The expression of mouse gene P1A in testis does not prevent safe induction of cytolytic T cells against a P1A-encoded tumor antigen.

Tumor antigen P815AB is recognized by cytolytic T lymphocytes (CTL) on mouse mastocytoma P815. This antigen is encoded by P1A, a gene activated in several tumors but silent in normal tissues except for testis and placenta. Notwithstanding the expression of P1A in testis, we found that male mice mounted P815AB-specific CTL responses as efficiently as females. The responding males remained fertile and no autoimmune lesions were observed in their testes. By immunohistochemistry with a rabbit antiserum directed against the P1A protein, we identified spermatogonia as the testicular cells expressing P1A. The absence of MHC class-I molecules on spermatogonia could be one of the mechanisms of protection against testicular autoimmunity, as the antigenic peptide should not be displayed at the cell surface. Human genes MAGE, BAGE and GAGE, which also code for tumor antigens recognized by autologous CTL, are not expressed in normal tissues other than testis. The results obtained in mice with antigen P815AB suggest that immunization of human males with such antigens will not generate autoimmune side-effects. Although P1A is strongly expressed in placenta, we also found that gestation did not prevent generation of CTL responses against antigen P815AB, and that such CTL responses did not affect gestation outcome. We identified labyrinthine trophoblasts as the placental cells expressing P1A. Again, the absence of MHC class-I molecules on these cells provides a plausible explanation for placental protection, although other mechanisms may also play a role.

The tumor protein MAGE-1 is located in the cytosol of human melanoma cells.

The MAGE-1 protein has been localized to the cytosol of human melanoma culture cells (MZ2-MEL.43) by subcellular fractionation. Its distribution between nuclear, mitochondrial, microsomal and cytosolic fractions was established by SDS-PAGE and immunoblotting with a rabbit antiserum and compared to that of markers for the main cell compartments. The immunoreactive material was recovered in the cytosolic fraction as a polypeptide migrating at 42 kDa, which has been further identified as the MAGE-1 protein because it comigrates with the product of cell-free transcription-translation of the MAGE-1 cDNA. Location to the cytosol was confirmed by immunofluorescence microscopy.

Identification and characterization of the tumor-specific P1A gene product.

In murine mastocytoma P815, gene P1A directs the expression of antigens P815A and B which are the target of a T cell-mediated rejection response in syngeneic animals. This gene is expressed at a high level in various tumors, but is silent in normal tissues except testis and placenta; its activation is thus possibly related to malignant transformation. An anti-synthetic peptide rabbit antiserum reacted by immunoblotting with a cellular protein migrating near 40 kDa on SDS-PAGE. The immunoreactive protein was detected only in lysates from cells which express antigen P815A: P1.HTR mastocytoma cells and, after transfection with cosmids carrying the P1A gene, the antigen-loss variant P0.HTR cells and DAP-3 H-2Ld fibroblasts. The identity of this protein as the P1A gene product was confirmed by cell-free transcription-translation of the P1A cDNA, the product of which also migrated near 40 kDa in SDS-PAGE and was captured by protein A-Sepharose in the presence of the antiserum. Subcellular fractionation by differential and isopycnic centrifugation indicated that the P1A protein is associated with cytoplasmic membranes demonstrating a broad distribution with respect to size and density. Immunofluorescence microscopy also revealed a cytoplasmic signal, particularly intense in small vesicles, which coincides with that produced by an anti-mouse type I collagen guinea pig antiserum except near the cell periphery where the P1A signal is weaker. We conclude that the P1A protein is bound to membranes of the secretory pathway, at a concentration which goes increasing from the endoplasmic reticulum to secretion vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

The tum- antigens P91A and P198 derive from proteins located in the cytosolic compartment of cells.

