Feedback regulation of CTP:phosphocholine cytidylyltransferase translocation between cytosol and endoplasmic reticulum by phosphatidylcholine

The mechanism for the increased association of CTP:phosphocholine cytidylyltransferase (CT) with membranes of hepatocytes derived from choline-deficient, compared with choline-supplemented rats, has been investigated. The cells were maintained in culture for 4 h in a choline- and methionine-deficient medium. (Methionine is required for synthesis of phosphatidylcholine (PC) via methylation of phosphatidylethanolamine.) Afterward, the cells were incubated +/- choline for various times up to 4 h. In the presence, but not in the absence, of choline there was a translocation of CT activity from membranes to cytosol. During this time period there was no change in the amounts of unesterified fatty acids or diacylglycerol recovered from the hepatocytes. In addition, there was no evidence for a difference in the incorporation of 32P into CT or other cytosolic proteins isolated from hepatocytes +/- choline. In contrast, there was a highly significant correlation between the concentration of PC in the membranes and the increased activity of CT in the cytosol (R = 0.98) and the decreased activity in the membranes (R = 0.93). The concentration of PC could alternatively be altered by incubation of the choline-deficient hepatocytes with methionine or lyso-PC. With either of these supplementations highly significant correlation coefficients were observed between the concentration of PC in membranes and decreased activity of CT in membranes or increased activity in cytosol. The concentration of PC was reduced in the endoplasmic reticulum, but not the Golgi membranes, isolated from choline-deficient compared with choline-supplemented livers. The data suggest that the amount of PC in the endoplasmic reticulum feedback regulates the amount of CT associated with this membrane.     

Immunological studies on CTP:phosphocholine cytidylyltransferase from the livers of normal and choline-deficient rats

Chickens were immunized with the purified low-molecular-weight form of CTP:phosphocholine cytidylyltransferase from rat liver cytosol. The antiserum was obtained and fractionated to yield immunoglobulin. The antibodies specifically inhibited the enzymatic activity of the partially purified low-molecular-weight form of the enzyme from pH 6.0 to 8.5. Antibodies against the low-molecular-weight form of the enzyme cross-reacted with the high-molecular-weight form of the enzyme from cytosol as well as with the cytidylyltransferase associated with the microsomal fraction. The antibodies were used for the immunochemical determination of the amount of cytosolic phosphocholine cytidylyltransferase in the livers of normal and choline-deficient rats. The amount of enzyme in rat liver cytosol was not changed for at least 18 days of choline deficiency. The decrease in specific activity of the enzyme in choline-deficiency may be caused by factors other than adaptive changes in the level of enzyme.     

Subcellular distribution and regulation of hepatic bilirubin UDP-glucuronyltransferase

We have investigated the subcellular location and regulation of hepatic bilirubin UDP-glucuronyltransferase, which has been presumed to be located largely in the smooth endoplasmic reticulum. Purity of subcellular membrane fractions isolated from rat liver was assessed by electron microscopy and marker enzymes. Bilirubin UDP-glucuronyltransferase activity was measured by radiochemical assay using a physiologic concentration of [14C]bilirubin, and formation rates of bilirubin diglucuronide and monoglucuronides (C-8 and C-12 isomers) were determined. Activity of the enzyme was widely distributed in subcellular membranes, the majority being found in smooth and rough endoplasmic reticulum, with small amounts in nuclear envelope and Golgi membranes. No measurable activity was found in plasma membranes or in cytosol. Synthesis of bilirubin diglucuronide as a percentage of total conjugates and the ratio of C-8/C-12 bilirubin monoglucuronide isomers formed were comparable in all membranes, suggesting that the same enzyme is present in all locations. However, the regulation of bilirubin UDP-glucuronyltransferase activity differed among intracellular membranes; enzyme activity measured in the presence of the allosteric effector uridine 5'-diphospho-N-acetylglucosamine exhibited latency in smooth endoplasmic reticulum and Golgi membranes, but not in rough endoplasmic reticulum and nuclear envelope. Since rough membranes comprise 60% of hepatocyte endoplasmic reticulum and bilirubin UDP-glucuronyltransferase activity in vitro is maximal in this membrane fraction under presumed physiologic conditions, it is likely that the rough endoplasmic reticulum represents the major site of bilirubin glucuronidation in hepatocytes.     

