Endoplasmic Reticulum Of Mammalian Cells Research Articles

Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells.

The formation of intra- and interchain disulphide bonds constitutes an integral part of the maturation of most secretory and membrane-bound proteins in the endoplasmic reticulum. Evidence indicates that members of the protein disulphide isomerase (PDI) superfamily are part of the machinery needed for proper oxidation and isomerization of disulphide bonds. Models based on in vitro studies predict that the formation of mixed disulphide bonds between oxidoreductase and substrate is intermediate in the generation of the native intrachain disulphide bond in the substrate polypeptide. Whether this is how thiol oxidoreductases work inside the endoplasmic reticulum is not clear. Nor has it been established which of the many members of the PDI superfamily interacts directly with newly synthesized substrate proteins, because transient mixed disulphides have never been observed in the mammalian endoplasmic reticulum during oxidative protein folding. Here we describe the mechanisms involved in co- and post-translational protein oxidation in vivo. We show that the endoplasmic-reticulum-resident oxidoreductases PDI and ERp57 are directly involved in disulphide oxidation and isomerization, and, together with the lectins calnexin and calreticulin, are central in glycoprotein folding in the endoplasmic reticulum of mammalian cells.

Expression of a functionally active human hepatic UDP-glucuronosyltransferase (UGT1A6) lacking the N-terminal signal sequence in the endoplasmic reticulum

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a membrane glycoprotein of the endoplasmic reticulum playing a key role in drug metabolism. It is synthesized as a precursor with an N-terminal cleavable signal peptide. We demonstrate that deletion of the signal peptide sequence does not prevent membrane targeting and integration of this human isoform when expressed in an in vitro transcription-translation system, as shown by N-glycosylation, resistance to alkaline treatment and protease protection. Furthermore, UGT1A6 lacking the signal peptide (UGT1A6Δsp) was targeted to the endoplasmic reticulum in mammalian cells as shown by immunofluorescence microscopy and was catalytically active with kinetic constants for 4-methylumbelliferone glucuronidation similar to that of the wild-type. These results provide evidence that the signal peptide is not essential for the membrane assembly and activity of UGT1A6 suggesting that additional topogenic element(s) mediate(s) this process.

Rabbit cardiac and skeletal myocytes differ in constitutive and inducible expression of the glucose-regulated protein GRP94.

The glucose-regulated protein GRP94 is a stress-inducible glycoprotein that is known to be constitutively and ubiquitously expressed in the endoplasmic reticulum of mammalian cells. From a rabbit heart cDNA library we isolated four overlapping clones coding for the rabbit homologue of GRP94 mRNA. Northern blot analysis shows that a 3200 nt mRNA species corresponding to GRP94 mRNA is detectable in several tissues and it is 5-fold more abundant in the heart than in the skeletal muscle. Hybridization analysis in situ shows that GRP94 mRNA accumulates in cardiac myocytes, whereas in skeletal muscles it is not detectable in myofibres. A monoclonal antibody raised by using a 35 kDa recombinant GRP94 polypeptide as immunogen detects a single reactive polypeptide of 94 kDa in a Western blot of liver and heart homogenates and does not react with skeletal muscle homogenates. Conversely, GRP94 mRNA and protein are detectable in both cardiac and skeletal muscle myocytes of fetal and neonatal rabbits. After 24 h of endotoxin administration to adult rabbits, GRP94 mRNA accumulation increases 3-fold in both heart and skeletal muscle and it is followed by a comparable increase in protein accumulation. However, hybridization and immunohistochemistry in situ do not reveal any change in the expression of GRP94 mRNA and protein in skeletal muscle myocytes after endotoxin treatment. Thus skeletal muscle fibres display a unique regulation of the GRP94 gene, which is up-regulated during perinatal development, whereas in the adult animal it is apparently silent and not responsive to endotoxin treatment.

A novel dynamin-like protein associates with cytoplasmic vesicles and tubules of the endoplasmic reticulum in mammalian cells.

Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.

Biochemical characterization of the Cys138Arg substitution associated with the AB variant form of GM2 gangliosidosis: evidence that Cys138 is required for the recognition of the GM2 activator/GM2 ganglioside complex by beta-hexosaminidase A.