To characterize the proteins P91Ap and P198p, of which mutants generate the tum- antigens P91A and P198, respectively, rabbit antisera were raised with ovalbumin-coupled synthetic peptides that correspond to their respective C terminus. In immunoadsorption tests using immobilized protein A the antisera recognized the translation products synthesized by rabbit reticulocyte lysates programmed with the SP6 polymerase transcripts of the P91A and P198 cDNA. The presence of the two proteins was demonstrated by SDS-PAGE and immunoblotting in all the mouse cells and organs examined. P91Ap is a constituent of the cytosol; despite a remarkable homology to the Drosophila diphenol oxidase DOX-A2, it separates from murine catechol oxidase activity in rate zonal sedimentation analysis. P198p is a ribosomal constituent, or a factor firmly linked to both the free and membrane-bound ribosomes. These subcellular localizations strengthen other evidence that the antigens presented to T lymphocytes by class I products of the major histocompatibility complex derive from proteins of the cytosol, or in direct contact with it.

A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E.

We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.

Identification of tumour rejection antigens recognized by T lymphocytes.

On the basis of the results reviewed here, there are two major mechanisms whereby tumour rejection antigens may arise. The first mechanism is mutational. Point mutations occurring in a large variety of genes may produce new antigenic peptides, either by providing them with the ability to bind to MHC class I molecules or by providing them with a new epitope (Fig. 2). The second mechanism is the activation of a gene that is silent in normal tissues and for which no strong natural tolerance has been established. Plausible candidates for the mutational mechanism are the "tumour specific transplantation antigens" observed on methylcholanthrene induced tumours and tumours induced by ultraviolet light. The diversity of these antigens appears to be very large, like that of the tum- antigens. Moreover, these tumours have been obtained with high doses of carcinogens, which are proven mutagens. On the other hand, a P815 tumour rejection antigen appears to arise through the activation of a silent gene, and it may turn out that this is the rule for most tumour rejection antigens. It is our hope that other genes coding for mouse and human tumour rejection antigens will soon be identified, so that it will become clear whether the activational mechanism is the rule or the exception. In our view, this is a crucial issue. Insofar as tumour rejection antigens result from mutations, they may be highly specific for every individual tumour. The tumour specific nature of these antigens would then be easily ascertained. However, active immunization of cancer patients would require that a tumour cell line be obtained from each patient, a most unpractical prospect. If, on the other hand, production of tumour rejection antigens results from the activation of a normal gene, then there is a good probability that the same gene may be activated in many different tumours, being perhaps preferentially shared by tumours of the same histological type. This would probably not result in the expression of the same antigen in all these tumours, because the patients would differ in their presenting molecules, which are determined by their HLA haplotype. However, a subset of the tumours expressing the same "tumour rejection" gene should share the same class I restricting element, so that all of these patients could be immunized with a cell that would express the gene and carry the appropriate HLA molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

The human liver growth hormone receptor.

Human livers, obtained from donors at the time of transplant, were homogenized in 0.25 M sucrose and fractionated by differential centrifugation. The specific binding of [125I] human (h) GH to total particulate fractions from 18 livers varied from 0.4-5.1% of the total radioactivity/100 micrograms protein. Binding affinity was 2.0 +/- 0.3 X 10(9) M-1, and binding capacity ranged from 14-53 fmol/mg protein. A different proportion of receptors occupied by endogenous hGH did not explain the large variation in binding. Binding sites were specific for hGH. Dissociation of the hormone-receptor complex was extremely slow. No specific binding of [125I]hPRL was observed. Specific binding of insulin was found in fractions from all livers and varied less than hGH binding. Cross-linking of [125I]hGH to plasma membrane and microsome receptors yielded two major autoradiographic bands corresponding to an estimated mol wt of 103,000 for the receptor, with a possible subunit of 54,000. Human liver primary fractions were characterized. The binding of hGH and insulin displayed a nucleo-microsomal distribution pattern in the primary fractions; 54.2% and 27.9% of the hGH-binding activity were found in the microsomes and the nuclear fraction, respectively, whereas insulin binds equally to nuclear and microsomal elements. Our findings suggest that hGH-binding sites are present in the plasma membrane and also in one or more intracellular compartments, whereas a high proportion of insulin receptors is associated with the plasma membrane.

