Protein mapping in rat basophilic leukaemia cells.

This review discusses some of our recent studies on rat basophilic leukaemia (RBL) cells, a model cell line for mast cell function. Our interest in these cells is a consequence of the role played by mast cells in the allergic response. Thus far we have described the identification of several spots, and their pI/M(r) co-ordinates. Here we describe how we can further decipher the mast cell proteome using a variety of staining/immuno-blotting procedures. We demonstrate the sensitivity of staining procedures and immuno-blotting using an anti-phosphotyrosine antibody. Our aim is to contribute to the ever-expanding two-dimensional gel and phosphoprotein databases currently available.

Immunohistochemical localization of myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase in flattened membrane expansions produced by cultured oligodendrocytes.

Oligodendrocytes, the cells responsible for myelin sheath formation in the central nervous system, were isolated from primary dissociated mixed glial cultures prepared from newborn mouse forebrain, and further cultured in a serum-free defined culture medium. Single and double indirect immunofluorescence using antibodies against the myelin glycolipids, galactocerebroside and sulfatide, and the myelin proteins, myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase, was used to investigate the composition of the flat membrane extensions produced by some oligodendrocytes in culture. Galactocerebroside and sulfatide were both expressed on the external surface of the plasma membrane of oligodendrocyte cell bodies and processes and also the membrane expansions. Neither myelin basic protein nor 2',3'-cyclic nucleotide 3'-phosphohydrolase were expressed on the external surface of oligodendrocytes. Myelin basic protein could be localized to the cell body and the membrane expansions but not the major and fine processes. The localization of these myelin components suggests that the expansions have characteristics of the mature myelin membrane. 2',3'-Cyclic nucleotide 3'-phosphohydrolase was found to be localized in the cell body, and in total contrast to myelin basic protein, in the major processes and the fine interconnecting processes, but not the membrane expansions. In some of the cells 2',3'-cyclic nucleotide 3'-phosphohydrolase was present at the outer extremities of the flat membrane sheets, giving the appearance of an extending growth region. Our results thus clearly show that 2',3'-cyclic nucleotide 3'-phosphohydrolase is localized within oligodendrocytes in discrete regions of plasma membranes and suggest that this protein has a possible role in the early stages of myelin formation.

The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the corpus callosum, subcortical white matter, and spinal cord in infants dying from sudden infant death syndrome.

The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) has been determined in corpus callosum, subcortical white matter, and spinal cord of infants whose death was attributed to the sudden infant death syndrome (SIDS), and compared with enzyme activity in other cases in which the cause of death was not associated with respiratory distress. In nearly half the SIDS cases, CNPase activity and oligodendroglial cell numbers were reduced before the onset of myelination, but only in the corpus callosum. In other SIDS cases, enzyme activity and cell numbers were the same as in non-SIDS cases. If the expression of CNPase activity reflects glioblast differentiation to oligodendrocytes with myelinating potential, then this transformation is abnormal in certain SIDS cases, as also evidenced in cases of prolonged neonatal respiratory insufficiency and gives rise to a subsequent deficit of myelin in the corpus callosum.

Phospholipid exchange activity in developing rat brain.

Phospholipid exchange activity has been determined in the supernatant fraction of rat brain from birth through to maturity by measuring the protein-catalysed transfer of total and individual 32P-labelled phospholipids from microsomal membranes to mitochondria, and the transfer of [14C]phosphatidylcholine from liposomes to mitochondria. Transfer activity has also been compared in brain and liver supernatant. Overall phospholipid exchange activity in the brain increased only slightly with age. The activity at birth was 75% of the adult value. However, the transfer of individual phospholipids showed markedly different trends during postnatal brain development. The transfer of phosphatidylinositol (PI) and ethanolamine phospholipids increased postnatally to a maximum at 9 days of age, with lowest values in adult brain. Phosphatidylcholine (PC) transfer increased from 9 days to reach maximum values in the mature brain. The transfer of sphingomyelin was highest immediately after birth. PI transfer activity was higher in brain than liver, while PC and ethanolamine phospholipid transfer activity was higher in liver. The heterogeneity of phospholipid exchange proteins in central nervous system tissue is reflected in the developmental changes in exchange activity towards individual phospholipids. The various exchange proteins appear to have separate induction mechanisms. The presence of exchange-protein activity from birth in the rat indicates the functional importance of phospholipid transport during cell acquisition and membrane proliferation. Activity is not primarily associated with membrane formation such as the formation of the myelin sheath, and therefore is more likely to be involved in the process of phospholipid turnover.

The hydrolysis of the alkenyl group from enthanolamine-plasmalogen by oligodendroglial cell-enriched fractions.

