Isolation and characterization of axolemma-enriched fractions from discrete areas of bovine CNS

Myelinated axons were isolated by flotation from bovine pons, middle cerebellar peduncle, cervical spinal cord and three regions of the subcortical white matter. The myelinated axons were osmotically and mechanically shocked, followed by fractionation on a linear 15% sucrose to 45% sucrose density gradient. Axolemma-enriched fractions (AEF) found in the 28% to 32% sucrose region of the gradient from brainstem and cord white matter had high acetylcholinesterase (AChE) while little or nil AChE activity was found in corresponding AEF derived from the subcortical white matter. Morphologically, the subcortical white matter from all regions contained a heterogeneous population of well-myelinated to thinly myelinated axons, while brainstem and cord regions contained a more homogeneous population of well-myelinated axons. Histochemical analysis of AChE localized this enzyme to axonal elements. The AEF derived from any white matter source had similar polypeptide compositions. AEF derived from subcortical white matter contained two-fold more myelin basic protein and a three-fold greater content of 2 3 cyclic nucleotide 3 phosphodiesterase (CNP) compared with AEF derived from well myelinated white matter. We conclude that the purity of the AEF is related to the degree of myelination of the white matter from which the AEF is derived. Homogeneously well myelinated white matter (pons, cerebellar peduncle, cervical spinal cord) yields the highest purity AEF, as judged by the low CNP and myelin basic protein content and highest enrichment in AChE specific activity.     

The base-exchange enzyme activities of sarcolemma and sarcoplasmic reticulum from rat heart

The Ca2+ dependent incorporation of [14C]ethanolamine, L-[14C]serine and [14C]choline into phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, respectively, were investigated in membrane preparations from rat heart. The ethanolamine and serine base-exchange enzyme-catalyzed reactions were associated with the sarcolemma and sarcoplasmic reticulum. There was a 17.2-fold and 6.8-fold enrichment, respectively, of the serine and the ethanolamine base-exchange enzyme activities in the sarcolemma compared to the starting whole homogenate. The sarcoplasmic reticulum was enriched in the ethanolamine and serine base-exchange enzyme activities. The choline base-exchange enzyme activity of all membranes fractions was negligible compared to the ethanolamine or serine base-exchange enzyme activities. The apparent Km for the ethanolamine and serine base-exchange enzyme in sarcolemma was 14 microM and 25 microM, respectively. The pH optimum for these base-exchange activities was 7.5-8.0. There was a dependence upon Ca2+ for these reactions with a 1 or 4 mM concentration required for maximal activity. The properties of the sarcoplasmic reticulum base-exchange enzymes were similar to the sarcolemmal base-exchange enzymes.     

Sidedness of Phosphatidylcholine-Synthesizing Enzymes in Rat Brain Microsomal Vesicles

The sidedness of CDP-choline:1,2-diradylglycerol choline phosphotransferase (EC 2.7.8.2) and of the choline base-exchange activity has been studied in rat brain microsomal vesicles. Proteases (trypsin and pronase) and mercury-dextran have been used as reagents for membrane surface components. All of them could inactivate both enzymes to a good extent, without affecting the morphology or the permeability to sucrose of the vesicles. It is therefore concluded that CDP-choline:1,2-diradylglycerol choline phosphotransferase and the choline base-exchange activity are localized on the outer surface of rat brain microsomal vesicles.     

Formation of phosphatidylethanol in rat brain by phospholipase D

The mechanism of phosphatidyl [14C]ethanol formation was studied in rat brain microsomal fraction. Phospholipase D and base-exchange enzymes were assayed with [14C]ethanol as substrate. Phospholipase D was found to catalyse the formation of phosphatidylethanol. The reaction was dependent on sodium-oleate as activating factor. Phosphatidylethanol formation by phospholipase D has previously only been reported to occur in plant tissues. Stimulation of base-exchange enzymes with calcium in the presence of [14 C]ethanol did not induce any formation of phosphatidylethanol. These findings indicate that phosphatidylethanol formation in ethanol intoxicated rats is catalysed by phospholipase D.     

