Correlation and composition of the plasmalogen and diacyl forms of phosphatidylethanolamine in subcellular brain fractions of the trout Salmo irideus and the frog Rana temporaria

In myelin, nuclear, microsomal, mitochondrial and synaptosomal fractions from the brain of the trout and frog, studies have been made on the composition of fatty acids and fatty aldehydes of the plasmalogen form and fatty acids of the diacylic form of phosphatidylethanolamin. It was shown that alongside with the increase of the relative content of the plasmalogen form of phosphatidylethanolamin in subcellular fractions of the brain in the frog, especially in the myelin, changes also take place in the composition of fatty acids (the increase in the content of polyenic acids, especially of arachidonic one) and fatty aldehydes (the increase in the degree of unsaturation). Brain myelin of coldblooded vertebrates exhibits similarity with myelin from higher vertebrates in its high content of plasmalogens with a high degree of unsaturation of fatty acids and fatty aldehydes.     

The Na+,K+-ATP-ase Activity and Lipids of Trout and Rat Brain Cell Membranes at the Pressure of 101 Atmospheres

The effect of elevated heliox pressure (101 ATA) on activity of Na⁺,K⁺-ATP-ase and some characteristics of fatty acid composition was studied in membrane phospholipids of trout and rat brain synaptosomal and mitochondrial fractions. The Na⁺,K⁺-ATP-ase activity was shown to decrease by 25% in both fractions of the rat brain; in mitochondrial fraction of the trout brain it decreased by 47%, while in synaptosomal fraction, only by 11%. It has also been established that under experimental conditions, the unsaturation index of fatty acids of phosphatidylcholine and phosphatidylethanolamine of trout synaptosomes decreased, with no changes in these lipids in mitochondrial fraction. The phosphatidylcholine unsaturation index in rats did not practically change in both fractions, while in rat phosphatidylethanolamine it increased in mitochondrial fraction and slightly decreased in synaptosomal fraction. Thus, under conditions of high pressure the reduction of the enzyme activity is also determined, specifically, by peculiarities of the phospholipid fatty acid composition in the subcellular fractions studied. A possibility of changes of the enzyme activity as a result of transition of its lipid component from the liquid crystalline to the gel state under effect of an enhancement of lipid peroxidation under conditions of elevated pressure is discussed.     

Ratio and composition of the plasmalogen and diacylated forms of phospholipids in subcellular fractions of the avian brain

Studies have been made on the specific content of plasmalogen and diacylated forms of phosphatidylethanolamine and phosphatidylcholine in subcellular fractions (myelin, nuclei, microsomes, mitochondria, synaptosomes) from the brain of pigeons, as well as in the myelin fraction from the brain of the crow Corvus cornix and the hawk Accipiter gentelis. Fatty acid composition and fatty aldehyde composition of these two main phospholipids of the brain were studied in the subcellular fractions obtained. It was shown that plasmalogen forms of phospholipids are localized in birds mainly in the myelin fraction which exhibits the highest plasmalogen concentration as compared to the same fraction of all the vertebrates investigated. With respect to fatty acid and fatty aldehyde composition, as well as to the degree of their unsaturation, myelin plasmalogens from birds are similar to those from other cold-blooded and warm-blooded animals. This fact indicates that high relative content of plasmalogens together with their high unsaturation account for normal functional activity of myelin membranes in all vertebrates.     

Effect of hypothermia on subcellular distribution and various physico-chemical properties of cathepsin D in rat brain

The total cooling ef rats down to the rectal temperature 30 degrees and 20 degrees C does not change significantly the ratio of the relative specific activity of cathepsin D in subcellular fractions of the rat brain. The gel chromatographic analysis of heterogeneity of cathepsin D molecular forms in subcellular fractions established the presence of a high-molecular (in the fractions of lysosome and microsome mitochondria) and a low-molecular (in the fractions of lysosome and cytosol mitochondria) enzyme forms. Under hypothermia (20 degrees C) in the brain cytosol fraction there arises a minor zone of the cathepsin D activity corresponding to the high-molecular enzyme form.     

