An improved assay for steroid glucuronyltransferase in rat liver microsomes

A simple and economical method of assaying rat liver microsomal estrone and testosterone glucuronyltransferase activity has been developed. Liver microsomes were activated by pretreatment with Lubrol WX. The incubation was carried out at 37°C for 30 min and contained 30–600 μm steroid, 1–2 mm UDP-glucuronic acid, 10 mm MgCl₂, and 80–150 μg of microsomal protein. Enzyme activities showed a maximum at pH 8.8 with Tris-HCl buffer. After incubation the unreacted substrate was quantitatively removed by a single extraction with dichloromethane. The glucuronide was estimated by counting an aliquot of the aqueous phase in a liquid scintillation counter. The variation coefficients with estrone and testosterone as substrates were 6.0 and 4.0%, respectively.     

Hepatic microsomal glucuronidation of bilirubin in unilamellar liposomal membranes. Implications for intracellular transport of lipophilic substrates

It has been assumed that following hepatic uptake, bilirubin is bound exclusively to cytosolic proteins prior to conjugation by microsomal UDP-glucuronyl-transferase. Since bilirubin partitions into lipid rather than the aqueous phase at neutral pH, we postulated that bilirubin reaches the sites of glucuronidation by rapid diffusion within membranes. To examine this hypothesis, [14C]bilirubin was incorporated into the membrane bilayer of small unilamellar liposomes of egg phosphatidylcholine. Radiochemical assay of this membrane-bound substrate in a physiologic concentration, using native rat liver microsomes, demonstrated immediate formation of bilirubin glucuronides at a more rapid initial velocity than for bilirubin bound to the high-affinity sites of purified cytosolic binding proteins, i.e. glutathione S-transferases (p less than 0.025) or native liver cytosol (p less than 0.05). Kinetic analysis suggested that the mechanisms of substrate transfer from liposomal membranes and from purified glutathione S-transferases to microsomal UDP-glucuronyltransferase were similar. The exchange of 3H- and 14C-labeled bilirubin substrate between binding proteins and liposomal membranes was then investigated using Sepharose 4B chromatography. As the concentration of bilirubin was increased relative to that of protein, net transfer of substrate from the protein to the membrane pool was observed. These findings indicate that bilirubin is efficiently transported by membrane-membrane transfer to hepatic microsomes, where it undergoes rapid conjugation. Bilirubin entering hepatocytes may partition between membrane and cytosolic protein pools, but as intracellular bilirubin concentration increases, the membrane pool is likely to provide a greater proportion of the substrate for glucuronidation.     

Binding of products originating from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomal membrane.

The binding of products derived from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomes was studied. To this end arachidonic acid labelled with tritium at the positions of the double bonds was given to rats and allowed to incorporate into the membrane lipids of the liver cell. When liver microsomes containing labelled arachidonic acid were incubated aerobically in the NADPH-dependent system, a marked production of malonic dialdehyde (MDA) occurred and, concomitantly, there was a consistent release of radioactivity from the microsomes into the incubation medium. The addition of EDTA to the incubation medium prevented, to a large extent, both the MDA formation and the release of radioactivity. Chromatographic studies showed that the bulk of the radioactivity released from the incubated microsomes is not MDA. In the incubated microsomes, the radioactivity decreased in total lipids, while it increased by about 15 times in the non-lipoidal residue. A similar increase in radioactivity was seen in microsomal protein, while no increase was observed in microsomal RNA (the radioactivity was negligible in both the incubated and the non-incubated samples). It seems therefore that products originating from lipoperoxidation of arachidonic acid covalently bind to the microsomal protein. In order to investigate whether alterations similar to those observed in the in vitro peroxidation of liver microsomes could be detected in the in vivo intoxication with carbon tetrachloride, rats given labelled arachidonic acid as above, were poisoned with CCl4. Sixty minutes after poisoning, the radioactivity present in the microsomal lipids was generally lower in the intoxicated rats than in the controls, while the labelling of the non-lipoidal residue and of the protein was higher in the CCl4-poisoned rats.     