The UDP-N-acetylglucosamine-asparagine-sequon N-acetyl-beta-D-glucosaminyltransferase activity in preparations of rough endoplasmic reticulum from regenerating rat liver.

The UDP-N-acetylglucosamine-asparagine-sequon N-acetylglucosaminyltransferase activity in preparations of rough endoplasmic reticulum from rat liver is less than 1% of that in preparations from rabbit liver. The activity of the enzyme is increased about 20-fold in preparations from regenerating rat liver within 48h after partial hepatectomy. A smaller, but still marked, increase (8-10-fold) occurs in preparations of rough endoplasmic reticulum from sham-operated rats.     

Subcellular localization and enzymatic properties of rat liver phosphatidylinositol-4-phosphate kinase

The phosphatidylinositol-4-phosphate kinase activity in rat liver showed a subcellular distribution different from that of phosphatidylinositol kinase. It was preferentially associated with plasma membrane-rich subcellular fractions, while no or minimal activity could be ascribed to mitochondria, lysosomes, Golgi membranes or the endoplasmic reticulum. The plasma membrane enzyme phosphorylated endogenous and exogenously added phosphatidylinositol 4-phosphate at comparable initial rates. The phosphorylation of endogenous substrate was strongly inhibited by Triton X-100, while the phosphorylation of added substrate was enhanced, suggesting that endogenous phosphatidylinositol 4-phosphate was readily available to the enzyme in unperturbed plasma membranes. The total activity of phosphatidylinositol-4-phosphate kinase in rat liver was only 1/20 that of phosphatidylinositol kinase. The enzyme activity showed an unusually broad pH-optimum in the neutral range. Mg2+ was the preferred divalent cation and Km towards ATP was about 3-fold higher than the corresponding value for phosphatidylinositol kinase.     

Biogenesis of very-low-density lipoproteins in rat liver. Intracellular distribution of apolipoprotein B

The hepatic subcellular distribution of apolipoprotein B (apo B) was studied quantitatively by using an enzyme immunoassay developed for apo B and by immunoadsorption-precipitation of [3H]leucine-labelled apo B. Over 50% (of 0.59 microgram/mg protein) of the apo B was located in the microsomal fraction. Further subfractionation of the microsomes revealed that 47% of the microsomal apo B was in the Golgi apparatus, while another 43% was associated with the rough endoplasmic reticulum. The smooth endoplasmic reticulum accounted for only 4% of the total. When rat livers were labelled with [3H]leucine for 10 min, the rough endoplasmic reticulum accounted for 80% of the total immunoadsorbed precipitable apo B radioactivity while the smooth accounted for 20%, with no contribution from the Golgi. However, only 8.7% of the total radioactive immunoadsorbed precipitable apo B was lipoprotein-associated, the remainder being membrane-bound. Lipoprotein-associated apo B radioactivity in the smooth endoplasmic reticulum accounted for 40%, with the rough contribution attributed at 50% and the Golgi at 9%. We concluded that (a) there are two major pools of apo B in rat liver microsomes; (b) although the apo B mass may be negligible in the smooth endoplasmic reticulum, the latter does play a role in lipoprotein biogenesis. The possible function of apo B associated with membranes of the microsomes is also discussed.     

The initial synthesis of proteins during development. Phosphoenolpyruvate carboxylase in rat liver at birth

1. A specific antibody, prepared by immunizing rabbits with phosphoenolpyruvate carboxylase (EC 4.1.1.32) purified from adult rat liver, was used to study the appearance of this enzyme in livers from developing rats. 2. Although some inactive precursor of the enzyme may be present in foetal liver, the amount is not sufficient to account for the enzyme appearance at birth. 3. The rate of phosphoenolpyruvate carboxylase synthesis relative to other cytosol proteins increases 20-fold from the foetus to the 1-day-old rat. The high rate of synthesis was maintained at least until 3 days after birth. 4. There was no measurable degradation of phosphoenolpyruvate carboxylase during the first day after birth. During this period the hepatic enzyme content increased 12-fold. 5. When phosphoenolpyruvate carboxylase attained a constant activity in the liver of rats 2 days after birth the half-time of degradation was approx. 13h. 6. We suggest that the pattern of changes occurring during appearance of phosphoenolpyruvate carboxylase is similar to substrate-induced enzyme induction in bacteria.     