The function of the GM2 activator protein is to act as a substrate-specific cofactor in the hydrolysis of GM2 ganglioside by beta-hexosaminidase A. Mutations in the gene encoding it result in the AB variant form of GM2 gangliosidosis. One such mutation, Cys138 Arg, results in the mutant protein being retained and degraded in the endoplasmic reticulum of mammalian cells. In order to characterize the biochemical effects of this substitution, we expressed the mutant protein in transformed bacteria. We first compared the wild-type protein produced by two bacterial expression methods, one requiring protein refolding, with activator purified from the medium of transfected CHO cells. The "activity" and circular dichroism spectrum (alpha-helical content) of all three proteins were similar, justifying the use of refolded activator from transformed bacteria in structure/function studies. Second, the mutant protein was expressed in both bacterial systems and in each retained approximately 2% of the wild type's specific activity. The presence of even this small amount of activity in the mutant protein coupled with a calculated alpha-helical content nearly identical to the wild type, strongly suggest that no major tertiary or secondary structural changes, respectively, had occurred due to the mutation. However, we demonstrate that its heat stability at 60 degrees C is reduced 14-fold, suggesting some localized change in tertiary structure. The loss of a disulfide loop was confirmed by reacting the mutant protein with Ellman's reagent. A kinetic analysis detected a large increase in the apparent K(m) of beta-hexosaminidase A for the mutant; however, there was no apparent change in Vmax. A fluorescence dequenching assay was used to evaluate the ability of the mutant protein to transport lipids and bind GM2 ganglioside. These assays detected no difference between the wild-type and mutant proteins, indicating that the Cys138 Arg substitution has no effect on these functions. We conclude that the mutation specifically affects a domain in the activator protein that is responsible for the recognition of the activator-GM2 ganglioside complex by beta-hexosaminidase A.

Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations.

Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, < 2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intra-chain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.

Similar processes mediate glycopeptide export from the endoplasmic reticulum in mammalian cells and Saccharomyces cerevisiae.

Glycopeptides are transported from the lumen of the yeast endoplasmic reticulum (ER) to the cytosol and in contrast to secretory proteins do not enter ER-to-Golgi transport vesicles. In a cell-free system, this process is ATP- and cytosol-dependent. While yeast cytosol promotes the export of glycopeptides from mammalian ER in vitro, glycopeptide release cannot be detected in the presence of mammalian cytosol. We demonstrate that this is due to an N-glycanase activity in mammalian cytosol rather than lack of glycopeptide transport activity in mammalian microsomes. Monitoring the amount of glycopeptide enclosed in ER membranes we show the cytosol- and ATP-dependent release of glycopeptide from mammalian microsomes. The fact that glycopeptide export can be achieved with ER and cytosol derived from heterologous sources indicates that glycopeptide export from the ER is an important process conserved during evolution.

Dissociation of coatomer from membranes is required for brefeldin A-induced transfer of Golgi enzymes to the endoplasmic reticulum.

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the "EAGE"-peptide of beta-COP, inhibit BFA-induced redistribution of beta-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing beta-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only approximately 50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTP(gamma)S. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

Characterization of a Fusion Protein between Human Cytochrome P450 1A1 and Rat NADPH-P450 Oxidoreductase inEscherichia coli

A cDNA of fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase was genetically engineered and expressed inEscherichia coliDH5α cells under the control of an inducibletacpromoter (Y. J. Chun, T. Shimada, and F. P. Guengerich, (1996)Arch. Biochem. Biophys.330, 48-58).E. colimembranes of transformed cells showed much higher P450 1A1-dependent monooxygenase and NADPH-P450 reductase activities than pCW control vector or P450 1A1 expression vector-transformed cells. EthoxyresorufinO-deethylase and methoxyresorufinO-demethylase were 22-fold and 11-fold higher than the control activity, respectively. α-Naphthoflavone and β-naphthoflavone strongly inhibited P450 1A1 activity of the fusion protein, with α-naphthoflavone being more potent than β-naphthoflavone. Divalent cations (e.g. Ca2+and Mg2+) increased P450 1A1 activity as well as NADPH-P450 reductase activity. These results demonstrate that this fusion protein inE. colimembrane may be a useful model for elucidating details of protein-protein interactions between P450 and NADPH-P450 reductase in the endoplasmic reticulum of mammalian cells.

Protein Folding within and Protein Transport into Mammalian Microsomes are Differentially Affected by Photoaffinity Labeling of Microsomes with 8-Azido-ATP

Transport of presecretory proteins into mammalian microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with 8-azido-ATP. Typically, protein folding within the lumen of the endoplasmic reticulum of mammalian cells depends on ATP and the member of the Hsp70 protein family, BiP. Here we addressed the question of whether protein transport into and folding within microsomes are differentially affected by photoaffinity labeling of microsomes with 8-azido-ATP. Folding of heterodimeric luciferase to the native state was more azido-ATP-sensitive compared to transport of the precursors of the two subunits. Therefore, we conclude that the microsomal protein which is responsible for the ATP-dependence of protein folding in the endoplasmic reticulum is sensitive to photoaffinity labeling with 8-azido-ATP and that this microsomal protein is distinct from the microsomal ATP-binding protein which is involved in protein transport.