Subcellular fractionation of epithelial cells from toad urinary bladder. 1. Assay of marker enzymes and differential centrifugation.

We have undertaken the analytical fractionation of epithelial cells from toad urinary bladder, a tissue extensively used to study epithelial transport of ions and water. In an attempt to establish markers for the main subcellular organelles, a number of enzymes were assayed in cell homogenates. The nearly ubiquitous plasma membrane marker 5'-nucleotidase, and the transferases that donate N-acetylglucosaminyl, galactosyl, and sialyl residues to glycoproteins and glycolipids in the Golgi complex were not detectable. Glucose-6-phosphatase activity was low in relation to that of nonspecific phosphatases and, therefore, not suitable for identifying the endoplasmic reticulum. Like the cytosolic enzyme lactate, dehydrogenase, catalase was essentially found in the high-speed supernatant, with a noteworthy part of aminopeptidase (substrate, leucyl-beta-naphthylamide) and NAD glycohydrolase. Other enzymes, including cytochrome c oxidase, acid phosphatase, acid N-acetyl-beta-glucosaminidase, alkaline phosphatase, alkaline phosphodiesterase I, nucleoside diphosphatase (substrate ADP), oligomycin-resistant Mg++-ATPase, and mannosyltransferase (acceptor, dolichylphosphate) were fairly active and largely sedimentable. After differential centrifugation, cytochrome oxidase, acid phosphatase, and acid N-acetyl-beta-glucosaminidase were typically associated with the large granule fraction, whereas the other sedimentable enzymes exhibited a broad distribution profile overlapping the nuclear, large granule, and microsome fractions. Their behavior in density equilibrium centrifugation is examined in a companion paper.

Translocation and proteolytic processing of nascent secretory polypeptide chains: two functions associated with the ribosomal domain of the endoplasmic reticulum.

Rat liver microsomes were subfractionated by isopycnic centrifugation in sucrose gradient. The subfractions were assayed for translocation and proteolytic processing of nascent polypeptides in a rabbit reticulocyte lysate programmed with total RNA from human term placenta. The distribution of the translocation and processing of prelactogen through the gradient correlated with that of the microsomal RNA (ribosomes). Microsomes became inactive upon incubation with elastase, but the proteolyzed membranes recovered their activity by recombination with the soluble and active fragment of the docking protein (SRP-receptor) from dog pancreas. When this fragment was combined with the gradient subfractions, or with the subfractions inactivated by incubation with elastase, the density profile of the translocation activity remained similar to that of RNA. Thus, its distribution cannot be accounted for merely by that of the docking protein; another membrane constituent, still unidentified, is both necessary for translocation of polypeptides and restricted to the rough portions of the endosplamic reticulum. Signal peptidase was assayed in the absence of protein synthesis, by use of preformed prelactogen and detergent-disrupted microsomes. Its density distribution was also similar to that of RNA. Several components of the endosplamic reticulum now appear to be segregated within restricted areas on either side of the membrane, and to make up a biochemically distinct domain. We propose to call it the ribosomal domain in consideration of its contribution to protein biosynthesis by bound ribosomes. This domain probably accounts for a greater part of the membrane area at the cytoplasmic than at the luminal surface, as postulated earlier to explain how enzymes of the cytoplasmic surface are relatively less abundant in the rough microsomes than those of the luminal surface [Amar-Costesec A. & Beaufay H. (1981) J. Theor. Biol. 89, 217-230].

[Endoplasmic reticulum: anatomy of a biologic membrane]. / Réticulum endoplasmique: anatomie d'une membrane biologique.