Stimulation of hepatic phosphatidylcholine biosynthesis in rats fed a high cholesterol and cholate diet correlates with translocation of CTP: phosphocholine cytidylyltransferase from cytosol to microsomes

A new model system for the study of phosphatidylcholine biosynthesis is presented. Young rats were fed a diet that contained 5% cholesterol and 2% cholate. After 6 days there was a 2-fold increase in the concentration of plasma phospholipid (243 mg/dl compared to 132 mg/dl for control animals) and a 3-fold increase in the concentration of plasma phosphatidylcholine. The rate of phosphatidylcholine biosynthesis was measured after injection of [Me-3H]choline into the portal veins. The incorporation of tritium into choline, phosphocholine and betaine by liver was similar for experimental and control animals, whereas there was a 3-fold increased incorporation into phosphatidylcholine of the cholesterol/cholate-fed rats. The activities of the enzymes of phosphatidylcholine biosynthesis in cytosol and microsomes were assayed. The only change detected was in the cytosolic and microsomal activities of CTP: phosphocholine cytidylyltransferase which were increased more than 2-fold in specific activity. When total cytidylyltransferase activity per liver was determined, a dramatic translocation of the enzyme to microsomes was observed. The control livers had 24% of the cytidylyltransferase activity associated with microsomes, whereas this value was 61% in the livers from cholesterol/cholate-fed rats. When the cytosolic cytidylyltransferase was assayed in the presence of phospholipid, the enzyme was stimulated several-fold and the difference in specific activity between control and cholesterol/cholate-fed rats was abolished. The increased activity in cytosol appears to be the result of a 2-fold increase in the amount of phospholipid in the cytosol from cholesterol/cholate-fed rats. The data strongly support the hypothesis that the special diet stimulates phosphatidylcholine biosynthesis by causing a translocation of the cytidylyltransferase from cytosol to microsomes where it is activated.     

Subcellular localization of the enzyme that forms mannosyl retinyl phosphate from guanosine diphosphate [14C]mannose and retinyl phosphate.

The subcellular distribution of the enzyme catalysing the conversion of retinyl phosphate and GDP-[14C]mannose into [14C]mannosyl retinyl phosphate was determined by using subcellular fractions of rat liver. Purity of fractions, as determined by marker enzymes, was 80% or better. The amount of mannosyl retinyl phosphate formed (pmol/min per mg of protein) for each fraction was: rough endoplasmic reticulum 0.48 +/- 0.09 (mean +/- S.D.); smooth membranes (consisting of 60% smooth endoplasmic reticulum and 40% Golgi apparatus), 0.18 +/- 0.03; Golgi apparatus, 0.13 +/- 0.03; and plasma membrane 0.02.     

Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver

Trafficking and sorting of lipids during transport from the endoplasmic reticulum to the Golgi apparatus was studied using a cell-free system from rat liver. Transitional elements of the endoplasmic reticulum were prepared from liver slices prelabeled with [14C]- or [3H]acetate as the donor fraction. Non-radioactive Golgi apparatus were immobilized on nitrocellulose as the acceptor. When reconstituted, the radiolabeled donor retained a capacity to transfer labeled lipids to the non-radioactive Golgi apparatus acceptor. Transfer exhibited two kinetically different components. One was stimulated by ATP, facilitated by cytosol and inhibited by guanosine 5'-O-(thiotriphosphate) and N-ethylmaleimide. In parallel with protein transport, the ATP-dependent lipid transfer occurred with a temperature transition at about 20 degrees C. The other was not stimulated by ATP, did not require cytosol, was acceptor unspecific, was unaffected by inhibitors and, while temperature dependent, did not exhibit a sharp temperature transition. The ATP-independent transfer was non-vesicular. In contrast, the ATP-dependent transfer was vesicular. Transition vesicles isolated by preparative free-flow electrophoresis, when used as the donor fraction, transferred lipids to Golgi apparatus acceptor with a 5-6-fold greater efficiency than that exhibited by the unfractionated transitional endoplasmic reticulum. Formation of transition vesicles was ATP-dependent. Transferred lipids were chiefly phosphatidylcholine and cholesterol. Membrane triglycerides, major constituents of the transitional endoplasmic reticulum membranes, were both depleted in the transition vesicle-enriched fractions and not transferred to Golgi apparatus suggestive of lipid sorting prior to or during transition vesicle formation. The characteristics of the ATP plus cytosol-dependent transfer were similar to those for protein transfer mediated by transition vesicles. Thus, the 50-70-nm vesicles derived from transitional endoplasmic reticulum appear to function in the trafficking of both newly synthesized proteins and lipids from the endoplasmic reticulum to the Golgi apparatus.     

Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum

After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of [3H] choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells. The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation. The limiting step in phosphatidylcholine biosynthesis was the formation of CDP-choline catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) as monitored by the decrease in phosphocholine labeling following phospholipase C treatment of cells prelabeled with [3H]choline. The specific activity of homogenate cytidylyltransferase was increased about 1.6-fold in phospholipase C-treated cells. Specific activity of the membrane fraction was increased 2-fold, whereas cytosolic specific activity decreased in phospholipase C-treated cells. The activation of cytidylyltransferase was concomitant with translocation of the enzyme from the cytosol to the membrane fraction. The latter was further fractionated using a Percoll gradient that allowed an efficient separation between endoplasmic reticulum and other subcellular membranes. In control cells, particulate cytidylyltransferase activity co-migrated with the endoplasmic reticulum and ribosome markers and not with the plasma membrane. Also, in treated cells, the stimulation of cytidylyltransferase activity occurred at the endoplasmic reticulum level and did not involve either the external cell membrane or other cellular organelles including the Golgi apparatus, lysosomes, or mitochondria. Thus, our results demonstrate that a stimulus acting on the plasma membrane promotes the translocation of the soluble form of cytidylyltransferase specifically to the endoplasmic reticulum.     

Phosphatidylethanolamine levels and regulation of phosphatidylethanolamine N-methyltransferase

The activity of phosphatidylethanolamine (PE) N-methyltransferase in liver microsomes, measured using endogenous microsomal PE as a substrate, was elevated 2-fold in the choline-deficient state. However, methyltransferase activity assayed in the presence of a saturating concentration of phosphatidyl-N-mono-methylethanolamine or microsomal PE was unchanged by choline deficiency. Accompanying the increase in methyltransferase activity in liver homogenates and microsomes were increased PE concentrations and an increased PE to phosphatidylcholine ratio. The concentration of other phospholipids was unchanged. Immunoblot analysis of choline-deficient and choline-supplemented rat liver microsomes using a rabbit polyclonal anti-PE N-methyltransferase antibody revealed that the amount of enzyme protein was unaltered. The regulation of methyltransferase by PE levels was also investigated in cultured hepatocytes obtained from choline-deficient rat livers. Supplementation of deficient hepatocytes with 200 microM methionine resulted in a 50% reduction in cellular PE levels over a 12-h period. PE N-methyltransferase activity assayed with endogenous PE was also reduced by 50%, but phosphatidyl-N-monomethylethanolamine-dependent activity was unchanged. A 4-h supplementation with choline did not affect PE levels or methyltransferase activity. Either methionine or choline supplementation resulted in net synthesis of cellular phosphatidylcholine. Immunoblotting of membranes from methionine-supplemented hepatocytes revealed no change in enzyme protein, a further indication that enzyme mass was constitutive, and activity was regulated by the concentration of PE.     

Albumin secreted by rat liver bypasses Golgi apparatus cisternae

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [³H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.     