The Role of Phosphatidylserine Decarboxylase in Brain Phospholipid Metabolism

In brain, phosphatidylethanolamine can be synthesized from free ethanolamine either by a pathway involving the formation of CDP-ethanolamine and its transfer to diglyceride, or by base-exchange of ethanolamine with existing phospholipids. Although de novo synthesis from serine has also been demonstrated, the metabolic pathway involved is not known. The enzyme phosphatidylserine decarboxylase appears to be involved in the synthesis of much of the phosphatidylethanolamine in liver, but the significance of this route in brain has been challenged. Our in vitro studies demonstrate the existence of phosphatidylserine decarboxylase activity in rat brain and characterize some of its properties. This enzyme is localized in the mitochondrial fraction, whereas the enzymes involved in base-exchange and the cytidine pathway are localized to microsomal membranes. Parallel in vivo studies showed that after the intracranial injection of L-[G-3H]serine, the specific activity of phosphatidylserine was greater in the microsomal fractions than in the mitochondrial fraction, whereas the opposite was true for phosphatidylethanolamine. When L-[U-14C]serine and [1-3H]ethanolamine were simultaneously injected, the 14C/3H ratio in mitochondrial phosphatidylethanolamine was 10 times that in microsomal phosphatidylethanolamine. The results demonstrate that serine is incorporated into the base moiety of phosphatidylethanolamine primarily through the decarboxylation of phosphatidylserine in brain mitochondria. A minimal value of 7% for the contribution of phosphatidylserine decarboxylase to whole-brain phosphatidylethanolamine synthesis can be estimated from the in vivo data.     

Myelination of prospective large fibres in chicken ventral funiculus

In mammals, the oligodendrocyte population includes morphological and molecular varieties. We reported previously that an antiserum against the T4-O molecule labels a subgroup of oligodendrocytes related to large myelinated axons in adult chicken white matter. We also reported that T4-O immunoreactive cells first appear in the developing ventral funiculus (VF) at embryonic day (E)15, subsequently increasing rapidly in number. Relevant fine structural data for comparison are not available in the literature. This prompted the present morphological analysis of developing and mature VF white matter in the chicken. The first axon-oligodendrocyte connections were seen at E10 and formation of compact myelin had started at E12. Between E12 and E15 the first myelinating oligodendrocytes attained a Schwann cell-like morphology. At hatching (E21) 60% of all VF axons were myelinated and in the adult this proportion had increased to 85%. The semilunar or polygonal oligodendrocytes associated with adult myelinated axons contained many organelles indicating a vivid metabolic activity. Domeshaped outbulgings with gap junction-like connections to astrocytic profiles were frequent. Oligodendrocytes surrounded by large myelinated axons and those surrounded by small myelinated axons were cytologically similar. But, thick and thin myelin sheaths had dissimilar periodicities and Marchi-positive myelinoid bodies occurred preferentially in relation to large myelinated axons. We conclude that early oligodendrocytes contact axons and form myelin well before the first expression of T4-O and that emergence of a T4-O immunoreactivity coincides in time with development of a Type IV phenotype. Our data also show that oligodendrocytes associated with thick axons are cytologically similar to cells related to thin axons. In addition, the development of chicken VF white matter was found to be similar to the development of mammalian white matter, except for the rapid time course.     

An image analysis morphometric method for the study of myelinated nerve fibers from mouse trigeminal root

