Rough Microsomal Fraction Research Articles

Subcellular Fractionation of Rough Microsomes

When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough microsomes are introduced here, as are methods for evaluating the purity and intactness of an isolated rough microsomal fraction.

Purification and Partial Molecular Characterization of GRP94, an ER Resident Chaperone

GRP94 is a resident glycoprotein of the endoplasmic reticulum (ER) and a member of the hsp90 family of molecular chaperones. Current experimental evidence indicates that GRP94 functions in an as yet undefined manner in protein folding and assembly in the ER. We report a rapid, high-yield GRP94 purification procedure that yields milligram quantities of homogeneous protein suitable for structural and biochemical analyses. Beginning with a rough microsome fraction derived from porcine pancreas, GRP94 was isolated by partial detergent extraction, anion exchange, and gel filtration chromatography. With this procedure, approximately 3 mg of homogeneous GRP94 can be prepared from a 25-g pancreas. Heterogeneity in the migration of purified GRP94 on native and denaturing PAGE was observed and demonstrated to reflect variability in the N-linked glycosylation state of the protein. Analysis by native and two-dimensional nonreducing/reducing gels indicated that the protein exists as a dimer of noncovalently associated subunits. The membrane localization of GRP94 in isolated pancreatic microsomes was assessed by alkali and detergent extraction. By comparison with the resident ER integral membrane protein TRAPα, GRP94 exists as a soluble, lumenal protein.

Changes in GTP-dependent fusion and ras-related proteins in membranes from rat hepatocellular carcinomas

The fusion capacity of rough endoplasmic reticulum membranes isolated from dissected liver tumor nodules of aflatoxintreated rats was determined by cell free assay to be greater than that of homologous membranes from control liver. In a first attempt to understand the reason for this difference we compared the content of ras-related proteins in rough microsomal fractions and other cell fractions of both dissected tumor nodules and control liver. Using [α-32P]GTP blot overlay and densitometric analysis, homogenate, Golgi and rough endoplasmic reticulum fractions from dissected tumor nodules were observed to contain increased amounts of [α-32P]GTP binding to ras-related proteins when compared to homologous control fractions. Western blot analysis indicated that ras content was also increased in the tumor fractions. [α-32P]GTP-blot overlay using double-dimensional SDS-polyacrylamide gel electrophoresis confirmed quantitative differences in the amount of [α-32P]GTP binding to ras-related proteins between fractions from tumor and control tissues and indicated a surprising number of such proteins in each fraction. The data suggest that the changes in ras-related proteins could, in part, account for the enhanced GTP-dependent fusion capacity observed for the tumor-derived membranes.

Changes in GTP-dependent fusion and ras-related proteins in membranes from rat hepatocellular carcinomas

The fusion capacity of rough endoplasmic reticulum membranes isolated from dissected liver tumor nodules of aflatoxintreated rats was determined by cell free assay to be greater than that of homologous membranes from control liver. In a first attempt to understand the reason for this difference we compared the content of ras-related proteins in rough microsomal fractions and other cell fractions of both dissected tumor nodules and control liver. Using [ α- 32P]GTP blot overlay and densitometric analysis, homogenate, Golgi and rough endoplasmic reticulum fractions from dissected tumor nodules were observed to contain increased amounts of [ α- 32P]GTP binding to ras-related proteins when compared to homologous control fractions. Western blot analysis indicated that ras content was also increased in the tumor fractions. [ α- 32P]GTP-blot overlay using double-dimensional SDS-polyacrylamide gel electrophoresis confirmed quantitative differences in the amount of [ α- 32P]GTP binding to ras-related proteins between fractions from tumor and control tissues and indicated a surprising number of such proteins in each fraction. The data suggest that the changes in ras-related proteins could, in part, account for the enhanced GTP-dependent fusion capacity observed for the tumor-derived membranes.

Casein kinase localization in the endoplasmic reticulum of the ROS 17/2.8 cell line.

