Cholesterol synthesis in rat liver peroxisomes. Conversion of mevalonic acid to cholesterol

The key regulatory enzyme of cholesterol, dolichol, and isopentenyl adenosine biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is a 97-kilodalton transmembrane glycoprotein which was believed until recently to reside exclusively in the endoplasmic reticulum of mammalian cells. However, several recent publications have shown that the enzyme in liver cells is present not only in the endoplasmic reticulum but also within peroxisomes. In an effort to clarify the role of peroxisomal HMG-CoA reductase, highly purified (95%) rat liver peroxisomes from cholestyramine-treated rats were incubated with RS-[2-14C]mevalonic acid plus cytosolic proteins and then tested for the presence of newly synthesized cholesterol. For comparison, highly purified microsomes from the same liver preparation were incubated at several protein concentrations under the same conditions. A three-step procedure was employed to resolve the newly synthesized cholesterol from the complex mixture of sterol intermediates in cholesterol biosynthesis. After termination of the reaction and addition of a [3H]cholesterol standard, the incubation products were extracted and separated by thin layer chromatography into a number of fractions. The fraction containing C-27 sterols was further resolved by reverse-phase high pressure liquid chromatography. After acetylation, the products were then separated by silicic acid high pressure liquid chromatography. Confirmation of the identity of newly synthesized cholesterol was obtained by recrystallization with added non-radioactive cholestenyl acetate standard. The results indicate that highly purified rat liver peroxisomes are able to convert mevalonic acid to cholesterol in the presence of cytosolic fraction in vitro. An abstract of these results has been published (Krisans, S. K., Thompson, S. L., Burrows, R., and Laub, R. J. (1986) J. Cell Biol. 103, 525 (abstr.).     

Biosynthesis of dolichol by rat liver peroxisomes

The ability of peroxisomes and microsomes to synthesize dolichol from [3H]mevalonate, [3H]isopentenyl-P2 or [3H]farnesyl-P2 in vitro was investigated. It was found that isoprenoid biosynthesis also occurs in peroxisomes and that this process demonstrates properties differing from those of isoprenoid biosynthesis by microsomes. The pH optimum in peroxisomes was 8.0 and, in contrast to microsomes, the peroxisomal biosynthesis was largely insensitive to detergents. After treatment with proteolytic enzymes, microsomes lost their capacity to incorporate [3H]mevalonate into dolichol, whereas proteolysis of intact peroxisomes did not influence their corresponding rate of incorporation. The soluble content of peroxisomes was separated from the membranes and found to demonstrate half of the biosynthetic capacity of the intact organelle. Fasting and cholestyramine treatment decreased only the microsomal incorporation of [3H]mevalonate into dolichol, while treatment with clofibrate, di-2-ethylhexyl phthalate or phenobarbital increased microsomal, but decreased peroxisomal labeling. After injection of [3H]mevalonate into the portal vein of rats, high initial labeling of dolichol was recovered both in isolated microsomes and peroxisomes, whereas when [3H]glycerol was administered, peroxisomal phospholipids became labeled later than the corresponding microsomal constituents. These results support the conclusion that dolichol is synthesized both in peroxisomes and the endoplasmic reticulum, but that the biosynthetic processes at these two locations have different properties.     

Very long chain fatty acid beta-oxidation by rat liver mitochondria and peroxisomes

Crude mitochondrial fractions were isolated by differential centrifugation of rat liver homogenates. Subfractionation of these fractions on self-generating continuous Percoll gradients resulted in clearcut separation of peroxisomes from mitochondria. Hexacosanoic acid beta-oxidation was present mainly in peroxisomal fractions whereas hexacosanoyl CoA oxidation was present in the mitochondrial as well as in the peroxisomal fractions. The presence of much greater hexacosanoyl CoA synthetase activity in the purified preparations of microsomes and peroxisomes compared to mitochondria, suggests that the synthesis of coenzyme A derivatives of very long chain fatty acids (VLCFA) is limited in mitochondria. We postulate that a specific VLCFA CoA synthetase may be required to effectively convert VLCFA to VLCFA CoA in the cell. This specific synthetase activity is absent from the mitochondrial membrane, but present in the peroxisomal and the microsomal membranes. We postulate that substrate specificity and the subcellular localization of the specific VLCFA CoA synthetase directs and regulates VLCFA oxidation in the cell.     

