Chinese hamster ovary cells deficient in peroxisomes lack the nonspecific lipid transfer protein (sterol carrier protein 2)

Chinese hamster ovary cells deficient in intact peroxisomes were compared with wild type cells for the presence of the nonspecific lipid transfer protein (nsL-TP; sterol carrier protein 2). With the immunoblotting technique using the affinity purified antibody against rat liver nsL-TP, this protein was shown to be present in the homogenates from wild type cells, but could not be detected in mutant cells. In agreement with a previous study using livers from Zellweger patients it appears that there is a positive, as yet unknown, correlation between peroxisomes and the occurrence of nsL-TP in the cell. As a control using the affinity-purified antibody against the phosphatidylinositol transfer protein from bovine brain, levels of this protein were found to be normal in mutant cells. By use of metrizamide density gradients, nsL-TP was shown to cosediment with a membrane fraction different from peroxisomes. A protein of 58,000 daltons cross-reactive with the antibody against nsL-TP did cosediment with the peroxisomes in wild type cells and possibly with a "peroxisomal remnant" in the mutant cells. Incubation of wild type and mutant cells with L-[3-14C]serine showed that the biosynthesis of phosphatidylserine and the subsequent conversion into phosphatidylethanolamine was comparable in both cell types. This indicates that nsL-TP is not involved in the translocation of phosphatidylserine from the endoplasmic reticulum to the mitochondria as the site of decarboxylation.     

The occurrence of soluble and membrane-bound non-specific lipid transfer protein (sterol carrier protein 2) in rat tissues

The occurrence and subcellular distribution of the non-specific lipid transfer protein (nsL-TP; sterol carrier protein 2) in rat tissues was investigated by the immunoblotting technique using the affinity purified antibody against rat liver nsL-TP. Highest levels of the protein were found in the homogenates of liver, lung and adrenals, whereas it could hardly be detected in brain. In other tissues (i.e., testis, kidney, heart and intestine) the protein was present at intermediate concentrations. Analysis of subcellular fractions obtained by differential centrifugation demonstrated that in all tissues except for the liver, nsL-TP was predominantly present in the particulate fractions. In the particulate fractions of all tissues, an immunoreactive 58 kDa-protein was detected. Density centrifugation of a nuclear-free homogenate from liver and testis indicated that the 58 kDa-protein did, and nsL-TP did not, cofractionate with catalase. This suggests that in these tissues the bulk of nsL-TP is extraperoxisomal. Membrane-bound nsL-TP in testis was sensitive to trypsin treatment, suggesting that it is exposed to the cytosol. Release of nsL-TP by washing the membranes with 0.1 M Na2CO3 (pH 11.5), indicated that nsL-TP is a periferal protein. It was shown by chromatofocussing that nsL-TP extracted from membrane fractions was more basic than nsL-TP present in the cytosol fraction from rat liver (isoelectric point of 8.7).     

The amino acid sequence of rat liver non-specific lipid transfer protein (sterol carrier protein 2) is present in a high molecular weight protein: evidence from cDNA analysis

The affinity-purified antibody against rat liver non-specific lipid transfer protein (nsL-TP; sterol carrier protein 2) was used to screen a lambda-gt11 rat liver cDNA library. Positive cDNA clones were further identified by Southern blot analysis and sequenced. The largest cDNA clone consisted of 1851 bp starting at the 5' end with an open reading frame of 1545 bp. The 369 bp located at the 3' end of this open reading frame corresponded with the amino acid sequence of nsL-TP.     