Phosphophoryns (PPs) are phosphoproteins specific to the dentin matrix and are the major noncollagenous matrix proteins in rat incisor dentin. It has been hypothesized that their phosphate groups are important in dentin mineralization. PPs have many sequences which are substrates for membrane-associated endogenous messenger-independent kinases. The objective of this study was to localize the protein kinases involved in phosphorylating the PPs. Osteoblast-like ROS 17/2.8 cells, which secrete extracellular matrix phosphoproteins, were lysed. After removal of the nuclei and mitochondria by low-speed centrifugation, the membrane associated organelles were isolated at higher speed from the cytosol. The Golgi vesicle and rough microsome fractions were collected from 29-43.7% sucrose density gradients. Each fraction was tested for casein kinase II (CKII) activity using an in vitro phosphorylation assay with PPs as substrate. To characterize and confirm the nature of the components of the sucrose gradient fractions, the activities of specific enzymes such as N-acetylglucosamine galactosyltransferase and cytochrome c reductase, which are exclusively associated with the Golgi and rough microsomes, respectively, were determined. Electron microscopy of the isolated fractions confirmed the enzyme assay characterizations. CKII activity capable of phosphorylating the PP was found in the rough microsome fraction but not in the Golgi. Thus, phosphorylation of the secreted phosphoproteins would appear to take place in the endoplasmic reticulum as a cotranslational event.

The effect of thyroid hormone treatment on the gene expression and enzyme activity of rat liver sodium-potassium dependent adenosine triphosphatase.

The effects of thyroid hormone (T3) treatment on liver Na,K-adenosine triphosphatase (Na,K-ATPase) at the levels of subunit messenger RNA (mRNA), enzymatic activity, and enzyme content were studied in euthyroid rats injected for 5 consecutive days with T3. Northern and slot blot analyses of polyadenylated mRNA revealed that T3 treatment coordinately increases the level of mRNA encoding the alpha 1- and beta 1-subunits, approximately 4- and 3-fold, respectively, above basal levels. To determine whether this increase in the subunit mRNA consequently results in an increase in the synthesis of the enzyme, a modified liver cell fractionation procedure was developed, and the subcellular fractions from control and T3-treated livers were examined biochemically. Western blot analysis and Na,K-ATPase assay demonstrated that T3 treatment resulted in a 2-fold increase in both the amount and activity of the enzyme. Furthermore, the Western blot analysis of endoglycosidase-H-treated membrane fractions revealed an increase in the amount of the precursor beta-subunit in the T3-treated liver rough microsomal fraction, suggesting that an increase in subunit synthesis contributes at least partially to the increase in the rat liver Na,K-ATPase by T3 treatment.

The effect of thyroid hormone treatment on the gene expression and enzyme activity of rat liver sodium-potassium dependent adenosine triphosphatase

The effects of thyroid hormone (T3) treatment on liver Na,K-adenosine triphosphatase (Na,K-ATPase) at the levels of subunit messenger RNA (mRNA), enzymatic activity, and enzyme content were studied in euthyroid rats injected for 5 consecutive days with T3. Northern and slot blot analyses of polyadenylated mRNA revealed that T3 treatment coordinately increases the level of mRNA encoding the alpha 1- and beta 1-subunits, approximately 4- and 3-fold, respectively, above basal levels. To determine whether this increase in the subunit mRNA consequently results in an increase in the synthesis of the enzyme, a modified liver cell fractionation procedure was developed, and the subcellular fractions from control and T3-treated livers were examined biochemically. Western blot analysis and Na,K-ATPase assay demonstrated that T3 treatment resulted in a 2-fold increase in both the amount and activity of the enzyme. Furthermore, the Western blot analysis of endoglycosidase-H-treated membrane fractions revealed an increase in the amount of the precursor beta-subunit in the T3-treated liver rough microsomal fraction, suggesting that an increase in subunit synthesis contributes at least partially to the increase in the rat liver Na,K-ATPase by T3 treatment.

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).

Localization of the FK506-binding protein, FKBP 13, to the lumen of the endoplasmic reticulum

The function of the immunophilins, FKBP 12 and FKBP 13, which are binding proteins for the immunosuppressant drug FK506 and rapamycin, remains poorly defined, although it has been suggested that immunophilins and immunophilin-like proteins may play a role in protein sorting/folding and intracellular calcium ion regulation. As a first step towards understanding the function of FKBP 13, we studied its subcellular localization by immunoblotting of well-defined subcellular fractions from a canine pancreatic homogenate and immunocytochemical analysis of an overexpressed cloned cDNA for FKBP 13. Whereas FKBP 12 fractionated entirely into the cytosol, virtually all FKBP 13 was found in the rough microsomal fraction which consisted of highly purified rough endoplasmic reticulum (ER), along with several well-characterized ER markers [the immunoglobulin heavy-chain binding protein (BiP), grp 94 and ribophorin I]. Moreover, FKBP 13 co-banded with the ER markers on isopycnic sucrose gradients. By immunofluorescence, the overexpressed cDNA for FKBP 13 in Hela cells gave an ER-staining pattern highly similar to that of known ER proteins. Addition of the ligand FK506 did not appear to alter the distribution of FKBP 13. Separation of the ER luminal contents and membrane revealed FKBP 13 to be a luminal ER protein. Since the lumen of the ER is where the folding of membrane and secreted proteins occurs, as well as a major site of intracellular calcium storage, it seems possible that FKBP 13 may be involved in one of these functions.