Membranes of rat liver peroxisomes

Summary Membranes of liver peroxisomes from rats fed with clofibrate were purified in a discontinuous gradient using a zonal rotor. The preparation consists of round or oval vesicles mostly devoid of nucleoids with a diameter ranging from 70–700 nm; open sheets are found very infrequently. Mitochondrial profiles as well as vesicles containing cytochemically demonstrable glucose 6-phosphatase are scarce; accordingly, glucose 6-phosphatase is nearly undetectable biochemically. Monoamine oxidase is absent in peroxisomal membranes. Cytochrome b₅ is found in a concentration of 0.3 nmoles/mg protein, an order of magnitude comparable to the content of endoplasmic reticulum membranes. Reduction of this cytochrome with palmitoyl-CoA is possible only after recombination of the membranes with the soluble peroxisomal matrix fraction.     

Fatty acid oxidation by human liver peroxisomes.

A cyanide insensitive fatty acid oxidation system is detected in human liver and is shown to be localized in peroxisomes by subcellular fractionation in Metrizamide continuous density gradients. Fatty acyl-CoA oxidase, its characteristic enzyme, acts maximally on C₁₂–C₁₈ saturated fatty acids and on oleoyl-CoA and requires FAD. These results, together with the already established properties of the system in rat liver, support its potential contribution to lipid metabolism and to the hypolipidemic effect of Clofibrate and related drugs in humans.     

Degradation of uric acid to urea and glyoxylate in peroxisomes.

The distribution of enzymes involved in purine degradation in fish and crustaceous liver was examined by centrifugation in a sucrose density gradient. In mackerel, yellow mackerel, and prawn liver and mantis club hepatopancreas, uricase and allantoinase were located only in the peroxisomes and in the soluble fraction from broken peroxisomes, and allantoicase was located only in the peroxisomes. Uricase and allantoinase seem to be located in the peroxisomal matrix and allantoicase in the peroxisomal membrane. Adenase, guanase, and xanthine oxidase were present only in the soluble fraction of mackerel liver.     

Formation of chenodeoxycholic acid from 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid by rat liver peroxisomes

The oxidation of the side chain of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) into chenodeoxycholic acid has been studied in subcellular fractions of rat liver. The product was separated from the substrate by high pressure liquid chromatography and identified by gas-liquid chromatography-mass spectrometry. The highest specific rate of conversion was found in the heavy (M) and the light (L) mitochondrial fractions with the highest enrichment in the L fraction. Washing the M fraction reduced the side chain cleavage activity by 90%. The peroxisomal marker enzyme urate oxidase was reduced to the same extent. The activity found in the M fraction may thus be due to peroxisomal contamination. After centrifugation of the L fraction on a Nycodenz density gradient, the highest specific activity for side chain cleavage of DHCA (31 nmol X mg-1 X h-1) was found in the fraction with the highest peroxisomal marker enzyme activity. This fraction also catalyzed conversion of 3 alpha,7 alpha,12 alpha-5 beta-cholestanoic acid (THCA) into cholic acid at the highest rate (32 nmol X mg-1 X h-1). The peroxisomal oxidation of DHCA into chenodeoxycholic acid required the presence of ATP, CoA, Mg2+, and NAD in the incubation medium. The reaction was not inhibited by KCN. It is concluded that rat liver peroxisomes contain enzymes able to catalyze the cleavage of the side chain of both DHCA and THCA. The enzymes involved are similar to, but not necessarily identical to, those involved in the peroxisomal beta-oxidation of fatty acids.     