For the morphometric light microscopic study of myelinated fibers in mouse trigeminal root, it was necessary to write: (1) an entirely automatic analysis program for the myelinated axons inside the myelin sheath, based on the detection of the myelin sheaths, and (2) an interactive analysis program for the myelinated fibers outside the myelin sheath, due to the high density of compactness of the myelinated fibers based on an indirect fiber individualization by reconstructing them from their axons. In the latter, a semiautomatic correction method (drawing the profile contours with a light pen) allowed compensation for the failures of the automatic method, except for the smallest fibers, which represented 8% of the total. Using these programs, 95% of the axons could be measured and 92% of the myelinated fibers whose axons were analyzed could be measured. The area-equivalent diameter was independent of the detection method; it is a correct-size measurement parameter for axons and fibers that is unrelated to their shape. The projected diameter, an estimation of the perimeter obtained by measurement of the profile projections, depended upon the detection method because the profile contour was influenced by the detection method; it thus takes into account the profile shape. For myelinated fibers, whose analysis program used two detection methods (automatic and semiautomatic), there was an average difference of 16% between the projected diameters obtained with these two methods, whereas the equivalent diameter value was the same. The fiber circularity factor could not be precisely estimated because of the detection error; the axon circularity factor was more reliable since the axon detection was completely automatic.     

Putrescine <Emphasis Type="Italic">N</Emphasis>-methyltransferases—a structure–function analysis

Putrescine N-methyltransferase (PMT) is a key enzyme of plant secondary metabolism at the start of the specific biosynthesis of nicotine, of tropane alkaloids, and of calystegines that are glycosidase inhibitors with nortropane structure. PMT is assumed to have developed from spermidine synthases (SPDS) participating in ubiquitous polyamine metabolism. In this study decisive differences between both enzyme families are elucidated. PMT sequences were known from four Solanaceae genera only, therefore additional eight PMT cDNA sequences were cloned from five Solanaceae and a Convolvulaceae. The encoded polypeptides displayed between 76% and 97% identity and typical amino acids different from plant spermidine synthase protein sequences. Heterologous expression of all enzymes proved catalytic activity exclusively as PMT and K _cat values between 0.16 s⁻¹ and 0.39 s⁻¹. The active site of PMT was initially inferred from a protein structure of spermidine synthase obtained by protein crystallisation. Those amino acids of the active site that were continuously different between PMTs and SPDS were mutated in one of the PMT sequences with the idea of changing PMT activity into spermidine synthase. Mutagenesis of active site residues unexpectedly resulted in a complete loss of catalytic activity. A protein model of PMT was based on the crystal structure of SPDS and suggests that overall protein folds are comparable. The respective cosubstrates S-adenosylmethionine and decarboxylated S-adenosylmethionine, however, appear to bind differentially to the active sites of both enzymes, and the substrate putrescine adopts a different position.     

Phospholipid base exchange in human leukocyte membranes: quantitation and correlation with other phospholipid biosynthetic pathways

The biosynthesis of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) by base-exchange reactions, and of PC and PE by the CDP pathways, was assessed in the membrane phospholipids of human leukocytes (neutrophils, lymphocytes, T lymphocytes, non-T lymphocytes, and monocytes). Of the three base-exchange activities, ethanolamine exchange was the highest and choline exchange the lowest in each leukocyte membrane. In the CDP pathways, ethanolaminephosphotransferase (EPT) and cholinephosphotransferase (CPT) had comparable activities. Among subpopulations of leukocytes, T lymphocytes showed the highest levels of each enzyme activity, and neutrophils showed the least. In contrast to the enzymes of the CDP pathways, each base-exchange activity was directly proportional to the Ca2+ concentration, but markedly inhibited by Mg2+. Despite this Ca2+ dependence, the base-exchange activities were increased in a dose-dependent manner by calmodulin antagonists and, except for ethanolamine exchange, inhibited by the addition of calmodulin; EPT and CPT activities were only slightly inhibited by calmodulin antagonists and were unaffected by calmodulin. PE formation in both neutrophil and lymphocyte base-exchange reactions was enhanced in a dose-dependent manner by the presence of low concentrations of bioactive stimulants (zymosan, 0.05-0.2 mg/ml; Con A, 0.5-2 micrograms/ml), while EPT and CPT activities were not increased by these cell stimulants. Taken together, our data suggest that base-exchange activity, the biological significance of which has been hitherto unclear, may be related to cell activation; in contrast, the CDP pathways appear primarily to involve the constitutive biosynthesis of phospholipids. Our data further suggest that ethanolamine required for base-exchange reactions is a precursor of PE, N-transmethylation of which can serve as a source of cell activation, leading to production of arachidonic through PC by mediation of phospholipase A2 activity.     

