The biosynthesis of phosphatidylserine and phosphatidylethanolamine from L-[3-14C]serine in isolated rat hepatocytes

Incorporation of L-[3-14C]serine into phosphatidylserine (PS) and phosphatidylethanolamine (PE) has been studied in isolated rat hepatocytes. Ethanolamine inhibited the incorporation, indicating competition with serine in the base-exchange reaction. Choline, monomethylethanolamine, dimethylethanolamine and dimethyl-3-aminopropan-1-ol had no such effect. The observed rate of PS biosynthesis corresponded to 7-17 nmol/min per liver at 0.55 mM L-serine. The results indicate that only a small fraction (1/25 to 1/70) of the PS pool equilibrates with the base-exchange enzyme, and that decarboxylation to PE occurs preferentially from this pool. The rate of PS synthesis and decarboxylation can therefore not be calculated by methods which assume random, homogeneous labelling of the total PS pool. The apparent rate of PS decarboxylation increased approx. 4-fold when L-serine increased from 0.5 to 2.25 mM, suggesting that decarboxylation of PS to PE might be regulated by the concentration of L-serine or by the amount of PS present in the hepatocyte cell membranes. Lauric, palmitic, stearic, oleic and linoleic acid decreased the rate of PS synthesis. At 0.5 mM, lauric and palmitic acid were most inhibitory. At 1.0 mM, linoleic acid was the least inhibitory fatty acid. The saturated hexaenoic and saturated tetraenoic species of PS contained 51 and 29%, respectively, of the incorporated L-[3-14C]serine. The combined monoene dienoic/diene dienoic fraction had the highest rate of synthesis judged by its relative specific activity. At 0.9 mM concentration, linoleic acid doubled the relative specific activity of the combined monoene dienoic/diene dienoic fraction of PS. Incorporation of L-[3-14C]serine into molecular species of PE resembled that into PS, both in the absence and presence of linoleic acid, suggesting that the phosphatidylserine decarboxylase (EC 4.1.1.65) has a low specificity towards the fatty acid composition of PS. The results indicate that biosynthesis of PS from L-serine occurs mainly by the base-exchange with only negligible contribution from direct incorporation of phosphatidic acid or diacylglycerol. Furthermore, the deacylation-reacylation pathway seem to contribute only little to the determination of the fatty acid composition of hepatocyte PS. Active PS turnover seems to be confined to a small fraction of the PS pool.     

The biosynthesis of phosphatidylserines by acylation of 1-acyl-sn-glycero-3-phosphoserine in rat liver

The conversion of 1-[14C]acyl-sn-glycero-3-phosphoserine into molecular species of [14C]phosphatidylserine was studied using rat liver homogenate and microsomal preparations in the absence of added fatty acyl moieties. In liver homogenates, 81% of the newly-formed phosphatidylserines were tetraenoic (arachidonoyl) species while saturated, monoenoic, dienoic, trienoic, pentaenoic, and hexaenoic (docosahexaenoyl) species each represented 2-5% of the total. A similar pattern of molecular species was produced in liver microsomes. The selectivity of the microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphoserine acyltransferase towards different acyl-CoA derivatives was also investigated. The relative suitability of the various acyl-CoA esters as substrates was found to be of the following order:20:4 = 18:2 greater than 18:1 greater than 16:0 = 18:0. These results with endogenous acyl donors suggest that the acylation of 1-acyl-sn-glycero-3-phosphoserine may partly account for the enrichment of liver phosphatidylserine in arachidonic acid but does not appear to be primarily responsible for the preponderance of docosahexaenoic acid in this phospholipid. The fatty acid specificity of the acyl-CoA: 1-acyl-sn-glycero-3-phosphoserine acyltransferase may contribute to the preferential formation of arachidonoyl phosphatidylserine.     

Calcium-dependent incorporation of choline into phosphatidylcholine (PC) by base-exchange in rat brain membranes occurs preferentially with phospholipid substrates containing docosahexaenoic acid (22:6(n-3))

The fatty acid composition of phosphatidylcholine (PC) formed by base-exchange was examined in rat brain membranes in vitro. The free choline incorporated into subspecies of PC by this phospholipase-D type activity can be distinguished from that which might enter indirectly via the last enzyme of the de novo pathway for phospholipid biosynthesis, cholinephosphotransferase, by its ionic requirements. Choline base-exchange in lysed synaptosomes is optimal when assayed at extracellular (mM) calcium concentrations and is blocked by magnesium. As much as 40% of the choline incorporated by base-exchange into rat brain membranes was recovered in subspecies of PC, representing no more than 10% of the total PC pool, which contained docosahexaenoic acid (22:6(n-3)). Docosahexaenoic acid is enriched in electrically-excitable membranes and its content in phospholipids of rat and human brain change during early development and increase with age.     