A new view on the role of phospholipids fatty acids: Possibility of electric charge transfer in the membrane monolayer

The fatty acid composition of phospholipids of subcellular fractions that are both generators and consumers of energy, mitochondria, synaptosomes, and myelin, has been studied in the brain and liver of several representatives of poikilothermal and homoiothermal vertebrates. The terrestrial poikilothermal animals have been shown to be close to homoiothermal animals by the main characteristics of membranes of the subcellular fractions, such as the content of saturated acids, the unsaturation index, and the ω3/ω6 acid ratio, but differ essentially from aquatic poikilotherms. The conclusion is made that the similarity in the fatty acid characteristics of the terrestrial poikilothermal and homoiothermal vertebrate membranes is due to their inhabitation in the oxygen-rich environment, while differences in intensity of the oxygen consumption are due to their different body temperatures. The models of structure of an arbitrary fragment of the lipid component of the studied trout membranes are presented, which illustrate a concept of a new functional role of fatty acids as participants of the electron transfer chain in the membrane.     

The effect of cold stress on ganglioside fatty acid composition and ganglioside-bound sialic acid content of rat brain subcellular fractions

The effect of cold stress on the ganglioside fatty acid composition and sialic acid content of brain subcellular fractions and homogenate of rats was studied, the animals were kept in a cold room with 12h light-dark cycles at 3 and 10 degrees C for 2 weeks. (1) The rat brain homogenate, synaptosomes and myelin of rats exposed to 3 degrees C contained significantly higher amounts of ganglioside-bound sialic acid per mg of protein than these fractions of control rats kept at 23 degrees C; the differences were less pronounced in rats exposed to 10 degrees C. (2) A small, but significant, diminution of relative palmitic acid content and an increase of stearic acid content was found to take place in gangliosides from rat brain synaptosomes, synaptosomal plasma membranes and homogenate as a result of the exposure of animals to 3 degrees C and to a lesser extent to 10 degrees C. (3) The content of unsaturated fatty acids in gangliosides from brain subcellular fractions was approximately the same in cold exposed and control rats.     

Effects of chronic ethanol administration on the activities and relative synthetic rates of myelin and synaptosomal plasma membrane-associated sialidase in the rat brain

In an attempt to understand the possible mechanism of chronic ethanol-induced generation of asialoconjugates in the brain and consequent behavioral abnormalities, we have studied the effects of chronic ethanol feeding to rats on the plasma membrane sialidase status in the various subcellular fractions of the brain. We determined sialidase activity using 3H-monosialoganglioside (3H-GM3), 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4-MU-NeuAC) substrates and Amplex Red (Sialidase) kit. We determined the plasma membrane sialidase protein by Western blot using the anti-plasma membrane sialidase. We also determined its relative synthetic rate (RSR) by the 60 min incorporation of intracranially infused [35S]-methionine (50 microCi/100 g) into immunoprecipitable plasma membrane sialidase. Chronic ethanol administration stimulated the sialidase activity in the total brain homogenate as well as the myelin and synaptosomal membrane fractions, respectively, in all the three experimental models. Chronic ethanol also increased the concentration of the rat brain plasma membrane sialidase protein relative to that of glyceraldehyde-3-phosphate dehydrogenase by 2.4-, 1.62- and 1.51-fold in the total brain homogenate, myelin and synaptosomal membrane fractions, respectively. These increases in plasma membrane sialidase activity and its protein content were due to concomitant increases in their relative synthetic rates by 115% (p < 0.01) and 72% (p < 0.01) in the myelin and synaptosomal membrane fractions, respectively. Thus, our studies clearly show that chronic ethanol induced deglycosylation of brain gangliosides is in part, due to specific up-regulation of plasma membrane sialidase in the myelin and synaptosomal membrane fractions of the brain. This increase in plasma membrane sialidase may be responsible for chronic-ethanol-induced physiological and neurological impairment in the brain, presumably due to deglycosylation of gangliosides that are essential for crucial cellular and metabolic activities.     

