taurine-deficient kittens compared with those isolated from the control kittens. The percent distributions of taurine and [³⁵S]taurine in the fractions isolated from the cerebrum of control and deficient kittens were identical. Thus, in the face of a severe diet-induced deficiency of taurine in kitten brain, there appears to be no conservation of taurine by any particular subcellular pool of taurine. These studies provide no evidence for differences in compartmentation of taurine in cerebrum of taurine-deficient kittens compared with control kittens.     

Subcellular distribution and postnatal development of cholinergic marker proteins in the striatum of rat

The distribution of muscarinic cholinergic receptors, choline acetyl-transferase and acetylcholinesterase activities were measured in subcellular fractions of the rat striatum on the 5th and 15th days postnatally and in adulthood. The receptor density in the striatum of 5 and 15-day-old rats was 15%, respectively, of the adult value. Similar increases of the receptors could be detected in the synaptosomal and microsomal fractions in the postnatal life of rat. The activity of choline acetyltransferase on the same days was 15% and 28%. In the subcellular fractions, the enzyme activity was the highest in the microsomal fraction on both the 5th and 15th days postnatally. The activity of acetylcholinesterase in the homogenate was 6% of the adult value in the 5-day-old rat striatum, while in the synaptosomal fraction it was 11% and 47% of the adult value on the 5th and 15th days, respectively. Our results show that the development of the muscarinic cholinergic receptors precedes that of the two cholinergic enzymes in both 5 and 15-day-old rat striatum. This may suggest an early perikaryonal synthesis and the fast translocation of receptors to the axon terminals during ontogenetic development.     

SUBCELLULAR DISTRIBUTION OF TAURINE AND CYSTEINE SULPHINATE DECARBOXYLASE ACTIVITY IN OX RETINA

Abstract– Taurine levels have been determined in primary and secondary subcellular fractions of ox retina and pigment epithelium.
About the 79.5% of recovered taurine is located in the soluble fraction (S₃), while the remainder is associated with the particulate components. In the secondary subcellular fractions, taurine is primarily associated with the synaptosomal fraction.
Cysteine sulphinate decarboxylase is predominantly associated with particulate components of retinal cells. About the 50% of the recovered enzyme activity of crude mitochondria is present in the synaptosomal fraction.     

Rat liver contains age-regulated cytosolic 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid (Kdn).

Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid), a unique deaminated member of the sialic acid family, has emerged as a new building block of glycoconjugates from a wide variety of organisms, ranging from bacteria to mammals. In particular, the presence of Kdn has been demonstrated in different rat organs and tissues, but not in liver. Here we report on the detection and quantitation of Kdn in rat liver and on its variations with postnatal development and aging. We have previously established the optimal conditions for derivatization of Kdn with 1,2-diamino-4, 5-methylene-dioxybenzene (DMB), and detection by reverse-phase HPLC. Analysis of whole liver homogenates and different subcellular fractions reveals that Kdn is fundamentally present in the cytosolic fraction as nucleotide precursor. The expression of Kdn, Neu5Gc, and Neu5Ac changes unevenly with age. While the content of Neu5Ac, the major species, and Neu5Gc decreases to a different extent from newborn to old animals, Kdn content decreases from newborn to trace amounts in adult rats and increases again with aging. Thus, expression of Kdn, Neu5Gc, and Neu5Ac appears to be independently regulated.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]labelled material into subcellular fractions of 30-day-old rat brain was studied as a function of time, following intracerebral injection of [2-¹⁴C] mevalonic acid. As in the adult and 15-day-old brain, the microsomal fraction was indicated as the site of sterol synthesis. Unlike the 15-day-old animal, the myelin fraction from the 30-day-old rat was the predominately labelled fraction at 2 weeks after injection of the animal. Significant amounts of [¹⁴C]cholesterol were not present until about 4 h after injection. In order to ascertain whether different populations of cholesterol were being labelled, depending on the age of the animal injected, we compared the labelling of myelin and non-myelin components in animals injected at 15 or at 30 days of age, and sacrificed, respectively, from 14 to 29 days or from 1 to 28 days after injection. Our results indicated that there was an apparent shift of labelled sterol from non-myelin to myelin fractions at about 37–44 days of age.     