Hepatic lipase-mediated hydrolysis versus liver uptake of HDL phospholipids and triacylglycerols by the perfused rat liver

Hepatic triacylglycerol-lipase-mediated hydrolysis and liver uptake of high-density lipoprotein (HDL) lipid components were studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and active at the vascular bed. Human native HDL were labelled with tri-[3H]oleoylglycerol, [N-methyl-3H]dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl,2-[14C]linoleoylphosphatidylcholine (PLPC), 1-palmitoyl,2-[14C]linoleoylphosphatidyl-ethanolamine (PLPE) and 1-palmitoyl,2-[14C]palmitoylphosphatidylethanolamine (DPPE). (1) Relative degradation rates of phosphatidylethanolamine molecular species were 2- to 10-fold higher than those of phosphatidylcholine. Considering [14C] PLPC and [14C] PLPE as representative of HDL phosphatidylcholine and phosphatidylethanolamine, respectively, the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine generated after a 60 min perfusion were comparable. The enzyme showed a clear preference for the molecular species bearing an unsaturated fatty acid at the 2 position of glycerol; this was the most pronounced in the case of phosphatidylethanolamine molecular species. (2) Relative liver uptake of HDL-phosphatidylethanolamine was 4- to 5-fold higher than that of HDL-phosphatidylcholine, irrespective of the constitutive fatty acids. Nevertheless, mass estimation indicated that 3 times more molecules of phosphatidylcholine than of phosphatidylethanolamine were transferred. No correlation could be found between the relative degradation rates of phospholipids and their relative liver uptake, indicating a dissociation between the two processes. (3) Perfusate decay and relative liver uptake of labelled HDL-triacylglycerol were higher than that of any phospholipid class. No circulating radiolabelled free fatty acids accumulated in the perfusate, but they were found acylated into liver cell phospholipids and triacylglycerols. (4) A prior 10-12-min washout of the liver vascular bed with heparin removed over 80% of the hepatic lipase activity, as assessed by specific immunoinhibition. Hepatic lipase-depleted liver displayed impaired phospholipid hydrolysis and triacyglycerol uptake, whereas the transfer of HDL phospholipids to liver tissue was unaffected.     

Dietary lipid effects on microsome fatty acid composition of liver and brain, on liver glucose-6-phosphatase, and on brain 5'-nucleotidase activity in the rat

The effects of incorporation of dietary oils with different n6/n3 ratio and polyunsaturated fatty acids content into rat liver and brain microsomes has been studied. The investigation of membrane fatty acid composition of liver microsomes and that of brain microsomes gave different results. In particular, liver microsomes of rats fed fish oil showed a relatively higher content of 20:5n3 and 22:6n3, and a lower content of 20:4n6. Under these conditions, a reduced glucose-6-phosphatase activity was measured. Brain microsomal fatty acid composition was only slightly affected by dietary lipid intake. The 5'-nucleotidase activity of those particles was similar, although statistically different values were found in fish-oil-fed rats and in olive-oil-fed rats. The effects of membrane fatty acid composition on membrane-bound enzyme activity are discussed.     

The OLE1 gene of Saccharomyces cerevisiae encodes the delta 9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene

Strains of Saccharomyces cerevisiae bearing the ole1 mutation are defective in unsaturated fatty acid (UFA) synthesis and require UFAs for growth. A previously isolated yeast genomic fragment complementing the ole1 mutation has been sequenced and determined to encode the delta 9 fatty acid desaturase enzyme by comparison of primary amino acid sequence to the rat liver stearoyl-CoA desaturase. The OLE1 structural gene encodes a protein of 510 amino acids (251 hydrophobic) having an approximate molecular mass of 57.4 kDa. A 257-amino acid internal region of the yeast open reading frame aligns with and shows 36% identity and 60% similarity to the rat liver stearoyl-CoA desaturase protein. This comparison disclosed three short regions of high consecutive amino acid identity (greater than 70%) including one 11 of 12 perfect residue match. The predicted yeast enzyme contains at least four potential membrane-spanning regions and several shorter hydrophobic regions that align exactly with similar sequences in the rat liver protein. An ole1 gene-disrupted yeast strain was transformed with a yeast-rat chimeric gene consisting of the promoter region and N-terminal 27 codons of OLE1 fused to the rat desaturase coding sequence. Fusion gene transformants displayed near equivalent growth rates and modest lipid composition changes relative to wild type yeast control implying a significant conservation of delta 9 desaturase tertiary structure and efficient interaction between the rat desaturase and yeast cytochrome b5.     