Endoplasmic reticulum (ER) is a large membranous network containing a wide variety of lipid and protein constituents which play important roles in cellular physiology. In this review, selection of experimental results are presented which have shaped our concepts of the molecular organization of ER. The morphological approach--electron microscope examination of ultra-thin sections of a variety of cells--led to the dualistic distinction between rough ER and smooth ER. Consequently, various attempts were made to separate the 2 entities and to demonstrate that they are endowed with distinct functional properties. Studies on the biogenesis of ER showed that enzymes associated with this organelle turn over independently, which was interpreted in terms of ER being biochemically organized as a mosaic. The results of isopycnic centrifugation of rat liver microsomes led us to conclude that the ER is comprised of three biochemically distinct domains and that the distribution of integral proteins in the lateral plane of the membrane (lateral topography) is primarily determined by their transmembrane disposition. Data on the transverse topography and the mode of biogenesis of ER enzymes are confronted with the predictions of this model.

Subcellular topology of rat liver methionyl-, leucyl-, and arginyl-tRNA synthetases.

We have investigated the distribution of methionyl-, leucyl-, and arginyl- tRNA synthetases in primary liver fractions obtained by differential centrifugation of homogenates in isotonic sucrose: 78-93% of synthetase activities are recovered in the cytosolic fraction. Microsomes contain only 4.8%, 19.4%, and 6.4% of the methionyl-, leucyl-, and arginyl-tRNA synthetases activities, respectively. This proportion increases up to 11.3%, 26.1%, and 20.7%, respectively, when the homogenization medium is supplemented with 5 mM Mg2+ and 25 mM K+. The presence of protease inhibitors in the homogenization medium does not increase the proportion of synthetases recovered in microsomes. After subfractionation of microsomes by isopycnic centrifugation, the distributions of the 3 synthetases display a second peak overlapping that of at a density of 1.12. In addition, methionyl- and leucyl- tRNA synthetases display a second peak overlapping that of RNA. This suggests that a small proportion of these synthetases (0.7% and 5.71% of total activities, respectively) bind to the d domain of the ER. The Golgi complex, the plasma membranes, and the peroxisomes lack aminoacyl-tRNA synthetase activity. The 3 synthetases are readily detached from membranes when intact microsomes are washed with 250 mM sucrose alone or containing 5 mM PPi, or 320 mM KCl. The binding of methionyl-tRNA synthetases to microsomes was measured in vitro, at 4 degrees C, with a sample of the cytosolic fraction as a source of synthetase. Microsomes stripped of their bound polysomes display a binding capacity that is not significantly different from that of unstripped microsomes. Even in the presence of cations, the amount of synthetase bound to the membranes remained low by comparison with the cytosolic content.

No evidence for a defect in growth hormone binding to liver membranes in thalassemia major.

To test the hypothesis of a defect in GH-receptor interaction, which could explain the growth failure of thalassemic children, the binding of [125I]human (h) GH to membrane fractions prepared from liver biopsies was studied. Small amounts of liver were obtained from 6 girls and 11 boys with homozygous beta-thalassemia, aged 3-15 yr, all prepubertal, at the time of splenectomy. Specific binding of [125I]hGH ranged from 0.37-5.11% of the added radioactivity/100 micrograms liver membrane protein, with variations in both receptor number and binding affinity. This 14-fold variation in hGH binding to liver membranes of thalassemic children was comparable to that in membrane fractions of livers obtained from normal donors at the time of liver transplant. The binding of insulin to liver membranes from the thalassemic patients ranged from 9.8-17.9% of the added radioactivity/100 micrograms membrane protein and from 2.8-15.0%/100 micrograms membrane protein in the normal donors. Insulin and GH binding to liver membranes did not vary in a consistent way. A 3-fold difference was found in 5'-nucleotidase activity of the membrane fractions. Histological hepatic modifications were assessed with respect to siderosis and fibrosis. No correlation was found between these parameters and GH binding. These results suggest that possible membrane alterations are not the only reason for the variations in hGH binding. All patients had retarded growth, and all but 2 had low plasma insulin-like growth factor I levels. No relationship was found between the level of GH binding to liver membranes and the growth failure. Thus, a defect in GH binding to liver membranes is probably not the cause of the growth retardation of thalassemic children.