The rate of hydrolysis of the 1-0-alkenyl group of sn-1-alk-1?-enyl-2-acyl-glycerylphosphorylethanolamine (alkenyl, acyl-GPE; ethanolamine plasmalogen) by plasmalogenase is higher in oligodendroglial cell-enriched fractions from bovine brain compared with fractions enriched in neuronal perikarya and astroglia. The distribution of plasmalogenase activity in membrane fractions isolated from bovine oligodendroglia has been compared with that of 'marker' enzymes. The highest specific activity was in a fraction enriched in plasma membranes, whilst most activity was recovered in an endoplasmic reticulum membrane fraction. In bovine oligodendroglial cell homogenates, the enzyme had a neutral pH optimum, had no requirement for divalent cations and its activity towards 1-alkenyl-GPE (lysoplasmalogen) was half that with alkenyl, acyl-GPE. C(16) alkenyl groups were hydrolysed more rapidly than C(18) alkenyl groups. With (3)H-labelled alkenyl, acyl-GPE as substrate, radioactivity in released aldehydes appeared in fatty acids esterified in phospholipid while the oxidation of fatty aldehydes was blocked by the addition of NADH. An NAD-dependent aldehyde dehydrogenase was found to be present in oligodendroglia which exhibited highest activity towards C(14)?C(18) aldehydes (K(m), 2 ?M).

The ontogenic development of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the corpus callosum in relation to oligodendroglial proliferation, myelination and the distribution of fat-containing glial cells.

The development of CNP'ase activity in the corpus callosum of infants dying from different causes has been compared with myelin formation and oligodendroglial proliferation determined by quantitative histological methods. Cases were classified according to the distribution of neutral lipid in capillary endothelia (Class I), with some fat-containing glial cells (Class II) or extensive occurrence of fatty glia (Class III) and compared with Class O cases, showing no neutral lipid accumulation. For cases in Classes II and III--in which nearly all cases of cardiorespiratory insufficiency were classified--there is a deficit of oligodendroglia and myelin, although the ratio of myelin staining intensity to glial cell numbers is similar to Class O cases. The deficit in myelin is due almost entirely to a reduction in oligodendroglial cell numbers. CNP'ase activity is reduced to a greater extent than myelin and the ratio of CNP'ase to glial cell number is reduced before myelination commences. The defect in expression of CNP'ase activity may be indicative of abnormal glioblast transformation. Fatty glial cells are also acquired before myelination suggesting that the primary insult to oligodendroglia, which may be hypoxia, occurs at the time of their proliferation and differentiation. Nearly half the cases of unexplained death in infancy show deficits in CNP'ase activity, correlating with reduced cell numbers and myelin, and the occurrence of fatty glial cells, all of which could be caused by hypoxia around birth.

Biochemical changes in Cuprizone-induced spongiform encephalopathy. I. Changes in the activities of 2',3'-cyclic nucleotide 3'-phosphohydrolase, oligodendroglial ceramide galactosyl transferase, and the hydrolysis of the alkenyl group of alkenyl, acyl-glycerophospholipids by plasmalogenase in different regions of the brain.

Cuprizone (biscyclohexanone oxaldihydrazone) which is known to produce a status spongiosus and demyelination in the CNS was administered in the diet of weanling male mice at a concentration of 0.4% by weight for a period of six weeks before returning animals to a normal diet. Changes in body weight but not brain weight were reversible. Based on the decline in CNP'ase activity and the concentration of galactocerebroside, the loss of myelin was around 70% in those sections of the cerebrum with a high content of white matter while the cerebellum was less affected. The activity of oligodendroglial HFA-ceramide galactosyl transferase was also reduced. These biochemical parameters of myelination were increased after withdrawal of Cuprizone. Remyelination in the cerebrum but not the cerebellum was incomplete. The activity of plasmalogenase hydrolysing the alkenyl group of alkenyl, acyl-phospholipids increased 2-fold in those sections in which myelin loss was most severe. The increase preceded the greatest loss of myelin components (3 to 6 weeks on Cuprizone). The origin of the increased phospholipase activity in demyelinating tissue is discussed. Following myelination, there was a deficit in plasmalogenase activity particularly in the frontal cortex of the cerebrum, where the plasmalogen concentration was higher than in controls.

UDP-galactose:ceramide galactosyl transferase of isolated oligodendroglia.

The activity of UDP-galactose:ceramide galactosyl transferase (CGalT) has been studied in isolated oligodendroglia from bovine brain white matter and myelinating rat brain. The specific activity and activity per mg DNA are 4- and 10-fold higher in rat oligodendroglia compared with neuronal perikarya from rat brain, and is higher in oligodendroglia from myelinating rat brain compared with bovine oligodendroglia. In membranes isolated from oligodendroglia, the specific activity decreased in the order endoplasmic reticulum greater than plasma membrane greater than myelin.

Postnastal changes in the concentration of carnitine and acylcarnitines in the rat brain.