Serine base-exchange in rat liver microsomes: effect of phospholipids.

We enriched liver microsomes in lipid classes and molecular species disrupting membranes with octyl glucoside and reassembling them by detergent removal. Phosphatidylethanolamine incorporated into membranes better than phosphatidylserine or phosphatidylcholine. In addition, the degree of incorporation depended on the unsaturation of fatty acyl-chains. The enrichment of the membranes with phosphatidylserine or phosphatidylcholine inhibited serine base-exchange, whereas the addition of phosphatidylethanolamine usually stimulated it. The effect of exogenous lipids also depended on molecular species; egg yolk phosphatidylcholine and dipalmitoyl phosphatidylcholine inhibited base-exchange whereas the effect of palmitoyl-oleoyl phosphatidylcholine depended on the incorporated amount. The degree of unsaturation also modulated the effect of phosphatidylethanolamine.     

Coding nucleotide sequence of rat liver malic enzyme mRNA

The nucleotide sequence of the mRNA for malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) from rat liver was determined from three overlapping cDNA clones. Together, these clones contain 2078 nucleotides complementary to rat liver malic enzyme mRNA. The single open reading frame of 1761 nucleotides codes for a 585-amino acid polypeptide with a calculated molecular mass of about 65,460 daltons. The cloned cDNAs contain the complete 3'-noncoding region of 301 nucleotides for the major mRNA species of rat liver and 16 nucleotides of the 5'-noncoding region. Amino acid sequences of seven tryptic peptides (67 amino acids) from the purified protein are distributed through the single open reading frame and show excellent correspondence with the translated nucleotide sequence. The putative NADP-binding site for malic enzyme was identified by amino acid sequence homology with the NADP-binding site of the enoyl reductase domain of fatty acid synthetase.     

Lactosylceramide molecular species specificity of rat liver CMP-N-acetylneuraminate:lactosylceramide sialyltransferase

Six naturally occurring and three synthetic molecular species of lactosylceramide (LacCer) were used to examine the molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase in a Golgi-rich fraction of rat liver. The enzyme molecular species specificity was determined either in the presence of nonspecific lipid transfer protein or in the presence of detergents. Assays performed in the presence of transfer protein showed that for those lactosylceramide molecular species with either d18:1 or d18:0 long chain base the enzyme activity decreased linearly as the effective carbon number of the fatty acid increased. An increase in the carbon number of the long chain base decreased the activity of the enzyme twice as much as a corresponding increase in the carbon number of the fatty acid. On the other hand, when the enzyme activity was assayed in the presence of detergents, there was no significant difference in activity among the various molecular species of lactosylceramide based upon the carbon number of the fatty acid or on the presence of a double bond in the long chain base. However, the decrease in enzyme activity with an increase in the carbon number of the long chain base persisted. These results demonstrate that sialyltransferase has binding specificity with respect to the long chain base, but not the fatty acid. The apparent molecular species towards the fatty acid is related to the aqueous solubility of the various LacCer molecular species.     

Effect of dietary conjugated linoleic acid (CLA) on lipid composition, metabolism and gene expression in Atlantic salmon (Salmo salar) tissues

Dietary conjugated linoleic acid (CLA) affects fat deposition and lipid metabolism in mammals, including livestock. To determine CLA effects in Atlantic salmon (Salmo salar), a major farmed fish species, fish were fed for 12 weeks on diets containing fish oil or fish oil with 2% and 4% CLA supplementation. Fatty acid composition of the tissues showed deposition of CLA with accumulation being 2 to 3 fold higher in muscle than in liver. CLA had no effect on feed conversion efficiency or growth of the fish but there was a decreased lipid content and increased protein content after 4% CLA feeding. Thus, the protein:lipid ratio in whole fish was increased in fish fed 4% CLA and triacylglycerol in liver was decreased. Liver beta-oxidation was increased whilst both red muscle beta-oxidation capacity and CPT1 activity was decreased by dietary CLA. Liver highly unsaturated fatty acid (HUFA) biosynthetic capacity was increased and the relative proportion of liver HUFA was marginally increased in salmon fed CLA. CLA had no effect on fatty acid Delta6 desaturase mRNA expression, but fatty acid elongase mRNA was increased in liver and intestine. In addition, the relative compositions of unsaturated and monounsaturated fatty acids changed after CLA feeding. CLA had no effect on PPARalpha or PPARgamma expression in liver or intestine, although PPARbeta2A expression was reduced in liver at 4% CLA feeding. CLA did not affect hepatic malic enzyme activity. Thus, overall, the effect of dietary CLA was to increase beta-oxidation in liver, to reduce levels of total body lipid and liver triacylglycerol, and to affect liver fatty acid composition, with increased elongase expression and HUFA biosynthetic capacity.     