Distribution of UDP-glucuronyl transferase among cell fractions of rat liver

Microsomal UDP-glucuronyl transferases (uridine-5′-diphosphate-glucuronate glucuronyl transferase, acceptor unspecific, EC 2.4.1.17) function in detoxification of a wide range of aglycons containing phenolic, alcoholic, carboxylic or amino acceptor groups through formation of water-soluble glucuronic acid derivatives. To localize this activity within a specific cell component, purified rough-surfaced endoplasmic reticulum and Golgi apparatus fractions from rat liver were compared with total homogenates. The UDP-glucuronyl transferase specific activity of the Golgi apparatus fractions was less than or approximately equal to that of the total homogenate whereas this activity was concentrated 3–8 fold in the rough-surfaced endoplasmic reticulum fraction depending upon substrate. We conclude that the rough-surfaced endoplasmic reticulum is the major site of glucuronide formation within the microsome fraction of normal rat livers.     

Subcellular localization in normal and vitamin A-deficient rat liver of vitamin A serum transport proteins,albumin, ceruloplasmin and class I major histocompatibility antigens

The subcellular localization in rat liver cells of retinol-binding protein (RBP), prealbumin, ceruloplasmin, albumin, and class I transplantation antigen chains was investigated by radioimmunoassay determinations. The concentration of RBP was high in the rough and smooth endoplasmic reticulum (SER). The relative concentrations of prealbumin, ceruloplasmin and albumin were similar in the endoplasmic reticulum fractions and in the Golgi fraction. Neither of the proteins were found in significant amounts in the post-microsomal supernatant nor in the plasma membrane. The concentrations of the class I transplantation antigen chains were higher in the Golgi fraction than in the endoplasmic reticulum fractions. In the rough endoplasmic reticulum (RER) fraction ceruloplasmin and the class I antigens partially interact with high-molecular weight (MW) components, presumably membrane-bound glycosyltransferases. RBP, prealbumin and albumin seemed to be present in free form within the microsomal lumen. In vitamin A deficiency the RBP and to a lesser extent the prealbumin concentrations in the endoplasmic reticulum fractions were significantly increased, as compared to fractions from normal livers. This suggests that the presence of vitamin A is a prerequisite for the transport of RBP from the endoplasmic reticulum to the Golgi complex. The intracellular concentrations of albumin and ceruloplasmin were not significantly altered by vitamin A deficiency. In contrast, the amounts of the class I antigen heavy chains were found to be increased.     

Electrophoretic mobility of gamma-glutamyltransferase in rat liver subcellular fractions.Evidence for structure difference from the kidney enzyme.

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.     

Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver

Preparations enriched in part-smooth (lacking ribosomes), part-rough (with ribosomes) transitional elements of the endoplasmic reticulum when incubated with ATP plus a cytosol fraction responded by the formation of blebbing profiles and approximately 60-nm vesicles. The 60-nm vesicles formed resembled closely transition vesicles in situ considered to function in the transfer of membrane materials between the endoplasmic reticulum and the Golgi apparatus. The transition elements following incubation with ATP and cytosol were resolved by preparative free-flow electrophoresis into fractions of differing electronegativity. The main fraction contained the larger vesicles of the transitional membrane elements, while a less electronegative minor shoulder fraction was enriched in the 60-nm vesicles. If the vesicles concentrated by preparative free-flow electrophoresis were from material previously radiolabeled with [3H]leucine and then added to Golgi apparatus immobilized to nitrocellulose, radioactivity was transferred to the Golgi apparatus membranes. The transfer was rapid (T1/2 of about 5 min), efficient (10-30% of the total radioactivity of the transition vesicle preparations was transferred to Golgi apparatus), and independent of added ATP but facilitated by cytosol. Transfer was specific and apparently unidirectional in that Golgi apparatus membranes were ineffective as donor membranes and endoplasmic reticulum vesicles were ineffective as recipient membranes. Using a heterologous system with transition vesicles from rat liver and Golgi apparatus isolated from guinea pig liver, coalescence of the small endoplasmic reticulum-derived vesicles with Golgi apparatus membranes was demonstrated using immunocytochemistry. Employed were polyclonal antibodies directed against the isolated rat transition vesicle preparations. When localized by immunogold procedures at the electron microscope level, regions of rat-derived vesicles were found fused with cisternae of guinea pig Golgi apparatus immobilized to nitrocellulose strips. Membrane transfer was demonstrated from experiments where transition vesicle membrane proteins were radioiodinated by the Bolton-Hunter procedure. Additionally, radiolabeled peptide bands not present initially in endoplasmic reticulum appeared following coalescence of the derived vesicles with Golgi apparatus. These bands, indicative of processing, required that both Golgi apparatus and transition vesicles be present and did not occur in incubated endoplasmic reticulum preparations or on nitrocellulose strips to which no Golgi apparatus were added.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of glutathione-insulin transhydrogenase (protein-disulfide interchange enzyme) in pancreas using immunocytochemistry, immunodiffusion, and enzymatic activity assay techniques