Inositol 1,4,5-trisphosphate receptors. Localization in epithelial tissue.

Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.

Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti- ras monoclonal antibodies 142-24E05 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [α- 32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [α- 32P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [α- 32P] GTP-binding protein in the plasma membrane fraction. When anti- ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations.

Subcellular distribution of calcium-binding proteins and a calcium- ATPase in canine pancreas [published erratum appears in J Cell Biol 1990 Oct;111(4):1726

Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with "Stains-All." Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER.

Subcellular distribution of small GTP binding proteins in pancreas: identification of small GTP binding proteins in the rough endoplasmic reticulum.

Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[gamma-35S]) was assayed in each fraction. Enrichment of GTP[gamma-35S] binding was greatest in the interfacial "smooth" microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a [alpha-32P]GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP[gamma-35S] binding, as well as the small GTP binding proteins detected by the [alpha-32P]GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMs were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP[gamma-35S] binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.

Identification of a G protein in rough endoplasmic reticulum of canine pancreas.

Employing [32P]ADP-ribosylation by pertussis toxin we have identified a G protein that is located in the rough endoplasmic reticulum of canine pancreas and therefore termed it GRER. Identification of GRER is based on the following data. A 41-kDa polypeptide was the only polypeptide that was [32P]ADP-ribosylated by pertussis toxin in pancreas rough microsomes. Guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) and 1 mM ATP, 6 mM MgCl2, 10 mM NaF (AMF) inhibited ADP-ribosylation of this polypeptide. The [32P]ADP-ribosylated 41-kDa polypeptide was immunoprecipitated by antisera which specifically recognized the C-terminal residues of the alpha subunits of Gi and transducin, indicating that the 41-kDa polypeptide is immunologically related to the alpha subunits of heterotrimeric G proteins. Treatment with GTP gamma S resulted in a reduction in the sedimentation rate of the [32P]ADP-ribosylated, detergent-solubilized GRER. It also induced the release of the [32P]ADP-ribosylated 41-kDa polypeptide from rough microsomes in the absence of detergent, unlike ADP-ribosylated alpha subunits of plasma membrane-associated G proteins. These data are consistent with an oligomeric nature of GRER. The codistribution of GRER with an endoplasmic reticulum marker protein during subcellular fractionation and the lack of plasma membrane contamination of the rough microsomal fraction, combined with the isodensity of GRER with rough microsomes as well as the isodensity of GRER with "stripped" microsomes after extraction of rough microsomes with EDTA and 0.5 M KCl, localized GRER to the rough endoplasmic reticulum. Preliminary experiments suggest that GRER appears not to be involved in translocation of proteins across the rough endoplasmic reticulum membrane.

The formation of phosphatidic acid de novo: a comparison of activities in neuronal nuclei and microsomes isolated from immature rabbit cerebral cortex

The formation of phosphatidic acid from sn-glycerol 3-phosphate was studied in neuronal nuclear fraction N 1 and a microsomal fraction P 3, isolated from cerebral cortices of 15-day-old rabbits. Two assays were used, employing dithiothreitol, MgCl 2, NaF and (A) sn-glycerol 3-phosphate, [ 14C]oleate, ATP and CoA or (B) sn-[ 3H] glycerol 3- phosphate and oleoyl-CoA. In both assays fraction N 1 had specific rates of phosphatidic acid labelling (expressed per μmol phospholipid in the fraction) which were 5- to 6-times the corresponding values for P 3. In contrast to N 1 the formation of phosphatidic acid by fraction P 3 was more sensitive to inhibition at high concentrations of oleoyl-CoA and was greatly dependent upon the presence of NaF. In the absence of this salt, P 3 showed decreased phosphatidate formation and increased levels of radioactive monoacylglycerols. Using cerebral cortex, rough (R) and smooth (S) microsomal fractions were prepared, as was a microsomal fraction P from isolated nerve cell bodies. P had specific rates of phosphatidic acid labelling which were 2–3-times the values for P 3, but were about 50% of the N 1 values. This indicates a concentration of phosphatidate synthesis in the nucleus within the nerve cell. Specific rates for fraction R were higher and were similar to those of N 1. In S, P 3 and R the specific rates of phosphatidic acid synthesis paralleled specific RNA contents and indicated a location for phosphatidic acid synthesis within the rough endoplasmic reticulum.