Characteristics of renal UDP-glucuronyltransferase

Rat renal microsomes catalyzed the glucuronidation of l-naphthol, 4-methylumbelliferone and p-nitrophenol, whereas morphine and testosterone conjugation were not detected. In contrast, all five substrates were conjugated by hepatic microsomes; the activity was typically 5-10 times greater than with renal microsomes. Renal microsomal UDP-glucuronyltransferase toward l-naphthol was fully activated (six-fold) by 0.03% deoxycholate while the hepatic enzyme was fully activated (eight-fold) by 0.05% deoxycholate. Full activation of hepatic UDP-glucuronyltransferase occurred when microsomes had been preincubated at 0 C with deoxycholate for 20 min. This effect of preincubation was not observed with renal microsomes. The presence of 0.25M sucrose in the buffers during renal microsomal preparation resulted in a two-fold greater rate of l-naphthol conjugation in both unactivated and activated microsomes than renal microsomes prepared in phosphate buffers alone. Preparation of hepatic microsomes with or without 0.25M sucrose had no effect on UDP-glucuronyltransferase activity. Unactivated (-deoxycholate) renal enzyme was activated when incubations were done at a low pH (5.7), whereas fully activated (0.03% deoxycholate) renal microsomal UDP-glucuronyltransferase displayed a pH optimum at 6.5. Renal microsomal UDP-glucuronyltransferase activity toward l-naphthol, p-nitrophenol and 4-methylumbelliferone was induced by pretreatment of rats with beta-naphthoflavone and trans-stilbene oxide but not by phenobarbital or 3-methylcholanthrene. These data demonstrate that renal UDP-glucuronyltransferases are different from the hepatic enzymes with regard to biochemical properties, substrate specificity and in response to chemical inducers of xenobiotic metabolism.     

Formation of omega- and (omega-1)-hydroxyfatty acids and plausible formation of ketofatty acid from microsomal phospholipids

Rat liver microsomes labeled with spin-labeled phosphatidylcholine release the label into the aqueous phase during the aerobic incubation with NADPH (Biochem. Biophys. Res. Commun. (1979) 87, 300-307). To establish the chemical nature of the released moiety, microsomes were labeled with [14C]phosphatidylcholine. When the 14C-labeled microsomes were incubated with NADPH under aerobic conditions, a few percent of the radioactivity was liberated into the aqueous phase within 60 min. Thin-layer chromatographic analysis of the radioactive substance liberated showed the presence of hydroxylated fatty acids derived from the 2-position of glycerol moiety. About one-third of the fatty acids formed from [14C]phosphatidylcholine during the incubation were converted into hydroxy-derivatives. Gas chromatography/mass spectrometry analysis further confirmed an NADPH-dependent formation of 16-hydroxypalmitic acid, 15-hydroxypalmitic acid, and hydroxy-derivatives of other fatty acids from the phospholipids of the microsomal membrane. Evidence was also obtained indicating the formation of ketopalmitic acid.     

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.     

In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems

The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.     

An NADPH-induced change in lipid bilayer of rat liver microsomes as observed by spin-labeled phosphatidylcholine.

Lipid bilayer of rat liver microsomes was spin-labeled by incubating with liposomes of 1-acyl 2-(12-doxylstearoyl) glycero-3-phosphorylcholine. When NADPH was added to the labeled microsomes, there appeared a rapidly tumbling component of spin label in the EPR spectrum. NADH was less effective than NADPH. The appearance of the sharp signal was prevented under anaerobic conditions or in the presence of either carbon monoxide, phenyl isocyanide or cytochrome $\text{c}$. The appearance of the rapidly tumbling component in the EPR spectrum was found to be due to the release of spin moiety from the membrane into the aqueous phase. That the release was associated with superoxide anion formation or with lipid peroxidation is unlikely, since 1) superoxide dismutase had little effect, 2) addition of either α-tocopherol or EDTA did not inhibit the release. These observations suggest that electron transfer from NADPH to oxygen $\text{via}$ cytochrome P-450 system induces a physical perturbation in the lipid bilayer resulting in the release of its component into the aqueous phase.     

Functional Changes of Rat Brain Microsomal Membrane Surface After Learning Task Depending on Dietary Fatty Acids

Abstract: Biochemical characteristics of brain microsomal membranes were examined before and after the brightness-discrimination learning tasks in rats that were fed either safflower oil (α-linolenate-deficient) or perilla oil (α-linolenate-sufficient) diets. We detected small changes in the chain elongation system for polyunsaturated fatty acids in microsomes, whereas no significant difference was detected in the inositol trisphosphate-induced calcium release and ATP-induced calcium uptake profiles of microsomes between the two dietary groups. The calcium ion-induced aggregation rate of microsomes was determined in both groups. We found that the aggregation rate of microsomes in the safflower oil group was significantly greater than that in the perilla oil group. The difference in susceptibility of microsomal membrane phospholipids to phospholipase A₂ between the groups was obvious, and the amount of released fatty acids by phospholipase A₂ from the perilla oil group microsomes was nearly half of that from the safflower oil group microsomes after the learning task. Susceptibility of sialic acids on the brain microsomal membranes to exogenous sialidase was different only after the learning task in the safflower and perilla oil groups. These results suggest that the biochemical characteristics of membrane surfaces of brain microsomes are affected significantly by the learning task itself in a dietary oil-dependent manner.     