Endonuclear lipids in liver cells

Lipids are not only components of cell nucleus membranes, but are also found in the membrane-depleted nuclei where they fulfill special functions. We have investigated the lipid composition of membrane-depleted rat liver nuclei obtained by incubation with low Triton X-100 concentrations of 0.04% and 0.08%, which rendered them unaltered or hardly altered. Under these conditions, 26% of proteins and 22% of phospholipids were recovered. The main phospholipids were phosphatidylcholine > phosphatidylethanolamine > phosphatidylinositol = or > phosphatidylserine and sphingomyelin (in decreasing concentrations). The fatty acid components of total lipids and phosphatidylcholine were mainly unsaturated. Over 40% belonged to the n-6 series (arachidonic > or = 25% and linoleic 15%); approximately 40% corresponded to saturated acids and <10% were monoenoic. Endonuclear phosphatidylcholine was built up by 16 molecular species, the most abundant being 18:0-20:4 (32%), 16:0-20:4 (19%), 16:0-18:2 (13%), and 18:0-18:2 (11%). The fatty acid composition and phosphatidylcholine molecular species distribution in the membrane-depleted nucleus of rat liver showed patterns similar to the whole nucleus, mitochondria, microsomes, and homogenate of the parent liver cells, suggesting that endonuclear lipid pool composition is mainly determined by a liver organ profile.     

A study of the molecular species of diacylglycerol, phosphatidylcholine and phosphatidylethanolamine and of cholinephosphotransferase and ethanolaminephosphotransferase activities in the type II pneumocyte

The percent distributions of the molecular species of diacylglycerol, phosphatidylcholine and phosphatidylethanolamine in rat whole lung and type II pneumocytes were found to differ significantly. Diacylglycerol from the type II pneumocyte is enriched in the disaturated species and diminished in the polyenoic species compared to whole lung. Type II pneumocyte phosphatidylcholine is enriched in the disaturated species and diminished in all other species compared to whole lung. Relative to whole lung, type II pneumocyte phosphatidylethanolamine is greatly enriched in monoenoic and depleted in polyenoic fatty acid species. Analysis of the fatty acid composition of the molecular species in general indicated differences in relative amounts of fatty acids which were most pronounced in palmitic, palmitoleic, stearic and oleic acids, both within and between type II pneumocyte and whole lung glycerolipids. Significant differences between molecular species also existed within type II pneumocyte glycerolipids. In this cell type, phosphatidylcholine is enriched in disaturated and diminished in monoenoic species compared to diacylglycerol. Phosphatidylethanolamine is enriched in monoenoic and polyenoic species relative to diacylglycerol. In order to determine whether differences observed in type II pneumocyte glycerolipid molecular species were attributable to differences in the specificities of cholinephosphotransferase and ethanolaminephosphotransferase, the selectivity of these enzymes was examined. While cholinephosphotransferase showed diminished activity towards 1-stearoyl-2-linoleoyl-sn-glycerol, neither enzyme showed selectivity towards other tested diacylglycerols under a variety of conditions. Therefore, while in the type II pneumocyte significant amounts of phosphatidylcholine (particularly the disaturated species) and phosphatidylethanolamine may be synthesized de novo, enzymes responsible for remodeling (phospholipase A2 and acyltransferases) may play an important role in establishing the final molecular species composition of both phosphatidylcholine and phosphatidylethanolamine.     