SUBCELLULAR DISTRIBUTION OF 5-HYDROXYTRYPTAMINE IN RAT BRAIN DURING DEVELOPMENT: EFFECT OF DRUGS AND FASTING

Abstract— Distribution of brain 5-HT content between the high-speed supernatant and particulate fractions under normal and experimental conditions was studied in postnatal and adult rats. In adult and 35-day-old rats the 5-HT content of the supernatant fraction was about 25% of that of the total homogenate and significantly higher than that in 1, 7 and 21-day-old rats. In 1-day-old rats fasting caused an increase of 100% in the turnover, 50% in the content and no alteration in the subcellular distribution of brain 5-HT, which suggests that under normal conditions 5-HT stores may be filled near to capacity. After 5-hydroxytryptophan administration, the 5-HT content of the adult rat brain increased 3-fold and that of the supernatant fraction to 35% of 5-HT content of the total homogenate. In postnatal rats, the brain 5-HT content rose to an adult level and the supernatant 5-HT percentage to a markedly higher than adult level, probably because of the known higher than adult 5-hydroxytryptophan decarboxylase activity of brain capillaries. Administration of tranylcypromine to adult rats caused a 2.6-fold increase of brain 5-HT content and a slight increase of the supernatant 5-HT percentage. At various times after the administration of the MAO inhibitors (tranylcypromine or pargyline) and fasting to the 1-day-old rats, brain 5-HT content increased 4, 5 and 7-fold, respectively, and the supernatant 5-HT rose consistently and, as in the adult, to about 30% of the 5-HT content of the total homogenate. After pargyline following reserpine pretreatment, the 5-HT content of the adult and 1-day-old rat brain increased to 2–3 times the control level and that of the supernatant fraction to about 40% of the 5-HT content of the total homogenate. The adult values for 5-HT in the particulate fraction of the 1-day-old rats after the drug treatments are in sharp contradiction to the low endogenous 5-HT content and known lack of nerve terminals and synaptic vesicles in their brains, and suggest that after MAO inhibition brain 5-HT neurons may bind the amine by some other mechanism than the Mg²⁺-ATP-dependent, reserpine-sensitive granular storage.     

The occurrence of soluble and membrane-bound non-specific lipid transfer protein (sterol carrier protein 2) in rat tissues

The occurrence and subcellular distribution of the non-specific lipid transfer protein (nsL-TP; sterol carrier protein 2) in rat tissues was investigated by the immunoblotting technique using the affinity purified antibody against rat liver nsL-TP. Highest levels of the protein were found in the homogenates of liver, lung and adrenals, whereas it could hardly be detected in brain. In other tissues (i.e., testis, kidney, heart and intestine) the protein was present at intermediate concentrations. Analysis of subcellular fractions obtained by differential centrifugation demonstrated that in all tissues except for the liver, nsL-TP was predominantly present in the particulate fractions. In the particulate fractions of all tissues, an immunoreactive 58 kDa-protein was detected. Density centrifugation of a nuclear-free homogenate from liver and testis indicated that the 58 kDa-protein did, and nsL-TP did not, cofractionate with catalase. This suggests that in these tissues the bulk of nsL-TP is extraperoxisomal. Membrane-bound nsL-TP in testis was sensitive to trypsin treatment, suggesting that it is exposed to the cytosol. Release of nsL-TP by washing the membranes with 0.1 M Na2CO3 (pH 11.5), indicated that nsL-TP is a periferal protein. It was shown by chromatofocussing that nsL-TP extracted from membrane fractions was more basic than nsL-TP present in the cytosol fraction from rat liver (isoelectric point of 8.7).     

Intracellular distribution of fumarase in various animals

The subcellular distribution of fumarase was investigated in the liver of various animals and in several tissues of the rat. In the rat liver, fumarase was predominantly located in the cytosolic and mitochondrial fractions, but not in the peroxisomal fraction. The amount of fumarase associated with the microsomes was less than 5% of the total enzyme activity. The investigation of the intracellular distribution of hepatic fumarase of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp revealed that the amount of the enzyme located in the cytosol was comparable to that in the mitochondria of all these animals. The subcellular distribution of the enzyme in the kidney, brain, heart, and skeletal muscle of rat, and in hepatoma cells (AH-109A) was also investigated. Among these tissues, the brain was the only exception, having no fumarase activity in the cytosolic fraction, and the other tissues showed a bimodal distribution of fumarase in the cytosol and the mitochondria. The mitochondrial fumarase was predominantly located in the matrix. About 10% of the total fumarase was found in the outer and inner membrane, although it was unclear whether this fumarase was originally located in these fractions. No fumarase activity was detected in the intermembranous space.     