Sterol carrier and lipid transfer proteins

The discovery of the sterol carrier and lipid transfer proteins was largely a result of the findings that cells contained cytosolic factors which were required either for the microsomal synthesis of cholesterol or which could accelerate the transfer or exchange of phospholipids between membrane preparations. There are two sterol carrier proteins present in rat liver cytosol. Sterol carrier protein 1 (SCP1) (Mr 47 000) participates in the microsomal conversion of squalene to lanosterol, and sterol carrier protein 2 (SCP2) (Mr 13 500) participates in the microsomal conversion of lanosterol to cholesterol. In addition SCP2 also markedly stimulates the esterification of cholesterol by rat liver microsomes, as well as the conversion of cholesterol to 7 alpha-hydroxycholesterol - the major regulatory step in bile acid formation. Also, SCP2 is required for the intracellular transfer of cholesterol from adrenal cytoplasmic lipid inclusion droplets to mitochondria for steroid hormone production, as well as cholesterol transfer from the outer to the inner mitochondrial membrane. SCP2 is identical to the non-specific phospholipid exchange protein. While SCP2 is capable of phospholipid exchange between artificial donors/acceptors, e.g. liposomes and microsomes, it does not enhance the release of lipids other than unesterified cholesterol from natural donors/acceptors, e.g. adrenal lipid inclusion droplets, and will not enhance exchange of labeled phosphatidylcholine between lipid droplets and mitochondria. Careful comparison of SCP2 and fatty acid binding protein (FABP) using six different assay procedures demonstrates separate and distinct physiological functions for each protein, with SCP2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP2 does not specifically bind or transport fatty acid. The results described in the present review support the concept that intracellular lipid transfer is a highly specific process, far more substrate-specific than suggested by the earlier studies conducted using liposomal techniques.     

The low-affinity lipid binding site of the non-specific lipid transfer protein. Implications for its mode of action.

The non-specific lipid transfer protein (nsL-TP) from bovine liver was studied by using the following fluorescent lipid analogs: phosphatidylcholine species with a sn-2-pyrenylacyl-chain of different length [Pyr(x)PC], sn-2-pyrenyldecanoyl-labelled phosphatidylinositol [Pyr(10)PI], -phosphatidylinositol 4-phosphate [Pyr(10)PIP], -phosphatidylinositol 4,5-bisphosphate [Pyr(10)PIP2] and dehydroergosterol. These analogs provided information on the effect of hydrophobicity and charge on lipid binding and transfer by nsL-TP. Binding of the Pyr(x)PC species decreased with increasing sn-2 acyl-chain length. Under equilibrium conditions, the fraction of nsL-TP that carried a PC molecule did not exceed 8%, which is consistent with a low affinity binding site. Also nsL-TP-mediated transfer of the Pyr(x)PC species decreased with increasing sn-2 acyl-chain length and was highly correlated with spontaneous transfer. Binding of the phosphoinositides increased in the order Pyr(10)PI less than Pyr(10)PIP less than Pyr(10)PIP2, indicating that an increase in lipid negative charge stimulates binding. The transfer of the phosphoinositides, however, decreased in the same order, which suggests that a high negative charge impairs the dissociation of the phospholipid from nsL-TP. Cholesterol, at concentrations up to 50 mol% in the donor membrane, hardly affected binding and transfer of Pyr(6)PC, strongly suggesting that nsL-TP has no high binding affinity for cholesterol. In agreement with this, binding of dehydroergosterol to nsL-TP was not detectable. Despite this apparently negligible affinity, nsL-TP-mediated transfer of dehydroergosterol was in the same order as that of Pyr(6)PC. The results are interpreted to indicate that transfer of lipids by nsL-TP involves the formation of a putative low-affinity lipid-protein complex. This formation is enhanced when lipid hydrophobicity decreases or lipid negative charge increases. Based on the binding and transfer data, the mode of action of nsL-TP is discussed in terms of change in free energy.     

Hydroxymethylglutaryl CoA reductase and the modulation of microsomal cholesterol content by the nonspecific lipid transfer protein

The influence of membrane cholesterol content on 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) in rat liver microsomes was investigated. Microsomes were enriched in cholesterol by incubation with egg phosphatidylcholine-cholesterol vesicles and the nonspecific lipid transfer protein from rat liver. By this method, the microsomal cholesterol content was 2.5-fold enhanced up to final concentrations of 140 nmol cholesterol per mg microsomal protein. In another experiment, microsomes isolated from rats fed a cholesterol-rich diet were depleted of cholesterol by incubation with egg phosphatidylcholine vesicles and the transfer protein. Both cholesterol enrichment and depletion had virtually no effect on the microsomal HMG-CoA reductase activity. In another set of experiments, normal rat liver microsomes were incubated with human serum, resulting in a rise of microsomal cholesterol content. This was reflected in an increase of acyl-CoA:cholesterol acyltransferase activity but failed to have an effect on HMG-CoA reductase.     

Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells.