Changes in lipid composition and lipogenic enzyme activities in liver of lambs fed omega-6 polyunsaturated fatty acids

Twenty-four lambs (Ovis aries) were used in a 45-day finishing study to evaluate the effects of feeding diets high in linoleic acid (C(18:2), omega-6) on liver lipid composition and on lipogenic enzyme activities in subcellular fractions of liver. Lambs were fed either a 5% safflower oil (SO, high linoleic acid) supplemented diet or a control diet without added oil. SO feeding caused a reduction in the amount of serum and liver triacylglycerols and cholesterol, whereas the level of phospholipids in both tissues was hardly affected. In liver of SO-treated lambs an increase in the levels of C(18:2) and arachidonic acid (C(20:4), omega-6), together with a simultaneous decrease of saturated fatty acids, was observed. In comparison to rat liver, rather low activities of enzymes in the pathway for de novo fatty acid synthesis, i.e. acetyl-CoA carboxylase and fatty acid synthase, were found in lamb-liver cytosol. Both enzyme activities, as well as those of the NADPH-furnishing enzymes, were significantly reduced by SO feeding. In contrast, microsomal and especially mitochondrial fatty acid chain elongation activity, the latter being much higher than that of rat liver, were significantly increased in SO-treated lambs. In these animals, a stimulation of triangle up(9)-desaturase activity was observed in liver microsomes.     

C19 odd-chain polyunsaturated fatty acids (PUfas) are metabolized to C21-PUfas in a rat liver cell line, and curcumin, gallic acid, and their related compounds inhibit their desaturation

It was demonstrated that the rat liver cell line BRL-3A converted exogenous C19 odd chain-polyunsaturated fatty acids (PUFAs) into the corresponding C21- and C23-PUFAs as follows: 21:3n-8, 21:4n-8, 23:3n-8, and 23:4n-8 (from 19:3n-8); 21:4n-5, 21:5n-5, 23:4n-5, and 23:5n-5 (from 19:4n-5); 21:5n-2, 21:6n-2, 23:5n-2, and 23:6n-2 (from 19:5n-2). It presumed that these C19 PUFAs were converted through the mimic route to docosahexaenoic acid (22:6n-3) from eicosapentaenoic acid (20:5n-3). In addition, the characterization of the change of fatty acid composition of cellular lipids in rat liver cells were examined, using 19:4n-5 and several fatty acid desaturation inhibitors. Curcumin related compounds, curcumin, capsaicin, isoeugenol, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, and gallic acid esters with near five carbon numbered alcohol had great changes of fatty acid composition of cellular lipids based on inhibition of the A6 desaturation of C24-PUFAs in rat liver cells.     

ALTERATIONS IN LIPID MOLECULAR SPECIES OF THE MARINE EUSTIGMA TOPHYTE NANNOCHLOROSIS SP.1

The molecular species of triacylglycerol and monogalactosyl diacylglycerol from the marine eustigmatophyte Nannochloropsis were analyzed by high-performance liquid chromatography with a flame ionization detector. Four major molecular species of triacylglycerol composed of C 14:0, C 16:0, and C 16:1 fatty acids at different combinations were identified. Six molecular species of monogalactosyl diacylglycerol were detected. Three of them contained C 20:5 fatty acid in the sn-1 position, and one component accommodated C 20:5 fatty acid in both the sn-1 and sn-2 positions. Variations in the relative distribution of the molecular species were further monitored in Nannochloropsis cultures grown under different irradiance levels and temperatures. The relative distribution of 16: 0/16:1/16:0 triacylglycerol increased in cells grown in high light and in high temperature. Variations in cellular fatty acid composition in Nannochloropsis grown under different environmental conditions of irradiance level and temperature were attributed to alterations in relative cellular content of lipid classes as well as in the relative composition of lipid class molecular species.     

NADH-cytochrome c reductase, succinate cytochrome c reductase and phospholipids.

Phospholipid peroxidation of isolated rat liver inner mitochondrial membranes induced by either ascorbate or cysteine was accompanied by a release of flavins and coenzyme Q. A straight correlation between this release and the alteration of molecular species of phosphatidylcholine and phosphatidylethanolamine containing one saturated and one unsaturated fatty acid has been found. Peroxidation induced on molecular species of phosphatidylcholine and phosphatidylethanolamine containing only unsaturated fatty acids were accompanied by losses in enzyme activities of NADH-cytochrome c reductase and succinate cytochrome c reductase.     