In vivo phosphorylation of myelin basic proteins: single and double isotope incorporation in developmentally related myelin fractions

The phosphorylation of myelin basic proteins (MBPs) was studied in developing mouse brain. Based on our previous work we postulated that phosphorylation of MBPs takes place prior to their appearance in the myelin compartment as well as within the myelin sheath. To further test this hypothesis we utilized a subfractionation protocol that yields brain fractions enriched in myelin membranes of differing developmental stages. Incorporation of radioactive phosphate into MBPs was studied in each of the subcellular fractions. After 5- and 15-min incubations of isotope in vivo the highest specific radioactivities (SAs) of MBPs were found in the least mature myelin fractions. Incorporation of 32P in MBPs was greater into serine residues than threonine residues in all of the subcellular fractions studied. The relative turnover of MBP phosphates was studied in each of the subcellular myelin fractions using a time-staggered, double isotope methodology. The most rapid equilibration of MBP phosphates with the trichloroacetic acid (TCA)-soluble phosphate pool occurred in the most mature myelin fractions indicating that the highest turnover of MBP phosphates occurs in the most mature myelin fractions. The SAs and turnover rates of each of the four commonly observed mouse MBPs (14, 17, 18.5, and 21.5 kDa) were similar in any particular subfraction demonstrating that the MBP phosphotransferase system(s) acts on each of the MBPs in a similar manner.     

ACTIVITIES OF 3,4-DIHYDROXY-l-PHENYLALANINE AND 5-HYDROXY-l-TRYPTOPHAN DECARBOXYLASES IN RAT BRAIN: ASSAY CHARACTERISTICS AND DISTRIBUTION

Abstract— Optimal assay conditions for decarboxylation of 3,4-dihydroxy- l -phenylalanine (DOPA) and 5-hydroxy- l -tryptophan (5-HTP) were determined in homogenates of rat brain by use of a sensitive, precise microradiometric technique. The two activities exhibited widely different optima for pH, temperature and substrate concentrations. The activity of 5-HTP decarboxylase was stimulated 2-fold by added pyridoxal-5-phosphate and was relatively resistant to antagonists of pyridoxal-P. By contrast, the activity of DOPA decarboxylase was stimulated 20-fold by added coenzyme and could be completely inhibited by carboxyl trapping agents. DOPA decarboxylase activity in subcellular fractions of brain was associated predominately with the soluble fractions and its distribution in the various fractions closely paralleled that of lactic acid dehydrogenase. 5-HTP decarboxylase activity in brain was distributed almost equally between soluble and particulate fractions, and its distribution within the particulate fractions differed from that of succinic acid dehydrogenase. The two decarboxylases in brain exhibited a 7-fold divergence in relative specific activity when their respective distributions in subcellular fractions were compared. Similarly, the regional distributions of the two decarboxylases in rat brain did not parallel one another; e.g. there was a 4-fold difference between the ratio of the two activities in cerebellum and that found in the corpus striatum.     

A tracking marker for the first dimension of the two-dimensional gel electrophoresis of ribosomal proteins

Protein content of different subcellular fractions from chick brain is compared by using Lowry, TCA—Lowry and Bradford assay methods. Caution is urged in application of Bradford's method to general assay for protein concentration in subcellular fractions.     

Phospholipids and their fatty acids in mitochondria, synaptosomes and myelin from the liver and brain of trout and rat: a new view on the role of fatty acids in membranes

The content of different phospholipids (PL) and their fatty acid (FA) composition in subcellular fractions from the liver and brain of rat (Rattus rattus) and trout (Salmo irideus) were estimated. It was shown that despite higher content of unsaturated fatty acids in myelin compared to synaptosomes, the unsaturation index of the latter is equal or higher than that of myelin. The total content of PL polyunsaturated fatty acids (PUFA) was shown to be higher in membrane structures with more active ion transport (mitochondria). This feature seemed to be characteristic of membranes from both representatives of homoiotherms and poikilotherms studied. A possible role for PUFA information within the lipid monolayer of areas with different capacity to accept electrons and transport them along a sort of intermolecular 'tunnel' is discussed. The double bonds of PUFA in this area seem to be able to produce bonds similar to conjugated bonds.     