The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g., Leydig and Sertoli cells whereas it could not be detected in germ cells. This cellular localization of SCP2 was confirmed by Northern blotting. Immunocytochemical techniques revealed that in Leydig cells, immunoreactive proteins were concentrated in peroxisomes. Although SCP2 was also detected in Sertoli cells, a specific subcellular localization could not be shown. SCP2 was absent from germ cells. Analysis of subcellular fractions of Leydig cells showed that SCP2 is membrane bound without detectable amounts in the cytosolic fraction. These results are at variance with data published previously which suggested that in Leydig cells a substantial amount of SCP2 was present in the cytosol and that the distribution between membranes and cytosol was regulated by luteinizing hormone. The present data raise the question in what way SCP2 is involved in cholesterol transport between membranes in steroidogenic cells but also in non-steroidogenic cells.     

cDNA sequence and bacterial expression of mouse liver sterol carrier protein-2.

Sterol carrier protein-2 (SCP-2) is an intracellular protein of Mr 13,096. In vitro studies have shown that it is involved in the transport and metabolism of cholesterol. This protein is believed to participate in these activities by forming a stoichiometric complex with the sterol. Because these activities occur in different intracellular locations, i.e. mitochondria, peroxisomes, and cytosol, it can be predicted that SCP-2 targets to these sites. In this report we show that a mouse cDNA (785 base pairs) encodes a precursor form of SCP-2 containing a N-terminal presequence and an additional C-terminal residue. These additional amino acid residues are found in proteins targeted to the mitochondria and peroxisomes, respectively. These signals are not found in SCP-2 purified from rat liver cytosol which is believed to be a cytosolic form. Northern analysis shows that there are four species of mRNA which hybridize to a SCP-2-specific probe at 1.0, 1.7, 2.2, and 2.9 kilobases. Southern analysis shows that the gene is distributed over a large amount of DNA or that there are multiple genes. We have cloned the cytosolic/peroxisomal form of mouse SCP-2 into the Escherichia coli expression vector pKK233-2 and have expressed and purified recombinant mouse SCP-2, Mr 13,034. The purified recombinant SCP-2 is immunoreactive to rabbit anti-rat SCP-2 antibody. It also has biological activity equivalent to homogeneous rat liver SCP-2 in stimulating the microsomal conversion of 7-dehydrocholesterol to cholesterol and in the esterification of cholesterol by acyl-CoA cholesterol acyltransferase by rat liver microsomes.     

Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein

We have investigated the esterification by liver membranes of retinol bound to cellular retinol-binding protein (CRBP). When CRBP carrying [3H]retinol as its ligand was purified from rat liver cytosol and incubated with rat liver microsomes, a significant fraction of the [3H]retinol was converted to [3H]retinyl ester. Esterification of the CRBP-bound [3H]retinol, which was maximal at pH 6-7, did not require the addition of an exogenous fatty acyl group. Indeed, when additional palmitoyl-CoA or coenzyme A was provided, the rate of esterification increased either very slightly or not at all. The esterification reaction had a Km for [3H]retinol-CRBP of 4 +/- 0.6 microM and a maximum velocity of 145 +/- 52 pmol/min/mg of microsomal protein (n = 4). The major products were retinyl palmitate/oleate and retinyl stearate in a ratio of approximately 2 to 1 over a range of [3H]retinol-CRBP concentrations from 1 to 8 microM. The addition of progesterone, a known inhibitor of the acyl-CoA:retinol acyltransferase reaction, consistently increased the rate of retinyl ester formation when [3H]retinol was delivered bound to CRBP. These experiments indicate that retinol presented to liver microsomal membranes by CRBP can be converted to retinyl ester and that this process, in contrast to the esterification of dispersed retinol, is independent of the addition of an activated fatty acid and produces a pattern of retinyl ester species similar to that observed in intact liver. A possible role of phospholipids as endogenous acyl donors in the esterification of retinol bound to CRBP is supported by our observations that depletion of microsomal phospholipid with phospholipase A2 prior to addition of retinol-CRBP decreased the retinol-esterifying activity almost 50%. Conversely, incubating microsomes with a lipid-generating system containing choline, CDP-choline, glycerol 3-phosphate, and an acyl-CoA-generating system prior to addition of retinol-CRBP increased retinol esterification significantly as compared to buffer-treated controls.     