Nucleic acid synthesis in proliferating peroxisomes of rat liver as revealed by electron microscopical radioautography

Summary Thirty albino rats were fed with a diet containing 1, 2 or 4% di-(2-ethylhexyl)-phthalate (DEHP), a peroxisome-proliferating agent. Others were fed with normal diet as controls. Both groups were sacrificed at varying intervals from 3 days to 4 weeks. The livers were either removed and fixed in glutaraldehyde and osmium tetroxide or fixed in glutaraldehyde, incubated in a diaminobenzidine (DAB) medium, postfixed, embedded in Epon, and sectioned. Other tissues were incubated in Eaglés MEM containing either [³H]thymidine or [³H]uridine, fixed, embedded in Epon, sectioned, and radioautographed. Specimens were observed in a Hitachi H-700 electron microscope.The number of peroxisomes showing DAB reactivity increased in DEHP-fed animals as compared with normal controls In radioautograms of normal rats labelled with [³H]thymidine, no silver grains were, observed, whereas grains were observed over some nuclei, mitochondria and peroxisomes of DEHP-fed animals. In contrast, radioautograms of tissue labelled with [³H]uridine revealed a few grains in nuclei and mitochondria or endoplasmic reticulum of normal rats, although grains appeared in nuclei, mitochondria, endoplasmic reticulum and peroxisomes of DEHP-fed animals more frequently.From these results, it is concluded that [³H]thymidine and [³H]uridine were incorporated in the proliferating peroxisomes, suggesting that nucleic acid synthesis had taken place.     

Heterogenous staining ofd-amino acid oxidase in peroxisomes of rat liver and kidney

Summary The localization ofd-amino acid oxidase (d-AAOX) in rat liver and kidney has been investigated using the cerium technique for electron microscopy and a recent modification of it for light microscopy. In the liver a mosaic pattern with strongly and weakly stained cells together with some completely negative hepatocytes is observed. The staining is stronger and more uniform in periportal than in perivenous regions of the liver lobule. In the kidney the reaction is confined to the proximal tubules of the renal cortex with the rest of the nephron being negative. At the ultrastructural level in both liver and kidney a marked heterogencity is obseved in the intensity of reaction in peroxisomes of some neighbouring cells. Moreover, in some cells heavily and weakly stained peroxisomes are seen side by side. When Pipes buffer is used in the incubation medium thed-AAOX reaction in kidney peroxiosomes is aggregated in the central region of the matrix with weaker staining of the periphery. A similar result is obtained when the enzyme is localized by immunocytochemistry confirming a recent report by Usuda et al. (1986). The heterogeneous staining of peroxisomes ford-AAOX suggests that subpopulation of this organelle with specialized functions may exist not only in different tissues and cells but even within the same cell.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Comparison of fatty acid oxidation in mitochondria and peroxisomes from rat liver

Oxygen uptake with succinate or palmitoyl-CoA as substrates can be measured in rat liver mitochondria that have been isolated by sucrose density gradient centrifugation providing the fractions are diluted with a 30 mM phosphate buffer rather than with an isotonic medium. Separate assay procedures were used to measure peroxisomal and mitochondrial β-oxidation of palmitoyl-CoA in the fractions of a sucrose gradient used to separate these organelles. A preliminary estimate of the ratio of palmitoyl-CoA oxidation by the mitochondrial fraction relative to the surviving peroxisomes from livers of male rats was 3.2.     

Carnitine acyltransferases in rat liver peroxisomes

Carnitine acyltransferase activities, as well as acetyl-CoA, octanyl-CoA, and palmityl-CoA hydrolase activities, were assayed in mitochondrial, peroxisomal, and endoplasmic reticulum fractions after isopycnic density sucrose gradient fractionation of rat liver homogenates. Both the forward and reverse assays show that carnitine acetyltransferase and carnitine octanyltransferase are associated with peroxisomes, mitochondria, and endoplasmic reticulum, while carnitine palmityltransferase was detected in mitochondria. Palmityl-CoA and octanyl-CoA hydrolase activities were found in all but the leading edge of the peroxisome peak of the gradient. The palmityl-CoA hydrolase in peroxisomal fractions was due to lysosomal contamination since the activity coincided with the lysosomal marker, acid phosphatase. The substrate specificity for carnitine octanyltransferase activity was maximum with medium-chain-length derivatives (about 20 nmol/ min/mg protein) and decreased as the acyl length increased until very low activity (<1 nmol/min/mg protein) was obtained with palmityl-CoA. When acyltransferases in peroxisomes were assayed by measuring acylcarnitine formation, nearly theoretical amounts of acetylcarnitine and octanylcarnitine were formed, but lesser quantities of 12 and 14 carbon acylcarnitines and very low amounts of palmitylcarnitine were detected. The presence of a broad spectrum of medium-chain and short-chain carnitine acyltransferases in peroxisomes is consistent with a role for carnitine for shuttling short-chain and medium-chain acyl residues out of peroxisomes. Carnitine acyltransferase activity was not detected in peroxisomes from spinach leaves.     