Functional and structural membrane topology of rat liver microsomal glutathione transferase

The membrane topology of rat liver microsomal glutathione transferase was investigated by comparing the tryptic cleavage products from intact and permeabilized microsomes. It was shown that lysine-4 of microsomal glutathione transferase is accessible at the luminal surface of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids (positions 11–35) which comprises the likely membrane-spanning region. Reaction of cysteine-49 of the microsomal glutathione transferase with the charged sulfhydryl reagent DTNB (2,2′-dithiobis(5-nitrobenzoic acid))) in intact microsomes further supports the cytosolic localization of this portion of the polypeptide chain. The role of two other potential membrane-spanning/associated segments in the C-terminal half of the polypeptide chain was examined by investigating the association of the protein to the membrane after trypsin cleavage at lysine-41. Activity measurements and Western blot analysis after washing with high concentrations of salt, as well as after phase separation in Triton X-114, indicate that this portion of the protein also binds to the membrane. It is also shown that cleavage of the purified protein at Lys-41 and subsequent separation of the fragments obtained yields a functional C-terminal polypeptide with the expected length for the product encompassing positions 42–154. The location of the active site of microsomal glutathione transferase was investigated using radiolabelled glutathione together with a second substrate. Since isolated rat liver microsomes do not take up glutathione or release the glutathione conjugate into the lumen, it can be concluded that the active site of rat liver microsomal glutathione transferase faces the cytosolic side of the endoplasmic reticulum.     

Failure of expression of the phenobarbital-induced enhancement of UDP-glycosyltransferases in native, sealed endoplasmic reticulum vesicles from rat liver.

Conflicting data have been published regarding the effects of phenobarbital treatment on bilirubin UDP-glucuronyltransferase activity in native liver microsomes. Recent evidence suggests that the bilirubin UDP-glycosyltransferase system faces the interior of microsomal vesicles, and that expression of its activities in sealed microsomes may be rate-limited by transport of UDP sugars across the membrane. These observations raise the possibility that the reported variability in the effects of phenobarbital may reflect differences in integrity of the membrane in microsomal preparations. We examined the effect of phenobarbital on bilirubin UDP-glucosyltransferase and the UDP-glucuronyltransferase activities towards bilirubin, 4-nitrophenol, and 1-naphthol using native rat liver microsomes with verified vesicle integrity. Phenobarbital-induced microsomes in which the membrane permeability barrier was eliminated by pretreatment with detergent displayed markedly higher UDP-glycosyltransferase activities towards all tested substrates compared with activities in similarly disrupted microsomes from untreated rats. In contrast, none of the transferase activities tested were significantly enhanced by phenobarbital treatment when the enzymic activities were assayed in sealed microsomes. Addition to the enzyme assay mixture of UDPGlcNAc, a presumed physiological activator of the UDP-glucuronyltransferases, failed to expose the enhanced UDP-glucuronyltransferase concentration in phenobarbital-induced sealed microsomes. Our findings are consistent with the idea that transport of UDP sugar across the membrane may be rate-limiting for expression of UDP-glycosyltransferase activities in sealed microsomes. Quantitative assessment of membrane integrity is an essential prerequisite in experiments designed to study the regulation of the microsomal UDP-glycosyltransferase system.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

Characterization of antibodies to a rabbit hepatic UDP-glucuronosyltransferase and the identification of an immunologically similar enzyme in human liver

An antibody to a UDP-glucuronosyltransferase (UDPGT) isoenzyme which catalyzes the glucuronidation of p-nitrophenol (PNP) in rabbit liver was raised in sheep and used to identify immunologically similar UDPGTs in rabbit and human livers. Immunoblotting experiments showed that the antisera specifically recognized PNP UDPGT but not estrone UDPGT purified from rabbit liver. Sheep anti-rabbit liver PNP UDPGT IgG immunoprecipitated PNP, 1-naphthol, and 4-methylumbelliferone glucuronidation activities in rabbit and human liver microsomal preparations. In rabbit liver microsomes the antibody did not immunoprecipitate estrone or estradiol glucuronidation activities. In human liver microsomes, 4-aminobiphenyl but not estriol glucuronidation activities were immunoprecipitated, suggesting that the antibody recognizes a specific UDPGT (pI 6.2) in human liver microsomes.     