Pyridoxal phosphate and glutamate decarboxylase in subcellular particles of mouse brain and their relationship to convulsions

The subcellular distribution of pyridoxal phosphate (PLP) was studied in mouse brain, as well as the effect of pyridoxal phosphate-γ-glutamyl hydrazone (PLPGH—a convulsant drug which decreases both PLP levels and glutamate decarboxylase activity [GAD] in whole brain) upon both the PLP concentration and the GAD activity in subcellular fractions. An electron microscopic evaluation of the subcellular particles of control and PLPGH-treated animals was also carried out. The main findings were the following: (1) PLP was localized mainly in the supernatant and crude mitochondrial fractions; two-thirds of the amount present in the latter were located in the subfraction containing pure mitochondria, and the remainder was in the synaptosomal fraction. After osmotic disruption of synaptosomes, PLP was found in both the intrasynaptosomal mitochondria and the synaptoplasm. (2) Treatment of mice with PLPGH decreased levels of PLP in several brain fractions, this effect being much more notable in the soluble fractions than in the particulate fractions. After osmotic disruption of the synaptosomes, a specific decrease of PLP in the synaptoplasm was observed. (3) Treatment with PLPGH produced also an inhibition of GAD activity in most of the fractions studied, when this enzyme was assayed in the absence of PLP. In general, the inhibition was greater in those fractions in which levels of PLP were also affected. In synaptosomes, this correlation between the decreased levels of PLP and decreased activity of GAD occurred only in the synaptoplasm. (4) The activation of GAD by PLP added to incubation mixtures was much greater in those fractions from PLPGH-treated animals which displayed extensive inhibition of GAD, in comparison to the corresponding fractions from control animals. (5) No ultrastructural changes were detected in the subcellular fractions from treated animals. Our results show that the decreases of both the levels of PLP and the activity of GAD (as previously found in whole brain) actually occur in the synaptosomes, a finding that supports the hypothesis that the role of PLP in the mechanisms controlling excitability can be explained, at least in part, by its regulatory action on GAD activity, which in turn determines the rate of GABA synthesis at the nerve endings.     

Subcellular localization of enzyme activities involved in the metabolism of platelet-activating factor in rainbow trout leukocytes

The subcellular distribution of an alkyllyso-GPC: acetyl-CoA acetyltransferase (EC 2.3.1.67) and transacylase, two important enzyme activities involved in the remodeling pathway for the biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) have been examined in leukocytes isolated from the pronephros of the rainbow trout, Oncorhynchus mykiss. Contrary to mammalian systems, in which the acetyltransferase is localized to intracellular membranes, the subcellular distribution of an acetyltransferase activity in rainbow trout leukocytes was localized to the plasma membrane. Analysis of the acetyltransferase products by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed synthesis of two subclasses of PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. The transacylase activity in this study was detected in membrane fractions in two domains of the intermediate density region which also contained the NADH dehydrogenase activity, a marker enzyme for the endoplasmic reticulum. Acylation of lysoPAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) exhibited approximately 95% specificity for omega-3 fatty acids. Acylation patterns were not significantly different in either domain of the endoplasmic reticulum. A model is proposed herein for the metabolism of PAF in rainbow trout leukocytes.     