Ubiquinone biosynthesis in rat liver peroxisomes

The possibility that ubiquinone biosynthesis is present in rat liver peroxisomes was investigated. The specific activity of trans-prenyltransferase was 30% that of microsomes, with a pH optimum of around 8. trans-Geranyl pyrophosphate was required as a substrate and maximum activity was achieved with Mn(2+). Several detergents specifically inactivated the peroxisomal enzyme. The peroxisomal transferase is present in the luminal soluble contents, in contrast to the microsomal enzyme which is a membrane component. The treatment of rats with a number of drugs has demonstrated that the activities in the two organelles are subjected to separate regulation. Nonaprenyl-4-hydroxybenzoate transferase has about the same specific activity in peroxisomes as in microsomes and like the transferase activity, its regulation differs from the microsomal enzyme. The results demonstrate that peroxisomes are involved in ubiquinone biosynthesis, and at least two enzymes of the biosynthetic sequence are present in this organelle.     

Bile acid synthesis in rat liver peroxisomes: metabolism of 26-hydroxycholesterol to 3 beta-hydroxy-5-cholenoic acid

Rat liver peroxisomes have been found to oxidize 26-hydroxycholesterol, the product of cholesterol C-26 hydroxylation to 3 beta-hydroxy-5-cholenoic acid. Peroxisomes were purified by differential and equilibrium density centrifugation in a steep linear metrizamide gradient to greater than 95% purity. Purity of peroxisomes was determined by measurement of specific marker enzymes. The activities of cytochrome oxidase (a mitochondrial marker) and acid phosphatase (a lysosomal marker) in the purified peroxisome fractions were below the level of detection. Esterase activity indicated a 2-4% microsomal contamination. Subsequent to incubation of peroxisomes with [16,22-3H]-26-hydroxycholesterol, the reaction products were extracted, methylated, acetylated, and subjected to thin-layer, high pressure liquid, and gas-liquid chromatographic analyses. 3 beta-Hydroxy-5-cholenoic acid was the major identifiable metabolite of 26-hydroxycholesterol. Incubations of pure microsomal fractions (greater than 99%) with 26-hydroxycholesterol under the same conditions demonstrated that the production of 3 beta-hydroxy-5-cholenoic acid by peroxisomes was not attributable to microsomal contamination. This study demonstrates that peroxisomes participate in the side-chain oxidation of intermediates in bile acid synthesis.     

Chain-shortening of prostaglandin F2 alpha by rat liver peroxisomes

Liver peroxisomes were isolated from di(2-ethylhexyl)phthalate treated rats by isopycnic sucrose gradient centrifugation of a light mitochondrial fraction. Incubation of prostaglandin F2 alpha with purified peroxisomes resulted in conversion into a more polar product(s). In contrast, incubation with mitochondrial fractions and microsomal fractions under the same conditions did not result in any detectable conversion. The polar material obtained from a preparative incubation was purified by high performance liquid chromatography and characterized by radio-gas chromatography and gas chromatography-mass spectrometry. The structure of the polar compound was shown to be 5,7,11-trihydroxy-tetranorprost-9-enoic acid (tetranor-prostaglandin F1 alpha). Prostaglandin F2 alpha was thus chain-shortened by four carbon atoms.     

Acyl-Coenzyme A synthetase and fatty acid oxidation in rat liver peroxisomes.

Rat liver peroxisomes oxidized palmitate in the presence of ATP, CoA and NAD+, and the rate of palmitate oxidation exceeded that of palmitoyl-CoA oxidation. Acyl-CoA synthetase [acid: CoA ligase (AMP-forming); EC 6.2.1.3] was found in peroxisomes. The substrate specificity of the peroxisomal synthetase towards fatty acids with various carbon chain lengths was similar to that of the microsomal enzyme. The peroxisomal synthetase activity toward palmitate (40--100 nmol/min per mg protein) was higher than the rate of palmitate oxidation by the peroxisomal system (0.7--1.7 nmol/min per mg protein). The data show that peroxisomes activate long chain fatty acids and oxidize their acyl-CoA derivatives.