Determination of nonspecific lipid transfer protein in rat tissues and Morris hepatomas by enzyme immunoassay

Rat tissues contain a nonspecific transfer protein which in vitro mediates the transfer of diacylphospholipids as well as cholesterol between membranes. This protein appears identical to sterol carrier protein. A specific enzyme immunoassay for this protein was developed using antibodies raised in rabbits, against a homogeneous protein from rat liver. This assay was based on the very high affinity of the nonspecific lipid transfer protein for polyvinyl surfaces. A reproducible adsorption was achieved by presenting the protein to the surface in the presence of a large excess of bovine serum albumin. The adsorbed protein was detected with specific immunoglobulin (IgG) isolated by antigen-linked affinity chromatography and a goat anti-rabbit IgG-enzyme conjugate. Adsorption was proportional to the amount of protein present, giving rise to a linear standard curve. The enzyme immunoassay measured transfer protein levels in the range 0.2-2 ng. The highest concentrations of transfer protein were found in liver and intestinal mucosa. Levels in other tissues including brain, lung, kidney, spleen, heart, adrenals, ovary and testis were 5-10-fold lower than in liver. In the fast-growing Morris hepatoma 7777 the concentration of nonspecific lipid transfer protein was approximately one-tenth of that measured in the host liver, whereas a reduction of 65% was observed in the slow-growing Morris hepatomas 7787 and 9633. Subcellular distribution studies showed that approx. 70% of the transfer protein was present in the soluble supernatant fraction.     

Sterol carrier protein2-like activity in rat intestine

A sterol carrier protein2 (SCP2)-like activity has been demonstrated in rat intestinal mucosal homogenates and in isolated intestinal cells from both crypt and villus zones. The results indicate the presence of a protein with similar molecular weight and antigenicity to that of authentic SCP2 purified from rat liver cytosol. Like liver SCP2, mucosal cytosol stimulates pregnenolone production in rat adrenal mitochondria and acyl coenzyme A:cholesterol acyltransferase activity of liver and mucosal microsomes. The distribution of SCP2-like activity as determined by radioimmunoassay indicates high levels in mitochondria and cytosol and relatively lower levels in microsomes and in brush-border membranes. The widespread distribution of SCP2-like protein in the intestine is consistent with potential transfer functions in all phases of cholesterol processing.     

The involvement of fatty acid binding protein in peroxisomal fatty acid oxidation

Rat liver fatty acid-binding protein (FABP) can function as a fatty acid donor protein for both peroxisomal and mitochondrial fatty acid oxidation, since 14C-labeled palmitic acid bound to FABP is oxidized by both organelles. FABP is, however, not detected in peroxisomes and mitochondria of rat liver by ELISA. Acyl-CoA oxidase activity of isolated peroxisomes was not changed by addition of FABP or flavaspidic acid, an inhibitor of fatty acid binding to FABP, nor by disruption of the peroxisomal membranes. These data indicate that FABP may transfer fatty acids to peroxisomes, but is not involved in the transport of acyl-CoA through the peroxisomal membrane.     

Purification of nonspecific lipid transfer protein (sterol carrier protein 2) from human liver and its deficiency in livers from patients with cerebro-hepato-renal (Zellweger) syndrome

The nonspecific lipid transfer protein (i.e., sterol carrier protein 2) from human liver was purified to homogeneity using ammonium sulfate precipitation, CM-cellulose chromatography, molecular sieve chromatography and fast protein liquid chromatography. Its amino acid composition was determined and found to be very similar to that of the nonspecific lipid transfer protein from bovine and rat liver with, as main feature, the absence of arginine, histidine and tyrosine. By way of a specific enzyme immunoassay using affinity-purified antibodies, the levels of nonspecific lipid transfer protein were determined in human livers. Levels varied from approximately 150 ng nonspecific lipid transfer protein per mg 105,000 X g supernatant protein for juvenile and adult humans to 40 ng per mg supernatant protein for a young infant. Levels of nonspecific lipid transfer protein in livers of infants with cerebro-hepato-renal (Zellweger) syndrome were extremely low (i.e., 2 ng per mg supernatant protein). Immunoblotting revealed the presence of crossreactive proteins of molecular masses of 40,000 and 58,000. The 40 kDa and 58 kDa proteins occurred in control livers, whereas only the 40 kDa protein was present in Zellweger livers. As in rat the 58 kDa protein could be demonstrated in a peroxisomal preparation isolated from an adult liver. A possible link between the occurrence of nonspecific lipid transfer protein and the presence of peroxisomes is discussed.     