Activation of microsomal glutathione transferase activity by reactive intermediates formed during the metabolism of phenol

The activity of microsomal glutathione transferase was increased 1.7-fold in rat liver microsomes which carried out NADPH dependent metabolism of phenol. Known phenol metabolites were therefore tested for their ability to activate the microsomal glutathione transferase. The phenol metabolites benzoquinone and 1,2,4-benzenetriol both activated the glutathione transferase in microsomes 2-fold independently of added NADPH. However, NADPH was required to activate the enzyme in the presence of hydroquinone. Catechol did not activate the enzyme in microsomes. The purified enzyme was activated 6-fold and 8-fold by 5 mM benzenetriol and benzoquinone respectively. Phenol, catechol or hydroquinone had no effect on the purified enzyme. When microsomal proteins that had metabolized [14C]phenol were examined by SDS polyacrylamide gel electrophoresis and fluorography it was found that metabolites had bound covalently to a protein which comigrated with the microsomal glutathione transferase enzyme. We therefore suggest that reactive metabolites of phenol activate the enzyme by covalent modification. It is discussed whether the binding and activation has general implications in the regulation of microsomal glutathione transferase and, since some reactive metabolites might be substrates for the enzyme, their elimination through conjugation.     

Glucuronidation of 3'-azido-3'-deoxythymidine in human liver microsomes: enzyme inhibition by drugs and steroid hormones.

The molecular form of UDP-glucuronosyltransferase involved in the catalysis of 3'-azido-3'-deoxythymidine (AZT)-5'-O-glucuronide was characterized in human liver microsomes. The specific activity (1.3 nmol/min per mg protein) in transplantable liver was more than 2-times higher than in post-mortem fragments. Liver microsomes from patients suffering Crigler-Najjar syndrome, who are genetically deficient in bilirubin UDP-glucuronosyltransferase, could also glucuronidate AZT to a similar extent, thus indicating that this protein was not involved in that process. A genetically engineered V79 cell line stably expressing a cDNA which encodes a human isozyme active towards 1-naphthol was unable to glucuronidate AZT. Clinically used drugs, most of them being glucuronidated, were tested as potential inhibitors of the glucuronidation of AZT in human liver microsomes. The drugs chemically related to 2-phenylpropionic acid, naproxen and flurbiprofen, and the steroid compounds testosterone, estrone and ethynylestradiol strongly inhibited AZT glucuronidation. Codeine and morphine also decreased the reaction rate although to a lower extent. Except estrone which elicited a partial competitive inhibition, ethynylestradiol, flurbiprofen naproxen and testosterone could competitively inhibit AZT glucuronidation with an apparent Ki of 38, 50, 172 and 250 microM, respectively. The results suggest that these drugs were substrates of the same isozyme(s) involved in AZT glucuronidation. Probenecid was a weak inhibitor of the reaction (Ki 900 microM), only when non-disrupted microsomes were used. This drug may compete with the anion carrier system involved in the microsomal uptake of UDP-glucuronic acid.     

Properties of the aromatase system associated with the mitochondrial fraction of human placenta

The aromatase system associated with the mitochondrial fraction of human term placenta, present at 35–50% the specific activity of the microsomal enzyme, is substantially the same as the microsomal enzyme as determined by the following: 1) The rate of aromatization of androstenedione, 19-nortestosterone, and 16α-hydroxytestosterone in mitochondria was a nearly constant proportion of the microsomal rate; 2) Sensitivity to carbon monoxide was the same; 3) The magnitude of cytochrome P-450 Type I spectral interactions with androgen substrates was a constant proportion in mitochondria and microsomes; 4) Sensitivity to an antibody raised against hepatic microsomal NADPH-cytochrome c reductase was the same. When inner and outer mitochondrial membrane subfractions were prepared, the predominant aromatase activity was associated with the outer membrane preparation. This aromatase activity could not be accounted for by microsomal contamination as determined by inosine diphosphatase activity, a microsomal marker. After correction, the rate of aromatization in the outer membrane preparation was almost six times that in the inner membranes and three times that of the whole mitochondrial fraction     

The stimulation by sodium fluoride of plasma-membrane Ca2+ inflow in isolated hepatocytes. Evidence that a GTP-binding regulatory protein is involved in the hormonal stimulation of Ca2+ inflow.