The concentration of total and free carnitine (TC and FC) together with short-chain and long-chain acylcarnitines (SCAC and LCAC) has been determined in whole brain of female rats from birth to maturity. The concentration of TC and SCAC increases postnatally to reach a peak 10 days after birth. The concentration of LCAC is high relative to adult brain between 1 and 10 days of age before falling to a low level. The change in LCAC concentration correlates with previously described developmental changes of palmitoyl-CoA: carnitine acyl transferase activity and fatty acid oxidation. The ratio of LCAC/FC declines postnatally while the SCAC/FC ratio is high at birth and increases further in adult brain. The results are consistent with the concepts of carnitine participating in exchange of short-chain acyl groups and in the transfer of fatty acids into mitochondria as an alternative energy supply in neonatal rat brain.

Alcohol dehydrogenase-coupled spectrophotometric assay of plasmalogenase.

Plasmalogenase has been assayed by conversion of the fatty aldehydes, released by hydrolysis of the vinyl ether bond of plasmalogens, to long-chain alcohols by horse liver alcohol dehydrogenase. The reaction was followed spectrophotometrically by measuring the oxidation of NADH. The assay is sufficiently sensitive to enable plasmalogenase activity to be determined in isolated oligodendroglia and derived membranes and in brain microsomal membranes using 50-250 micrograms protein.

The quantitative determination of plasmalogen by its reaction with mercuric chloride.

The alk-1-enyl group of 1-alk-1(1)-enyl-2-acyl-glycerophospholipids (plasmalogens) rapidly combines with mercuric chloride. At 0 C, there was a 1:1 stoichiometry for Hg binding to the reactive enol group of plasmalogens. Aldehydes were not released, indicating that the alkenyl ether bond was not cleaved. Hg binding to less reactive double bonds in unsaturated fatty acids was not significant. Quantitative estimation of bound Hg afforded a rapid and sensitive assay for alkenylacyl lipids and gave values similar to those obtained with other methods of analysis. The proportion of plasmalogens in bovine myelin glycerophosphatides and in ethanolamine glycerophosphatide was 35 and 75%, respectively. Plasmalogens account for 23.3% of the total glycerophospholipid of rat erythrocytes.

The interaction of fatty acid synthetase with cytoplasmic protein in the control of the chain-length of fatty acids synthesised by the lactating rabbit mammary gland.

1. Fatty acid synthetase from rabbit mammary gland can be separated from the protein which modifies the chain-length at which fatty acids are released from the enzyme complex in the soluble fraction. This can be achieved by ultracentrifugation, precipitation with specific antibody or ammonium sulphate. 2. The chain-length modifying protein in the supernatant fraction from rabbit mammary gland was less active towards cow mammary gland fatty acid synthetase than rabbit mammary gland fatty acid synthetase in the synthesis of medium-chain fatty acids. The fatty acid synthetases from these two tissues are also immunologically non-identical. 3. It is proposed that there is a loose but specific interaction of rabbit mammary gland fatty acid synthetase with the chain-length modifying protein in regulating product chain length which is dependent on the concentration of interacting proteins. 4. The chain-lengthening effect of added malonyl-CoA decreases with increasing concentration of interacting proteins, but differences in the fatty acid chain-length with malonyl-CoA synthesised in situ by acetyl-CoA carboxylase and with added malonyl-CoA indicate that the product chain-length is sensitive to the availability of malonyl-CoA for enlongation in all but the most tightly coupled situations.

Comparison of the acylation of sn-glycerol 3-phosphate and membrane-bound lipid in the microsomal fraction from rabbit brain throughout maturation.

1. Age-related changes in the specific activity of palmitoyl-CoA synthetase, sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) and the esterification of [3H]palmitate into endogenous lipid in the microsomal fraction from rabbit brain have been determined throughout development. 2. The increased specific activity of sn-glycerol 3-phosphate acyltransferase at the onset of myelination (rising in parallel with other lipogenic enzymes) is consistent with a direct role of the acyltransferase in promoting the accumulation of cerebral lipid. In adult brain microsomes, although the specific activity was low, the total activity was only 20% lower than during active myelination. 3. Palmitoyl-CoA, synthesized by the palmitoyl-CoA synthetase in the microsomal membrane, was the preferred substrate for the esterification of sn-glycerol 3-phosphate. There was no evidence for a pool of palmitoyl-CoA formed from palmitate. 4. The esterification of [3H]palmitate into membrane-bound lipid remained high throughout development and may be part of an acyl-exchange cycle via lysophospholipids. [3H]palmitate was incorporated into both neutral lipids and phospholipids, while phosphatidic acid was the major product of sn-[1(3)-3H]-glycerol-3-phosphate esterification. 5. The microsomal fraction contained a pool of unesterified fatty acid, which was activated and esterified into sn-glycerol 3-phosphate.