Stimulation of cholinephosphotransferase activity by phosphatidylcholine transfer protein. Regulation of membrane phospholipid synthesis by a cytosolic protein

The effect of rat liver phosphatidylcholine transfer protein on the incorporation of CDP-choline and dioleoylglycerol into phosphatidylcholine catalyzed by rat liver microsomal CDP-choline: 1,2-diacyl-sn-glycerol cholinephosphotransferase was studied. In the presence of phosphatidylcholine transfer protein, the incorporation of CDP-choline into phosphatidylcholine was markedly stimulated. Phosphatidylcholine transfer protein isolated from either rat or bovine liver was capable of this stimulatory effect; in contrast, phosphatidylinositol transfer protein from rat liver had no effect on phosphatidylcholine synthesis. Kinetic analysis showed that microsomal phosphatidylcholine synthesis increased 2.4-fold after 1 min and reached a maximum of approximately 10-fold within 10 min in the presence of phosphatidylcholine transfer protein; in the absence of this protein phosphatidylcholine synthesis stopped after 2-4 min. These results suggest that phosphatidylcholine transfer protein permits phosphatidylcholine synthesis to proceed further. With the addition of phospholipid vesicles, as an acceptor membrane in the reaction mixture, there was a significant amount of protein-mediated transfer of synthesized phosphatidylcholine to the vesicles. Measurable transfer of synthesized phosphatidylcholine to vesicles could only be detected after a lag of 2-4 min. The stimulation of cholinephosphotransferase could be nearly abolished by increasing the amount of added phospholipid vesicles; concurrently, a greater transfer to the vesicles was observed. These results describe a new property of phosphatidylcholine transfer protein which may be of physiological significance in the regulation of phosphatidylcholine synthesis in mammalian tissues.     

The subcellular distribution of the nonspecific lipid transfer protein (sterol carrier protein 2) in rat liver and adrenal gland

The distribution of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) over the various subcellular fractions from rat liver and adrenal gland was determined by enzyme immunoassay and immunoblotting. This distribution is very different in each of these two tissues. In liver, 66% of the transfer protein is present in the membrane-free cytosol as compared to 19% in the adrenal gland. In the latter tissue, the transfer protein is mainly found in the lysosomal/peroxisomal and the microsomal fraction at a level of 1093 and 582 ng per mg total protein, respectively (i.e., 17% and 35% of the total), and to a lesser extent in the mitochondrial fraction (11% of the total). Of all the membrane fractions isolated, the microsomal fraction from the liver and the mitochondrial fraction from the adrenal gland have the lowest levels of the transfer protein (i.e., 168 ng and 126 ng per mg total protein, respectively). These low levels correlate poorly with the active role proposed for this transfer protein in the conversion of cholesterol into bile acids and steroid hormones in these fractions. Using immunoblotting, it was demonstrated that in addition to the transfer protein (14 kDa) a cross-reactive 58 kD protein was present in the supernatant and the membrane fractions of both tissues. Cytochemical visualization in adrenal tissue with specific antibodies against the nonspecific lipid transfer protein showed that immunoreactive protein(s) were present mainly in the peroxisome-like structures.     

Elevated cholesterol and decreased sterol carrier protein-2 in peroxisomes from AS-30D hepatoma compared to normal rat liver

Peroxisomes were isolated from AS-30D hepatoma and compared to normal rat liver cells for the purpose of investigating the cholesterol accumulation in the hepatoma cells. Cholesterol was found to be approximately 10-fold higher relative to protein in AS-30D peroxisomes as compared to peroxisomes from normal liver. The peroxisomes from the hepatoma cells were found to be more stable; catalase was not released from these peroxisomes during isolation or osmotic shock of the peroxisomal fraction. The elevated cholesterol level may stabilize the peroxisomal membrane. Sterol carrier protein-2 (SCP-2) levels were measured using a radioimmunoassay (RIA), which indicated the highest concentration of SCP-2 to be in peroxisomes. Hepatoma peroxisomes had a lower concentration of SCP-2 (2.5 micrograms/mg) than normal liver peroxisomes (8 micrograms/mg). Approximately half of all SCP-2 detected was found to be soluble in both hepatoma and normal rat liver cells. Immunoblots from both rat liver and AS-30D fractions demonstrated the presence of the 14-kDa form of SCP-2. The liver fractions also had a 57-kDa immunoreactive protein, which was barely detectable in the AS-30D fractions. The low abundance of the high molecular weight form of SCP-2 from hepatoma peroxisomes and the lower amounts of SCP-2 detected in the AS-30D peroxisomes may be related to the accumulation of cholesterol in the cells.     