Subcellular distribution of carbonic anhydrase and Na+,K+-ATPase in the brain of the hyt/hyt hypothyroid mice

Activities of carbonic anhydrase and Na⁺,K⁺-ATPase in tissue homogenates and in subcellular fractions from different brain regions were studied in inherited primary hypothyroid (hyt/hyt) mice. The body weight, the weight of different brain regions, and the plasma thyroxine and triiodothyronine levels of hyt/hyt mice were significantly lower than those of the age-matched hyt/+ controls. In tissue homogenates of cerebral cortex, brain stem and cerebellum of hypothyroid mice, the activity of carbonic anhydrase (units/mg protein) was 59.2, 57.6, and 43.2%, and the activity of Na⁺,K⁺-ATPase (nmol Pi/mg protein/min) was 73.7, 74.4 and 68.7%, respectively, of that in corresponding regions of euthyroid littermates. The decrease in enzyme activity in tissue homogenates was also reflected in different subcellular fractions. In cerebral cortex and brain stem, carbonic anhydrase activity in cytosol, myelin and mitochondrial fractions of hypothyroid mice was about 45–50% of that in euthyroid mice, while in cerebellum the carbonic anhydrase activity in these subcellular fractions of hyt/hyt mice was only 33–38% of that in hyt/+ controls. Na⁺,K⁺-ATPase activity in myelin fraction of different brain regions of hyt/hyt mice was about 34–42% of that in hyt/+ mice, while in mitochondria, synaptosome and microsome fractions were about 44–52, 46–53, and 66–68%, respectively of controls. These data indicate that the activity of both carbonic anhydrase and Na⁺,K⁺-ATPase was affected more in the myelin than other subcellular fractions and more in the cerebellum than cerebral cortex and brain stem by deficiency of thyroid hormones. A reduction in the activity of transport enzymes in brain tissues as a result of thyroid hormone deficiency during the critical period of development may underlie permanent nervous disorders in primary hypothyroidism.     

Distribution of proteins in different subcellular fractions of rat brain studied by two-dimensional gel electrophoresis

The subcellular distribution of proteins normally visible on two-dimension gels of rat brain tissue punches and crude brain homogenate was investigated using two-dimensional gel electrophoresis and computerized scanning densitometry. Seven enriched subcellular fractions (cytosol, mitochondria, microsomes, nucleus, crude synaptic vesicles, myelin and synaptic membrane) were generated from a crude extract of rat brain. Fifty microgram samples of the crude homogenate and each fraction were then taken and the proteins within these samples separated by two-dimensional gel electrophoresis. Proteins were stained with silver and the gels then analyzed by computerized scanning densitometry. Of 136 proteins visible on two-dimension gels of the crude homogenate that were quantitatively examined, a total of 73 (54%) were identified as being primarily located in a single subcellular fraction. The majority of these 73 proteins were found to be located primarily in either the cytosolic or mitochondrial fractions, while fewer proteins were identified as being primarily located in the microsomal, nuclear or crude synaptic vesicular subfractions. In contrast, the myelin and synaptic membrane fractions were found to be the primary location for only a single protein each that is clearly visible in the crude homogenate. In addition, gels of four of the subfractions (mitochondria, cytosol, nucleus and myelin) contained proteins that are not normally visible on gels generated using a crude extract. The subcellular location of a number of proteins found previously to be altered by specific experimental manipulations was also determined, providing further information on these proteins in brain. These results should prove useful in future experiments designed towards isolating and characterizing specific proteins of neurochemical interest.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]-labeIled material into subcellular fractions of 15-day-old rat brain was studied as a function of time after intracerebral injection of [2-¹⁴C]mevalonic acid. As previously shown for adult brain, the data indicated the microsomal fraction to be the site of sterol biosynthesis. The synaptosomal fraction exhibited a marked early uptake of [¹⁴C]-nonsaponifiable material. Total radioactivity in both myelin and myelin-like fractions remained low in comparison to that in the other subcellular fractions at all time periods examined. At 2 h after injection, labelled digitonin-precipitable material was demonstrable in all subcellular fractions. Examination of the [¹⁴C]-labelled nonsaponifiable material by thin-layer chromatography indicated the rapid appearance of labelled 4-desmethyl sterol in all subcellular fractions, with the most rapid appearance in the myelin fraction, followed in decreasing order by microsomal, synaptosomal, and mitochondrial fractions. Examination of [¹⁴C] digitonin-precipitable material from each fraction by the dibromide method demonstrated that although 4-desmethyl sterol appeared quickly, the formation of cholesterol was slow in all fractions, an effect that had been reported earlier for adult brain.     