Antibodies to rat pancreas Golgi subfractions: identification of a 58-kD cis-Golgi protein.

A 58-kD cis-Golgi protein has been identified by generating polyclonal antibodies against heavy (cis) Golgi subfractions. Total microsomes isolated from rat pancreatic homogenates were subfractionated to yield a rough microsomal fraction (B1) and three smooth membrane subfractions (B2-B4) enriched in cis-, middle, and trans-Golgi elements, respectively. The heavy (cis) subfraction, B2 (d = 1.17 g/ml), was fractionated by Triton X-114 phase separation, and the proteins recovered in the detergent phase were used to immunize rabbits. One of the anti-B2 antibodies obtained gave a "Golgi"-staining pattern when screened by immunofluorescence on normal rat kidney cells and mouse RPC 5.4 myeloma cells. In rat pancreatic exocrine cells the antibody reacted with the plasmalemma as well as elements in the Golgi region. By immunoelectron microscopy, the antigen recognized by anti-B2 IgG was found to be restricted to cis-Golgi elements in myeloma cells where it was concentrated in the fenestrated cis-most cisterna and in some of the tubules and vesicles located along the cis face of the Golgi complex. By immunoprecipitation and immunoblotting, the anti-B2 IgG exclusively recognized a 58-kD protein in myeloma cells. The anti-B2 IgG reacted with several proteins in solubilized pancreatic B2 membranes, including a 58-kD protein, but affinity-purified anti-58-kD IgG reacted exclusively with the 58-kD protein. These results suggest that the 58-kD protein is a specific component of cis-Golgi membranes.

Intracellular transport and processing of lysosomal cathepsin H

Intracellular transport and processing of lysosomal cathepsin H was investigated in the subcellular fractions of rat liver by pulse-labeling experiments with [ 35S]methionine in vivo . A newly synthesized procathepsin H with a molecular weight of 41 kDa first appeared in the rough microsomal fraction at 10 min postinjection of label. This procathepsin H moved from the microsomal fraction to the Golgi subfractions at 30 min. Then a processed single-chain form with 28 kDa appeared in the lysosomal fraction at 60 min and persisted as the predominant form after 180 min. These results suggest that the propeptide-processing of procathepsin H indeed takes place in lysosomes in the course of intracellular transport from endoplasmic reticulum via Golgi complex to lysosomes.

Intracellular transport and processing of lysosomal cathepsin B

Intracellular transport and processing of lysosomal cathepsin B was investigated in the subcellular fractions of rat liver by pulse-labeling experiments with [ 35S]methionine in vivo . A newly synthesized procathepsin B with a molecular weight of 39 kDa firstly appeared in the rough microsomal fraction at 10 min postinjection of label. This procathepsin B moved from the microsomal fractions to the Golgi subfractions at 30 min postinjection, and then a processed mature enzyme appeared in the lysosomal fraction at 60 min. These results suggest that the propeptide-processing of procathepsin B takes place in lysosomes in the course of intracellular transport from endoplasmic reticulum through Golgi complex to lysosomes.

Topological studies on rat liver microsomal cholesterol ester hydrolase

Lateral and transversal distribution of cholesterol ester hydrolase activity in rat liver microsomal membranes has been studied. Total cholesterol ester hydrolase activity was found predominantly (75%) in rough microsomes though specific esterase activities were similar in rough and smooth microsomal fractions. The transversal assymetry of the enzyme was examined using the criteria of protease sensitivity and latency of mannose-6-phosphate phosphatase. Cholesterol ester hydrolase resulted drastically inhibited by proteolysis with trypsin when microsomal integrity had been previously disrupted with sodium deoxycholate or sodium taurocholate. Under these conditions, most lumenal mannose-6-phosphate phosphatase activity was destroyed. However, cholesterol esterase was unaffected by preincubating microsomes with the detergent alone, which led to the complete expression of latent mannose-6-phosphate phosphatase or by preincubating them with trypsin, where less than a 15% of the lumenal mannose-6-phosphate phosphatase was lost. These findings suggest that cholesterol ester hydrolase activity is located on the lumenal surface of the hepatic microsomal vesicles.