The Human Immunodeficiency Virus Type 1 Vpu Protein: A Potential Regulator of Proteolysis and Protein Transport in the Mammalian Secretory Pathway

HIV-1 Vpu is a small transmembrane phosphoprotein of 16 kDa which performs critical roles in CD4 proteolysis and virus release. Previous studies have demonstrated that Vpu-induced degradation of CD4 occurs in the endoplasmic reticulum (ER), and that the proteolytic process is sequence specific requiring both the transmembrane and cytoplasmic domains of CD4. In the present study, we investigated the effects of Vpu expression on the intracellular membrane trafficking pathway of mammalian cells. In singly transfected cells, the HIV envelope glycoproteins and vesicular stomatitis virus glycoprotein (VSV G) were properly transported to the cell surface undergoing oligosaccharide modifications characteristic of their movement through the Golgi complex. In contrast, the cell surface delivery of glycoproteins was severely impeded in cells expressing Vpu. Biochemical analyses revealed that Vpu expression blocked the transfer of proteins from the ER–Golgi complex to the plasma membrane in a dose- and protein-dependent manner. Soluble gp120 exhibited extreme transport defects in the presence of Vpu, whereas transmembrane proteins (e.g., gp160, VSV) responded only moderately to wild-type Vpu. To gain insight into Vpu-mediated transport inhibition, we performed mutational analysis of the CK-2 phosphorylation sites (serines at 52 and 56) in the Vpu protein. CK-2 phosphorylation of Vpu has been shown to regulate the activity of the protein in reactions that involve the proteolysis of CD4 in the ER. We demonstrate here that the phosphorylation mutant is defective in both sequence-specific degradation of VRE-containing substrates and the transport inhibition of gp120 and VSV-G in the secretory pathway. Thus, these experiments have revealed that Vpu-mediated proteolysis and transport inhibition are mechanistically coupled requiring the same structural elements of the Vpu protein in both processes. We propose that the primary effect of Vpu expression is to impede the secretion process and then access glycoproteins bearing the VRE for Vpu-mediated proteolysis in the ER of mammalian cells.

Regulated Export of Cargo from the Endoplasmic Reticulum of Mammalian Cells

Vesicular traffic through the constitutive secretory pathway has previously been viewed as a nonselective, bulk-flow process in which transport of cargo via vesicular carriers is not subject to regulation (Pfeffer and Rothman 1987; Wieland et al. 1987). This view is now in transition with the realization that both membrane-associated (Balch et al. 1994) and soluble proteins (Mizuno and Singer 1993) are sorted and concentrated during export from the endoplasmic reticulum (ER) (Balch and Farquhar 1995). This new view raises many questions concerning the potential biochemical mechanisms mediating protein sorting and vesicle formation and their potential regulation.

Complete Golgi passage of glycotripeptides generated in the endoplasmic reticulum of mammalian cells

The tripeptide, N-octanoyl-Asn-[ 125I]Tyr-Thr-NH 2, which contains the acceptor sequence for N-glycosylation, is readily taken up by cell culture cells, glycosylated in the endoplasmic reticulum (ER), and secreted into the medium. Therefore such glycosylated tripeptides have been used as markers for the vesicular flow from the endoplasmic reticulum to the plasma membrane [(1987) Cell 50, 289–300; (1990) J. Biol. Chem. 265, 20027-20032]. We have now studied the pathway taken by the glycotripeptides in mammals in more detail. In the perfused rat liver, the glycotripeptides secreted to the medium were analyzed by digestion with exoglycosidases, and a significant fraction was found to contain the terminating sequence -Gal-Sial, which is generated by processing enzymes that reside in the late Golgi apparatus. Thus we conclude that these glycotripeptides have passed through the complete Golgi complex on their way from the ER to the cell surface.

Binding of BiP to an assembly‐defective protein in plant cells

SummaryThe binding protein (BiP) has been implicated as a mediator of protein folding and assembly in the endoplasmic reticulum of mammalian cells and has often been found in stable association with structurally defective proteins. To acquire information on the activity of BiP in plant cells, we have expressed in tobacco protoplasts the wild type form and an assembly‐defective form of bean phaseolin. Phaseolin (PHSL) is a soluble, trimeric, storage glycoprotein co‐translationally inserted into the lumen of the endoplasmic reticulum and then transported along the secretory pathway to the protein storage vacuoles. We have previously shown that a PHSL mutant in which the last 59 amino acids have been deleted (Δ363PHSL) is unable to form trimers and is retained in a pre‐Golgi compartment when synthesized in Xenopus oocytes. When transiently expressed in tobacco leaf protoplasts, wild‐type PHSL is correctly glycosylated and assembles efficiently and rapidly into trimers. Δ363PHSL is also correctly glycosylated but does not trimerize. Tobacco BiP and Δ363PHSL are co‐immunoselected using either anti‐PHSL or anti‐BiP antibodies. Under the same conditions, co‐immunoselection of BiP with wild‐type PHSL is not detectable. The BiP bound to Δ363PHSL can be released by treatment of the complex with ATP, indicating that the binding is related to the proposed function of BiP in protein folding and assembly in the endoplasmic reticulum. These data indicate that BiP stably binds structurally defective proteins in plant cells.