Ontogeny and Subcellular Distribution of Rat Brain Tele-Methylhistamine

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

Mechanism of the involvement of monoamine oxidase in the regulation of (Na+ + K+)-ATPase in rat brain

Increasing concentrations of dopamine fail to give a biphasic response to (Na+ + K+)-ATPase activity in various subcellular fractions of rat brain preincubated with monoamine oxidase inhibitors, viz. 1 X 10(-4) M clorgyline and 1 X 10(-4) M deprenyl. The product of the monoamine-oxidase-catalysed reaction with dopamine as substrate is 3-methoxy-4-hydroxyphenylacetaldehyde. An analogue of this product is 3-methoxy-4-hydroxybenzaldehyde. This analogue, when incubated with the subcellular fractions which had been preincubated with monoamine oxidase inhibitors and dopamine, gave a more pronounced biphasic response to (Na+ + K+)-ATPase activity than that observed in the fractions incubated with dopamine alone.     

Regulation by cytidine nucleotides of the acylation of sn-[14C]glycerol 3-phosphate. Regional and subcellular distribution of the enzymes responsible for phosphatidic acid synthesis de novo in the central nervous system of the rat

1. The regional and subcellular distribution of the incorporation of sn-[(14)C]glycerol 3-phosphate into rat brain lipids in vitro was investigated and compared with the relative specific activity of various chemical and enzyme markers. The similarity between the subcellular distribution of this incorporation and of NADPH-cytochrome c reductase activity indicated that the synthesis of phosphatidic acid via this route correlated with the presence of endoplasmic reticulum. 2. Experiments in which various amounts of the microsomal fraction were added to fixed amounts of nuclear, myelin, nerve-ending and mitochondrial preparations clearly demonstrated that the endoplasmic-reticulum contamination of these fractions was entirely responsible for the incorporation of sn-[(14)C]glycerol 3-phosphate. 3. The presence of CMP or CTP inhibited the incorporation of sn-[(14)C]glycerol 3-phosphate into the whole homogenate. Similar effects were observed with individual fractions, except for the mitochondria. With the mitochondrial fraction the effect of these cytidine nucleotides varied with the preparation, stimulating in some preparations and inhibiting with other preparations. The presence of CDP-choline stimulated the incorporation into the whole homogenate and to a lesser extent into the subcellular fractions. 4. These results indicate that the various organelles of the central nervous system are more dependent on endoplasmic reticulum for the production of glycerolipids de novo than has previously been appreciated.     

Subcellular distribution of the transmissible agent in Creutzfeldt-Jakob disease mouse brain

To determine the intracellular localization of the Creutzfeldt-Jakob disease (CJD) agent in mouse brain, cerebrum tissue of the mouse brain affected with the Fukuoka-1 strain was separated into six subcellular fractions (microsome, nerve ending, myelin, mitochondria, nucleus, and soluble fractions) by differential sucrose density gradient, and then the CJD infectivity of these fractions was examined. Serially diluted samples of each subfraction were inoculated intracerebrally into groups of BALB/c mice, and the infectivity was determined as to end point titration value, incubation period, and number of affected mice. On the basis of the protein content, the highest CJD infectivity was observed in the microsomal fraction. The nerve ending (synaptic plasma membrane) and myelin fractions were also infective. The mitochondria and nucleus fractions showed the lower infectivity. The infectivity of the soluble fraction was the lowest among the six subcellular fractions. From the findings obtained in this study two possibilities as to the intracellular localization of CJD agent were suggested: 1) the transmissible agent of CJD is closely associated with surface membranes of neuronal and/or glial cells, including their processes; 2) the CJD agent is diffusely present intracellularly, including in the surface membranes, but for manifestation of infectivity the agent needs membrane components as prerequisite factors.