Subcellular fractionation of epithelial cells from toad urinary bladder. 2. Isopycnic centrifugation and effect of density perturbants.

Cytoplasmic granules obtained from toad urinary bladder epithelial cells were brought to buoyancy in a linear sucrose gradient. The gradient was loaded either with untreated cytoplasmic granules, or with granules treated with Na pyrophosphate (PPi), with digitonin, or with PPi and digitonin in succession. The following enzymes were assayed in the gradient subfractions: oligomycin-insensitive Mg++-ATPase, alkaline phosphodiesterase I, alkaline phosphatase, acid N-acetyl-beta-glucosaminidase, cytochrome oxidase, nucleoside diphosphatase (substrate, ADP), aminopeptidase (substrate, leucyl-beta-naphthylamide), and mannosyltransferase (acceptor, dolichylphosphate). Comparison of the density distributions of enzymes in untreated and treated preparations led to the characterization of 4 distinct subcellular entities. In agreement with the properties of mitochondria from other cell types, cytochrome oxidase buoys at 1.18 within a narrow density range and its behavior is not significantly altered by PPi or digitonin. Under all conditions, acid N-acetyl-beta-glucosaminidase is recovered over a broad density range in the lower part of the gradient and appears as a qualified lysosomal marker. Mg++-ATPase, alkaline phosphodiesterase I, and alkaline phosphatase belong to a group with the distinguishing features of a low equilibrium density in native cytoplasmic granules and a marked shift (+0.03 density units) after digitonin treatment. Such properties are typical of the plasma membranes. Part of the aminopeptidase activity probably also belongs to plasma membrane-derived elements. Minor differences between alkaline phosphatase and the other 2 members of that group make it possible that their distribution domains in the membrane do not overlap or coincide.(ABSTRACT TRUNCATED AT 250 WORDS)

A membrane preparation that contains proteins characteristic of the rough endoplasmic reticulum.

We describe a procedure for disassembling rat liver rough microsomes, which allows the purification of the rough endoplasmic reticulum (ER) membrane. Membrane-bound ribosomes and adsorbed proteins are first detached by washing rough microsomes with 5 mM Na-pyrophosphate. In a second step, the vesicle membrane is opened by digitonin, with concomitant release of the luminal content. The purification is monitored at each step by electron microscopy, and by assaying chemical constituents (protein, phospholipid, RNA) and marker enzymes for the main subcellular organelles. The final membrane preparation is representative of the ER, since it contains 24.1% of the liver glucose 6-phosphatase with a relative specific activity of 14.2. Contaminants represent less than 5% of its protein content. SDS-polyacrylamide gel electrophoresis, followed by immunoblot analysis, reveals that the ribophorins I and II, two established markers of the rough (d) domain are still present in the final membrane preparation. It also contains the docking protein (or signal recognition particle receptor) and protein disulfide isomerase, and has conserved the functional capacity to remove co- and post-translationally the signal peptide of pre-secretory proteins. The membrane preparation is suitable for studies on the polypeptide composition of the d domain.

Analytical study of microsomes and isolated subcellular membranes from rat liver. IX. Nicotinamide adenine dinucleotide glycohydrolase: a plasma membrane enzyme prominently found in Kupffer cells.