Molecular analysis of SAR1 -related cDNAs from a mouse pituitary cell line

Vesicular transport between the endoplasmic reticulum (ER) and the Golgi in the yeast Saccharomyces cerevisiae requires a Ras-like, small GTP-binding protein, Sarip [1–3]. Whether a functional homologue operates in export from the ER in mammalian cells is unknown, nor is it clear if transport in other branches of the secretory pathway requires member(s) of a gene family. In this study, we used a PCR approach to examine the complexity of SAR1-related sequences expressed in mammalian cells that possess multiple secretory pathways. Amplification of cDNA sequences from rodent pituitary cells with primers corresponding to two conserved GTP binding domains of Sarlp yielded several clones with sequences homologous to Sarl and/or the closely related ADP-ribosylation factor (ARF) family. Of these, only two showed closer homologies to S. cerevisiae Sar1 than members of the ARF family and are designated as mSARa and mSARb. Northern blot analysis shows that mSARa is expressed in most tissues including liver, heart, brain, skeletal muscle and kidney. In contrast, mSARb is preferentially expressed in skeletal muscle and liver. The full-length cDNA of mSARa isolated from a mouse pituitary AtT-20 cDNA library encodes a protein of 198 amino acids, and is 61.6% identical to Sarlp from S. cerevisiae. Thus in contrast to the large rab family of GTP-binding proteins, vesicular transport in mammalian cells appears to be mediated by a relatively small number of Sar1-related proteins.

Visualizing the intracellular membrane system of yeast cells using a laser scanning confocal microscope

Progress has been made in developing new preparative techniques for electron microscopic visualization of the intracellular structures of yeast. In addition, development of the laser scanning confocal microscope (LSCM) has provided improved resolution for fluorescent microscopy. We asked whether the LSCM in combination with new preparative techniques could be used for comparable investigative research of the intracellular organizaton of the yeast cell.To investigate this possibility, a BioRad MRC600 LSCM equipped with a krypton/argon laser and integrated computer imaging capabilities, was used to study various dipliod strains of the yeast Saccharomyces cerevisiae. Cells were treated with the lipophilic, cationic fluorescent dye DiOC6 (3,3’-dihexyloxacarbocyanine iodide), which has been used to visualize intracellular membrane structures, and in particular the endoplasmic reticulum of mammalian cells and living yeast cells. Since one of our interests is the intracellular localization of proteins in the yeast cell, we utilized transformed yeast cells expressing a human gene encoding a protein that inappropriately accumulates in the endoplasmic reticulum (ER).

Binding protein BiP is required for translocation of secretory proteins into the endoplasmic reticulum in Saccharomyces cerevisiae.

The endoplasmic reticulum of mammalian cells contains a heat shock protein of approximately 70 kDa (hsp70) termed binding protein BiP that is thought to promote the folding and subunit assembly of newly synthesized proteins. To study BiP function, we placed the BiP-encoding gene from Saccharomyces cerevisiae under the control of a regulated promoter and examined the effects of BiP depletion. Reduction of BiP protein to about 15% of normal levels led to a profound reduction in secretion of alpha factor and invertase. At the same time, unglycosylated precursors of these proteins accumulated intracellularly. The predominant form of the invertase precursor had undergone signal sequence cleavage but accumulated as a soluble species in the cytosol. In contrast, the alpha-factor precursor was exclusively in the signal-uncleaved form. It sedimented with microsomal membranes and was exposed at the cytoplasmic face in a protease-resistant form. These findings suggest that, in yeast, BiP function is required for translocation of soluble proteins into the endoplasmic reticulum at a stage beyond the initial nascent chain-membrane association.

Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle

Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.

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.).

Use of a discrete diode array spectrophotometer for the quantitative and qualitative analysis of cytochrome P-450

Cytochrome P-450, the hemoprotein located in the endoplasmic reticulum of mammalian cells and responsible for the metabolism of xenobiotics, was qualitatively and quantitatively analyzed using the HP-8450A diode array spectrophotometer. The diode array instrument was compared to a conventional spectrophotometer and the advantages of the diode array instrument over conventional spectrophotometry with respect to the analysis of cytrochrome P-450 were discussed.