Dose-Dependent, K+-Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca2+-Dependent

The K+-stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7-9-day-old rats was studied at 16-18 days in vitro using HPLC analysis. Taurine efflux was dose-dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K+-stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3-5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0-5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.     

Taurine Interactions with Chick Retinal Membranes

Abstract: Binding of [³H]taurine to whole retinal membranes and to membranes obtained from retinal subcellular fractions was studied. [³H]Taurine bound to chick retinal membranes with high affinity and specificity. Two types of [³H]taurine binding associated to retinal membranes were observed, one with a K_D= 0.68 μM and the other one with a K_D,= 9.32 μM. Both types of binding were highly Na⁻-dependent. The Na⁺-dependent taurine binding was antagonized by strychnine. Bound [³H]taurine was effectively displaced by β-alanine but not by GABA or glycine. Taurine binding was preferentially localized in membranes obtained from the crude synaptosomal fraction, although it is also present in substantial amounts in all retinal membranes. A Na⁺-independent [³H]taurine binding exhibiting properties which might represent interaction with postsynaptic receptor sites could not be demonstrated in the chick retina.     

Quantification and regulation of the subcellular distribution of bile acid coenzyme A:amino acid N-acyltransferase activity in rat liver

Bile acid coenzyme A:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids glycine and taurine. To quantify total BAT activity in liver subcellular organelles, livers from young adult male and female Sprague-Dawley rats were fractionated into multiple subcellular compartments. In male and female rats, 65-75% of total liver BAT activity was found in the cytosol, 15-17% was found in the peroxisomes, and 5-10% was found in the heavy mitochondrial fraction. After clofibrate treatment, male rats displayed an increase in peroxisomal BAT specific activity and a decrease in cytosolic BAT specific activity, whereas females showed an opposite response. However, there was no overall change in BAT specific activity in whole liver homogenate. Treatment with rosiglitazone or cholestyramine had no effect on BAT activity in any subcellular compartment. These experiments indicate that the majority of BAT activity in the rat liver resides in the cytosol. Approximately 15% of BAT activity is present in the peroxisomal matrix. These data support the novel finding that clofibrate treatment does not directly regulate BAT activity but does alter the subcellular localization of BAT.     

Immunocytochemical localization of taurine in different muscle cell types of the dog and rat

The presence and distribution of the amino acid taurine in different muscle cell types of the dog and rat was examined by immunocytochemical methods. The light microscope study revealed that smooth muscle cells were similarly immunoreactive for taurine, whereas skeletal muscle fibres showed wide differences in taurine immunoreactivity among individual cells. Some skeletal fibres were strongly immunoreactive whereas others did not display immunolabelling. Mononucleated satellite cells, found adjacent to skeletal fibres in a quiescent stage, were also immunostained. Other myoid cells, such as testicular peritubular cells showed a cytoplasmic and a nuclear pool of taurine. By means of electron microscope immunolabelling, the subcellular localization of taurine was studied in vascular and visceral smooth muscle cells. Taurine was present in most subcellular compartments and frequently appeared randomly distributed. Taurine was localized on myofilaments, dense bodies, mitochondria, the plasma membrane and the cell nucleus. Moreover, the labelling density within individual smooth muscle cells was variable and depended on the state of contraction of each single fibre. Contracted cells showed a higher density of gold particles than relaxed cells. Unmyelinated nerve fibres, found adjacent to smooth muscle cells from the muscularis mucosae and the lamina propria of the stomach, were unstained or poorly stained.     

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat.

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.     