Enzymology of long-chain base synthesis by liver: characterization of serine palmitoyltransferase in rat liver microsomes

Serine palmitoyltransferase [palmitoyl-CoA:L-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50] catalyzes the initial and committed step in the biosynthesis of the long-chain bases of sphingolipids. A simple assay, based upon the incorporation of [3H]serine into the chloroform-soluble product 3-ketosphinganine, has been developed and demonstrated to be valid for analyzing this enzyme in rat liver microsomes. More than 75% of the serine palmitoyltransferase of rat liver was associated with the microsomal subfraction. The dependencies of activity on the incubation time, pH, temperature, other assay components (e.g., dithiothreitol, EDTA, and pyridoxal 5'-phosphate), and the concentrations of microsomal protein, L-serine, and palmitoyl-CoA were investigated. The requirement of pyridoxal 5'-phosphate for activity was established by formation of the apoenzyme by dialysis against cysteine, and recovery of full activity upon reconstitution with the coenzyme. Activities with fatty acyl-CoA's of varying alkyl chain length were distributed nearly symmetrically around a maximum at 16 carbons (palmitoyl-CoA) for the fully saturated substrates. Less activity was obtained with the CoA thioesters of cis-unsaturated fatty acids, but trans-9-hexadecenoyl-CoA yielded essentially the same activity as palmitoyl-CoA. Hence, this enzyme is capable of initiating the synthesis of the major long-chain bases, as well as compounds that may constitute the unidentified bases reported in analyses of mammalian sphingolipids.     

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.     

Comparative studies on lipogenic enzyme activities in the liver of human and some animal species

1. The activities of enzymes involved in fatty acid synthesis in the human liver (sample taken during abdominal surgery) and in the livers of some animals were studied. 2. Fatty acid synthase, ATP-citrate lyase and malic enzyme activities were found to be from 4 to 70-fold lower in human liver than in rat or bird livers. 3. The activities of hexose monophosphate shunt dehydrogenases in human liver were from half to almost equal to the corresponding activities in birds, but much lower than in rat liver. 4. The activities of all enzymes listed above in human and beef liver were very similar (except fatty acid synthase which was undetectable in the beef liver). 5. Very high activity of NADP-linked isocitrate dehydrogenase was found in livers of all species tested. 6. These results are discussed in relation to the role of the human liver in lipogenesis. 7. The activities of the enzymes generating NADPH in human liver taken during abdominal surgery were similar to the activities observed in the tissue obtained post mortem. 8. This suggested that post mortem tissue may be used as a reliable human material for some enzyme assays. 9. Thus we also examined the activity of malic enzyme in post mortem human kidney cortex, heart, skeletal muscle and brain. 10. Relatively high activity of NADP-linked malic enzyme has been observed in human brain.     

Presence of three acyl-CoA oxidases in rat liver peroxisomes. An inducible fatty acyl-CoA oxidase, a noninducible fatty acyl-CoA oxidase, and a noninducible trihydroxycoprostanoyl-CoA oxidase

Mammalian liver peroxisomes are capable of beta-oxidizing a variety of substrates including very long chain fatty acids and the side chains of the bile acid intermediates di- and trihydroxycoprostanic acid. The first enzyme of peroxisomal beta-oxidation is acyl-CoA oxidase. It remains unknown whether peroxisomes possess one or several acyl-CoA oxidases. Peroxisomal oxidases from rat liver were partially purified by (NH4)2SO4 precipitation and heat treatment, and the preparation was subjected to chromatofocusing, chromatography on hydroxylapatite and dye affinity matrices, and gel filtration. The column eluates were assayed for palmitoyl-CoA and trihydroxycoprostanoyl-CoA oxidase activities and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results revealed the presence of three acyl-CoA oxidases: 1) a fatty acyl-CoA oxidase with a pI of 8.3 and an apparent molecular mass of 145 kDa. The enzyme consisted mainly of 52- and 22.5-kDa subunits and could be induced by clofibrate treatment; 2) a noninducible fatty acyl-CoA oxidase with a pI of 7.1 and an apparent molecular mass of 427 kDa. It consisted mainly, if not exclusively, of one polypeptide component of 71 kDa; and 3) a noninducile trihydroxycoprostanoyl-CoA oxidase with a pI of 7.1 and an apparent molecular mass of 139 kDa. It consisted mainly, if not exclusively, of one polypeptide component of 69 kDa. Our findings are probably related to the recent discovery of two species of acyl-CoA oxidase mRNA in rat liver (Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furata, S., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8131-8137) and they probably also explain why in human peroxisomal beta-oxidation defects an accumulation of very long chain fatty acids is not always accompanied by an excretion of bile acid intermediates and vice versa.