A Survey of Plants for Leaf Peroxisomes

Leaves of 10 plant species, 7 with photorespiration (spinach, sunflower, tobacco, pea, wheat, bean, and Swiss chard) and 3 without photorespiration (corn, sugarcane, and pigweed), were surveyed for peroxisomes. The distribution pattern for glycolate oxidase, glyoxylate reductase, catalase, and part of the malate dehydrogenase indicated that these enzymes exist together in this organelle. The peroxisomes were isolated at the interface between layers of 1.8 to 2.3 m sucrose by isopycnic nonlinear sucrose density gradient centrifugation or in 1.95 m sucrose on a linear gradient. Chloroplasts, located by chlorophyll, and mitochondria by cytochrome c oxidase, were in 1.3 to 1.8 m sucrose.In leaf homogenates from the first 7 species with photorespiration, glycolate oxidase activity ranged from 0.5 to 1.5 mumoles x min(-1) x g(-1) wet weight or a specific activity of 0.02 to 0.05 mumole x min(-1) x mg(-1) protein. Glyoxylate reductase activity was comparable with glycolate oxidase. Catalase activity in the homogenates ranged from 4000 to 12,000 mumoles x min(-1) x g(-1) wet weight or 90 to 300 mumoles x min(-1) x mg(-1) protein. Specific activities of malate dehydrogenase and cytochrome oxidase are also reported. In contrast, homogenates of corn and sugarcane leaves, without photorespiration, had 2 to 5% as much glycolate oxidase, glyoxylate reductase, and catalase activity. These amounts of activity, though lower than in plants with photorespiration, are, nevertheless, substantial.Peroxisomes were detected in leaf homogenates of all plants tested; however, significant yields were obtained only from the first 5 species mentioned above. From spinach and sunflower leaves, a maximum of about 50% of the marker enzyme activities was found to be in these microbodies after homogenization. The specific activity for peroxisomal glycolate oxidase and glyoxylate reductase was about 1 mumole x min(-1) x mg(-1) protein; for catalase. 8000 mumoles x min(-1) x mg(-1) protein, and for malate dehydrogenase, 40 mumoles x min(-1) x mg(-1) protein. Only small to trace amounts of marker enzymes for leaf peroxisomes were recovered on the sucrose gradients from the last 5 species of plants. Bean leaves, with photorespiration, had large amounts of these enzymes (0.57 mumole of glycolate oxidase x min(-1) x g(-1) tissue) in the soluble fraction, but only traces of activity in the peroxisomal fraction. Low peroxisome recovery from certain plants was attributed to particle fragility or loss of protein as well as to small numbers of particles in such plants as corn and sugarcane.Homogenates of pigweed leaves (no photorespiration) contained from one-third to one-half the activity of the glycolate pathway enzymes as found in comparable preparations from spinach leaves which exhibit photorespiration. However, only traces of peroxisomal enzymes were separated by sucrose gradient centrifugation of particles from pigweed. Data from pigweed on the absence of photorespiration yet abundance of enzymes associated with glycolate metabolism is inconsistent with current hypotheses about the mechanism of photorespiration.Most of the catalase and part of the malate dehydrogenase activity was located in the peroxisomes. Contrary to previous reports, the chloroplast fractions from plants with photo-respiration did not contain a concentration of these 2 enzymes, after removal of peroxisomes by isopycnic sucrose gradient centrifugation.     