Tyrosinated,detyrosinated and acetylated tubulin isotypes in rat brain membranes. Their proportions in comparison with those in cytosol

The heterogeneity of -tubulin and the relative proportions of the tubulin isotypes were investigated in brain membranes of rats of 1, 25 and 180 days of age by using four anti--tubulin antibodies: a) the monoclonal DM1A antibody, specific for -tubulin; b) the monoclonal 6-11B-1 antibody, specific for acetylated tubulin; c) a polyclonal antibody (Glu antibody), specific for detyrosinated tubulin; and d) a polyclonal antibody (Tyr antibody), specific for tyrosinated tubulin. We found that rat brain membranes contain the three tubulin isotypes mentioned above. The proportions of tyrosinated and detyrosinated tubulin relative to total -tubulin were somewhat lower in membrane than in cytosol in animals of 25 and 180 days of age. At day one of development, the proportions in membrane were similar to those found in cytosol. With respect to the acetylated form, it was about 20 times higher in membrane than in cytosol at the three ages studied. The proportion of acetylated tubulin was determined in different subcellular fractions: myelin, synaptic vesicles, mitochondria, microsomes, and plasma membrane. While the amount of total tubulin differed between the different subcellular fractions, the proportion of acetylated tubulin relative to total -tubulin was constant and similar to that found in total membranes. The proportion of acetylated tubulin was also investigated in non-neural tissues (kidney, liver and lung). Although values for cytosol were about 10-fold higher than that found in brain cytosol, no detectable values for membranes could be obtained in these organs.     

Some mechanisms of antihypoxic action of taurine]

It is shown that preliminary taurine treatment prevents the disturbances of energy metabolism in the brain, heart and liver tissues of Wistar rats with acute hypoxic hypoxia. Administration of taurine restored to normal the parameters of adenine pool: the concentration of ATP increased within the cytoplasm, while that of ADP and AMP diminished; mitochondrial respiration proceeded more rapidly; the concentrations of pyruvate and malate decreased; isocitrate dehydrogenase activity, P/O and NAD/NADH ratios increased. Taurine treatment resulted in a decreased level of lipid peroxides in the rat tissues with hypoxia. The role of intracellular calcium content and biomembranes structure changes as the mechanisms of taurine action on energy metabolism and lipid peroxidation is discussed.     

Modulation of the GABA-benzodiazepine receptor complex by taurine in rat brain membranes

The interactions of taurine and its precursor hypotaurine with the GABA-benzodiazepine receptor complex were studied by investigating their effects on GABA and flunitrazepam binding in rat brain membranes. Taurine, and to a lesser degree also hypotaurine, displaced the high- and low-affinity GABA binding. The maximal binding capacities of both sites were decreased in the presence of taurine, while the binding constants remained the same, suggesting noncompetitive interactions. Taurine and hypotaurine affected flunitrazepam binding only at a very high concentration (50 mmol/l), whereas GABA (within the concentration range of 0.1–100 mol/l) significantly enhanced the binding. Taurine inhibited the GABA-stimulated binding dose-dependently. These modulatory effects of taurine on the GABA-benzodiazepine receptor complex could result from interactions with the GABA recognition site but not from direct actions on the benzodiazepine site.     

Glycogen synthase (casein) kinase-1: tissue distribution and subcellular localization

The distribution of glycogen synthase (casein) kinase-1 (CK-1) among different rat tissues and subcellular fractions was investigated. Using casein, glycogen synthase and phosphorylase kinase as substrates, CK-1 activity was detected in kidney, spleen, liver, testis, lung, brain, heart, skeletal muscle and adipose tissue. The distribution of CK-1 among different subcellular fractions of rat liver was; cytosol (72.1%), microsome (17.6%), mitochondria (9.6%) and nuclei (0.7%). CK-1 from rat tissues was shown to have a similarly wide substrate specificity as highly purified CK-1 from rabbit skeletal muscle. Such wide substrate specificity and distribution among different mammalian tissues and subcellular organelles indicate that CK-1 may be involved in the regulation of diverse cellular functions.     

Effect of ethanol on the DNA-polymerase activity in the liver subcellular fractions of adult and old rats

The effect of 5% ethanol on DNA polymerase activity in nuclei, mitochondria, microsomes and cytosol of intact and regenerating liver of adult and old rats has been studied. No changes in DNA polymerase activity were detected in subcellular fractions of adult rat liver. On the contrary, the increased activity of intact liver nuclei and decreased activity of regenerating liver microsomes was observed with ageing. These age-dependent peculiarities of DNA polymerase activity in response to 5% ethanol may be related to changes in the enzyme molecules or microenvironment associated with ageing.