Bilirubin may be transported within intracellular membranes of the hepatocyte and may undergo membrane-membrane transfer to gain access to the conjugating enzyme UDP-glucuronyltransferase in the endoplasmic reticulum. We have demonstrated previously that the lipid composition of liposomal membranes incorporating bilirubin substrate influences the rate of transfer and glucuronidation of bilirubin by hepatic microsomes. To examine the mechanism(s) of substrate transfer, we incorporated radiolabelled bilirubin into small unilamellar model membranes of egg phosphatidylcholine or natural phospholipids in the proportions present in native hepatic microsomes. The rate at which bilirubin was transferred to rat liver microsomes and glucuronidated was then examined in the presence of various endogenous compounds that promote membrane fusion. For bilirubin substrate in membranes of egg phosphatidylcholine, the addition of Ca2+ (2 mM) increased the microsomal glucuronidation rate, whereas retinol enhanced microsomal conjugation rates for bilirubin in membranes of both lipid compositions. When the transfer of [3H]bilirubin from dual-labelled liposomes to microsomes was enhanced by Ca2+ or retinol, there was no associated increase in [14C]phospholipid transfer. Thus it appears likely that bilirubin is transferred to the endoplasmic reticulum by rapid cytosolic diffusion or membrane-membrane collisions, rather than by membrane fusion; this process may be modulated by changes in the lipid microenvironment of the substrate or the effective intracellular concentrations of Ca2+ or retinol. The observation that polymyxin B induced concomitant membrane-membrane transfer of [3H]bilirubin and [14C]phospholipid suggests that under certain circumstances membrane fusion or aggregation may promote the movement of lipophilic substrates in hepatocytes.     

Vitamin K dependent carboxylase: subcellular location of the carboxylase and enzymes involved in vitamin K metabolism in rat liver

Vitamin K dependent carboxylation of an exogenous peptide substrate and endogenous protein substrates, vitamin K epoxidation, and reduction of vitamin K epoxide were measured in subcellular fractions from rat liver. The rough microsomal fraction was highly enriched in all four activities; lower levels were found in smooth microsomes. Mitochondria, nuclei, and cytosol had negligible activities. The addition of 0.2% Triton X-100 to intact microsomes resulted in a 10-20-fold stimulation in carboxylation of a peptide substrate. This marked latency suggests that the active site of the carboxylase may be accessible only from the lumen of the microsomal membrane. A lumen-facing orientation of the carboxylase was also supported by its inaccessibility to trypsin in intact microsomes contrasted with marked inhibition by trypsin in detergent-permeabilized microsomes. Vitamin K epoxidase and epoxide reductase activities were also inhibited by trypsin much more effectively in permeabilized than in intact microsomes, although some degree of exposure at the cytosolic surface was also indicated. These data suggest that carboxylation is an early event in prothrombin synthesis occurring primarily on the lumen side of the rough endoplasmic reticulum membrane. The location of the vitamin K epoxidation-reduction cycle enzymes is consistent with their possible role in the carboxylation reaction.     

Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol

The glycoproteins of microsomes and cytosol were studied. Various washing procedures did not release the proteins from the microsomes, and immunological tests demonstrated that the sialoproteins are not serum components. Low concentrations of deoxycholate and incubation in 0.25 M sucrose solution liberated a small amount of microsomal sialoprotein and this fraction exhibited a high degree of labeling of protein-bound N-acetylneuraminic acid. A part of the glycoprotein fraction could not be solubilized, even with a high concentration of the detergent. Thoroughly perfused rat liver contained sialoproteins in the particle-free supernate. The level of sialoprotein present could not be due to contamination with serum or broken organelles. The high in vivo incorporation of [3H]glucosamine into protein-bound sialic acid of Golgi membranes and cytosol was paralleled by a delayed and lesser rate of incorporation into the rough and smooth microsomal membranes. This incorporation pattern suggests the possibility that the glycoproteins of cytosol and Golgi may later be incorporated into the membrane of the endoplasmic reticulum.