The distribution of nicotinamide adenine dinucleotide (NAD) glycohydrolase in rat liver was investigated by subcellular fractionation and by isolation of hepatocytes and sinusoidal cells. The behavior of NAD glycohydrolase in subcellular fractionation was peculiar because, although the enzyme was mainly microsomal, plasma membrane preparations contained distinctly more NAD glycohydrolase than could be accounted for by their content in elements derived from the endoplasmic reticulum or the Golgi complex identified by glucose-6-phosphatase and galactosyltransferase, respectively. When microsomal and plasmalemmal preparations were brought to equilibrium in a linear-density gradient, NAD glycohydrolase differed from these enzymes and behaved like 5'-nucleotidase and alkaline phosphodiesterase I. NAD glycohydrolase was markedly displaced towards higher densities after treatment with digitonin. This behavior in density-gradient centrifugation strongly suggests that NAD glycohydrolase is an exclusive enzyme of the plasma membrane. NAD glycohydrolase differed clearly from other plasmalemmal enzymes when the liver was fractionated into hepatocytes and sinusoidal cells; its specific activity was considerably greater in sinusoidal cell than in hepatocyte preparations. Further subfractionation of sinusoidal cell preparations into endothelial and Kupffer cells by counterflow elutriation showed that NAD glycohydrolase is more active in Kupffer cells. We estimate that the specific activity of NAD glycohydrolase activity is at least 65-fold higher at the periphery of Kupffer cells than at the periphery of hepatocytes. As the enzyme shows not structure-linked latency and is an exclusive constituent of the plasma membranes, we conclude that it is an ectoenzyme that cannot lead to a rapid turnover of the cytosolic pyridine nucleotides.

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. II. Rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes.

Ribophorins I and II, two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum (ER) are thought to be part of the translocation apparatus for proteins made on membrane bound polysomes. To study the stoichiometry between ribophorins and membrane-bound ribosomes we have determined the RNA and ribophorin content in rat liver microsomes or in microsomal subfractions of different density (i.e., ribosome content). The specificity of antibodies against the ribophorins was demonstrated by Western blot analysis of rat liver rough microsomes separated by 2-dimensional gel electrophoresis. The ribophorin content of microsomal subfractions was determined by indirect immunoprecipitation and for ribophorin I by a radioimmune assay. In the latter assay a molar ratio of ribophorin I/ribosomes approaching one was calculated for total microsomes as well as in the gradient subfractions. We therefore suggest that ribophorins mediate the binding of ribosomes to endoplasmic reticulum membranes or play a role in co-translational process which depend on this binding, such as the insertion of nascent polypeptides into the membrane or their transfer into the cisternal lumen.

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions.

A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle.

Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver.

To establish on a quantitative basis the subcellular distribution of the enzymes that glycosylate dolichyl phosphate in rat liver, preliminary kinetic studies on the transfer of mannose, glucose, and N-acetylglucosamine-1-phosphate from the respective (14)C- labeled nucleotide sugars to exogenous dolichyl phosphate were conducted in liver microsomes. Mannosyltransferase, glucosyltransferase, and, to a lesser extent, N- acetylglucosamine-phosphotransferase were found to be very unstable at 37 degrees C in the presence of Triton X-100, which was nevertheless required to disperse the membranes and the lipid acceptor in the aqueous reaction medium. The enzymes became fairly stable in the range of 10-17 degrees C and the reactions then proceeded at a constant velocity for at least 15 min. Conditions under which the reaction products are formed in amount proportional to that of microsomes added are described. For N- acetylglucosaminephosphotransferase it was necessary to supplement the incubation medium with microsomal lipids. Subsequently, liver homogenates were fractionated by differential centrifugation, and the microsome fraction, which contained the bulk of the enzymes glycosylating dolichyl phosphate, was analyzed by isopycnic centrifugation in a sucrose gradient without any previous treatment, or after addition of digitonin. The centrifugation behavior of these enzymes was compared to that of a number of reference enzymes for the endoplasmic reticulum, the golgi complex, the plasma membranes, and mitochondria. It was very simily to that of enzymes of the endoplasmic reticulum, especially glucose-6-phosphatase. Subcellular preparations enriched in golgi complex elements, plasma membranes, outer membranes of mitochondira, or mitoplasts showed for the transferases acting on dolichyl phosphate relative activities similar to that of glucose- 6-phosphatase. It is concluded that glycosylations of dolichyl phosphate into mannose, glucose, and N-acetylglucosamine-1-phosphate derivatives is restricted to the endoplasmic reticulum in liver cells, and that the enzymes involved are similarly active in the smooth and in the rough elements.

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes.

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.