Hexokinases of tobacco leaves: Subcellular localization and characterization

Subeellular localization of the enzymes which phosphorylate hexoses was studied in photosynthesizing tobacco leaves by means of differential centrifugation and centrifugation in sucrose gradient. More than 80 % of the total hexokinase activity of leaf tissues were found to be associated with the particulate fraction of mitochondria; however, the ratio of the particulate hexokinase fraction to the soluble fraction was influenced by the extraction medium applied. The particulate hexokinases showed a high affinity to glucose (Km = 26.8 μM) and a relatively low affinity to fructose (Km = 17.6 mM). They had a broad pH optimum, because 81 % of the phosphorylating activity obtained for glucose and 75 % of the activity obtained for fructose occurred in pH range from 7.9 to 9.1 (Tris-HCl buffer). The hexokinases were Mg2+ dependent with the highest activity occurring at equimolar Mg2+ and ATP concentrations. Their activity was enhanced by KC1, NaCl, and (NH4)2SO4 at 30 to 120 mM concentrations.     

Changes in invertase activities precede ovary growth induced by gibberellic acid in

Developing pods of pea ( Pisum sativum L. cv. Alaska no 7) were used to study the enzymes of sucrose metabolism. Acid and neutral invertase (EC 3.2.1.26). sucrose synthase (SS, EC 2.4.1.13) and sucrose phosphate synthase (SPS, EC 2.4.1.14) have been localized in the soluble fraction. Acid invertase activity was also present in the insoluble fraction and in pea ovary apoplast. In pea pods, sucrose breakdown was dominated by the invertase pathway. SS specific activity only increased at late stages of parthenocarpic pod development, while SPS did so in pods obtained by pollination. Changes in time course of invertase activities have been correlated with the growth rate of fruits induced to develop either by fertilization or by exogenous application of giberellic acid (GA), 2,4-dichloro-phenoxy acetic acid (2,4-D) or 6-benzylaminopurine (6-BAP). The soluble neutral activities might be associated with pod elongation, while the acid ones were rather related to assimilate import by the induced fruits. Application of gibberellic acid to non-pollinated ovaries significantly enhanced the soluble neutral invertase activity before any ovary outgrowth was detected (up to 2 h after treatment). Within the same period of time. GA-treated ovaries showed a decrease in the acid invertase activity of the soluble fraction and an increase of the acid invertase activity in the apopiast. preceding in time the increment of the acid invertase activity associated with the insoluble fraction. Our results suggest that the early GA response may be mediated through a promotion of processes of protein secretion.     

Regulation of liver base-exchange activity by acidic phospholipids

Liver microsomes were enriched in liposomal acidic lipids by Ca²⁺-dependent fusion of liposomes at pH 7.0. The extent of fusion was monitored by the transfer of radioactive cholesteryl oleate. The enrichment of membranes in phosphatidylserine inhibited ethanolamine base-exchange, whereas the fusion with phosphatidylinositol inhibited both ethanolamine and serine base-exchange reactions. In contrast, these two phospholipids had scarce effects on choline base-exchange. Phosphatidic acid did not suppress any of the three base-exchange activities. Possible functional implications are discussed.Abbreviations DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - SHB suerose-HEPES buffer (0.25M sucrose, 3mM HEPES, pH 7.4)     

An improved procedure for the simultaneous purification in high yield of peroxisomes,lysosomes, and mitochondria from rat liver

Summary Peroxisomes, lysosomes, and mitochondria have been purified from rat liver by sucrose density gradient centrifugation without prior treatment of the animals with Triton WR-1339 or other detergents which cause hyperlipidemia. A crude organelle fraction was first prepared by differential centrifugation of a rat liver homogenate, this fraction contained approximately 70% of the mitochondrial, 40% of the peroxisomal, and 30% of the lysosomal marker enzymes measured in the homogenate. The crude organelle fraction was applied to the top of a sucrose density gradient and centrifuged. A clear separation of the organelles was obtained only when dextran was present in the gradients. Success or failure of the method was found to depend on the particular preparation of dextran used in the gradients. A method for subfractionating dextran was developed which yields dextran fractions that make the separations completely reproducible. Starting with a crude organelle fraction derived from 12 g of liver, approximately 85% of the mitochondrial, 70% of the peroxisomal, and 50% of the lysosomal activities were obtained as pure fractions. The organelle separation takes less than five hours to complete, it represents a substantial improvement over previous methods.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Activity of choline acetyltransferase and acetylcholinesterase in the goldfish optic tectum after disconnection

Activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were investigated in the goldfish optic tectum after disconnection of the optic afferents. Permanent disconnection was achieved by eye removal, and optic nerve crush produced a temporary disconnection until regeneration. There was a rapid loss in total activity per tectum for both enzymes under the two disconnection conditions. At longer intervals after optic nerve crush the levels of total activity for both enzymes returned toward control levels, as regeneration of the nerve proceeded. Total activity for both enzymes remained depressed after eye removal, however. Variable results were obtained in specific activity data, expressed per unit protein, although ther was a 10% loss in specific activity of CAT at early intervals after eye removal. The data are interpreted as consistent with the possibility that at least a fraction of the axons in the retinotectal pathway of goldfish are cholinergic, and parallel our previous observations showing similar rapid losses of nicotinic-cholinergic receptor activity in this system.     

Fractionation studies of the enzyme complex involved in teichoic acid synthesis.

The membrane-bound enzymes participating in the syntheses of the teichoic acid main chain and linkage unit have been solubilized with Triton X-100 and fractionated by sucrose density gradient centrifugation. Two main fractions were obtained: a heavy fraction, containing enzymes effecting synthesis of the main chain attached to the linkage unit, which was associated with only a small amount of lipid, and a light fraction which was rich in prenyl phosphate and catalyzed only linkage-unit synthesis. The separation by density was not based entirely on polypeptide chain length, as some of the shortest chains appeared in the denser fractions and some relatively high-molecular-weight peptides occurred in the lightest fraction. High activity for linkage-unit synthesis was observed in a fraction containing only a few peptides. Addition of ficaprenyl phosphate to the enzyme preparations had no stimulatory effect. It is concluded that the enzymes for main-chain and linkage unit syntheses frm one or more fairly tightly associated complexes and that polyprenyl phosphate is an integral firmly bound component of the complex in which the linkage unit is synthesized.     

A New Myelin-Deficient Mutant Hamster: Biochemical and Morphological Studies

Biochemical and morphological studies were done on a new trembling mutant hamster CBB. The yield of myelin from the mutant was 30 and 40% of the control at 46 and 140 days of age, respectively, but myelin composition and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase) activity were normal. Morphologically, about 18% of the axons were myelinated in the mutant optic nerve at 46 days of age, in which the myelinated fibers were those with larger diameters (more than 0.6 micron), while the control had a peak at 0.4 micron in diameter. The ultrastructure and thickness of compact myelin lamellae in the mutant were normal. Myelination and the structure of peripheral nerve myelin appeared normal. The results indicate that the essential defect is the delay and arrest of myelination in the CNS, which is probably caused by either a decreased rate of synthesis of myelin components in oligodendrocytes or a defect in the oligodendrocyte-axon recognition in smaller axons.     

Isolation of transverse tubules by fractionation of sarcoplasmic reticulum preparations in ion-free sucrose density gradients

A new method for the preparation of transverse tubules (T-tubules) from rabbit skeletal muscles is reported. When crude sarcoplasmic reticulum (SR) preparations were centrifuged on sucrose density gradients containing buffering ions (buffered gradients) 70-80% of the material sedimented as a single heavy band in the region of 43% sucrose. When this fraction (or crude SR) was recentrifuged on sucrose gradients prepared free of buffer or other ions (ion-free gradients) the heavy band dissociated into three fractions of different densities. The lightest fraction sedimented at 28% sucrose and was identified as T-tubules on the basis of its nitrendipine and ouabain binding properties. The enzymatic properties, cholesterol contents, and protein compositions of the fractions obtained when SR is centrifuged on buffered and ion-free sucrose density gradients were measured. The T-tubules were enriched in cholesterol and in marker enzymes for surface membranes while the other fractions were shown to be terminal cisternae and longitudinal cisternae on the basis of their (Ca2+,Mg2+)-ATPase activities and characteristic protein profiles.