Sterol carrier protein 2 and fatty acid-binding protein. Separate and distinct physiological functions

Sterol carrier protein 2 (SCP-2) participates in the microsomal conversion of lanosterol to cholesterol, in the conversion of cholesterol to cholesterol ester, and in intracellular cholesterol transfers. The stoichiometry of binding between cholesterol and SCP-2 is 1:1. However, reports have appeared attributing sterol carrier protein activity to a protein preparation identical to hepatic fatty acid-binding protein (FABP). Therefore, the present investigation was conducted to compare homogeneous preparations of FABP and SCP-2 with respect to their capacities to participate as carrier proteins in reactions involving sterols or fatty acids. The results show that SCP-2 and FABP have separate and distinct physiological functions, with SCP-2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP-2 does not specifically bind or transport fatty acid. As long as only small quantities of organic solvent (1.6 volume %) were used for substrate addition, the sterol delta 7-reductase liver microsomal assay for SCP-2 correlated well with the physiologically relevant assays employed in the reconstituted adrenal system. The sterol carrier protein activity previously attributed to rat hepatic FABP is explained by the presence of significant quantities of propylene glycol (15 volume %) or Tween 80 in the assay procedure.     

微粒体甘油三脂转运蛋白MTP的结构和功能研究概况

微粒体甘油三酯转运蛋白MTP(microsomal triglyceride transfer protein,MTP)首先是从牛的肝细胞微粒体碎片中分离获得的,其作用是加速甘油三脂(triglyceride,TG)、胆固醇(cholesteryl ester,CE)和磷脂酰胆碱(phosphatidylcholine,PC)的转运和细胞或亚细胞膜的生物合成。它后来在肝细胞和小肠的微粒体膜中发现[1],由于它的位置及其转运TG可以推测与血浆脂蛋白中极低密度脂蛋白(very low density lipoprotein,VLDL)和乳糜微粒(chylomicrons,CM)的组装过程有关。     

Mevalonate kinase is localized in rat liver peroxisomes.

Recent data suggest that rat liver peroxisomes play a critical role in cholesterol synthesis. Specifically, peroxisomes contain a number of enzymes required for cholesterol synthesis as well as sterol carrier protein-2. Furthermore, peroxisomes are involved in the in vitro synthesis of cholesterol from mevalonate and contain significant levels of apolipoprotein E, a major constituent of several classes of plasma lipoproteins. In this study we have investigated the subcellular localization of mevalonate kinase (EC 2.7.1.36; ATP:mevalonate-5-phosphotransferase). Mevalonate kinase is believed to be a cytosolic enzyme and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. Mevalonate kinase has been purified from rat liver cytosol and a cDNA clone coding for rat mevalonate kinase has also been isolated and characterized. In this study, utilizing monoclonal antibodies made against the purified rat mevalonate kinase, we demonstrate the presence of mevalonate kinase in rat liver peroxisomes and in the cytosol. Each of these compartments contained a different form of the protein. The pI and the Mr of the peroxisomal protein is 6.2 and 42,000, respectively. The pI and Mr of the cytosolic protein is 6.9 and 40,000, respectively. The peroxisomal protein was also significantly induced by a number of different hypolipidemic drugs. In addition, we present evidence for the unexpected finding that the purified mevalonate kinase (isolated from the cytosol and assumed to be a cytosolic protein) is actually a peroxisomal protein.     

Ultrastructural localization of a peroxisomal protein in rat liver using the specific antibody against the non-specific lipid transfer protein (sterol carrier protein 2)

An antibody against the non-specific lipid transfer protein from rat liver was purified by immunoabsorbent affinity chromatography. This antibody in conjunction with protein A-colloidal gold was used to localize the transfer protein in rat liver by electron microscopy. Labeling by this immunocytochemical technique was found to be mainly restricted to the peroxisomes; low labeling was observed in the cytoplasm. Subsequent analysis of isolated peroxisomes by immunoblotting indicated that the non-specific lipid transfer protein (mol. wt. 14800) was absent from this organelle and that a protein of molecular weight 58000 was responsible for the immunological response. Immunoblotting of the membrane-free cytosol showed the presence of both proteins. It remains to be established to what extent the non-specific lipid transfer protein in the cytosol and the high-molecular weight protein in the peroxisomes are related.