The localization of the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (GIT), in rat and mouse pancreas was studied by protein A-gold immunocytochemistry, immunodiffusion, and assay of enzymatic activity. Immunocytochemistry on tissue sections using antibody to GIT and protein A-gold complex indicated the presence of GIT in alpha and beta cells in islets as well as acinar cells. The beta cells in obese (ob/ob) hyperinsulinemic mice showed increased GIT immunoreactivity. In both alpha and beta cells, GIT immunoreactive sites were associated predominantly with secretory granules. In pancreas from rats injected with glibenclamide, the degranulated beta cells contained GIT immunoreactive sites on the cisternal surface of the rough endoplasmic reticulum (RER). In acinar cells, the RER, Golgi elements, condensing vacuoles, and zymogen granules possessed GIT immunoreactive sites as did mitochondria. Immunocytochemistry on sections of isolated subcellular fractions showed that GIT was associated with different membranes. The enzymatic activity of GIT was found in the following order: Golgi elements greater than mitochondria greater than microsomes greater than zymogen granules greater than cytosol. In Ouchterlony immunodiffusion tests, each subcellular fraction showed a precipitin band which was continuous with that of purified GIT, a result indicating the presence of immunologically identical GIT in all fractions.     

Characterization of polyisoprenyl phosphate phosphatase activity in rat liver

The polyisoprenyl phosphate dephosphorylating activity of rat liver has been investigated with regard to substrate specificity, subcellular distribution, and transmembrane orientation. Total liver microsomes were employed as a source of enzymatic activity against a variety of 32P-labeled substrates. Susceptibility to dephosphorylation followed the order solanesyl phosphate greater than alpha-cis-polyprenyl 19-phosphate = alpha-trans-polyprenyl 19-phosphate = dihydrosolanesyl phosphate greater than (S)-dolichyl 19-phosphate = (R)-dolichyl 19-phosphate = (R,S)-dolichyl 11-phosphate. There appeared to be no major effect of chain length from 11 to 20 isoprenes. Data obtained from inhibition studies using solanesyl [32P]phosphate as substrate were consistent with the substrate specificity studies and suggested that a single activity is responsible. With dolichyl [32P]phosphate as substrate, the phosphatase specific activity of the subcellular fractions prepared from rat liver was found to follow the sequence Golgi = smooth endoplasmic reticulum greater than plasma membrane greater than lysosomes = rough endoplasmic reticulum greater than nuclei greater than mitochondria. Transmembrane topography studies, using enzyme latency as a criterion, were consistent with an orientation of the active site facing the cytoplasm.     

Studies on the cellular toxicity of polychlorinated biphenyls (PCBs). I. Effect of PCBs on microsomal enzymes and on synthesis and turnover of microsomal and cytoplasmic lipids of rat liver- a morphological and biochemical study.

The acute effects of the PCB (polychlorinated biphenyls) mixture (Aroclor 1254) on microsomal enzymes and on synthesis and turnover of microsomal and cytoplasmic lipids of rat liver were investigated. Six daily i.p. injections of 25 and 50 mg PCB/kg body weight resulted in increased liver weight and liver to body weight ratios. When compared to controls PCB treatment resulted in a six-fold increase in amount of cytochrome P-450. Activities of NADPH-cytochrome c reductase, ethylmorphine demethylase and inosine diphosphatase were increased whereas glucose-6-phosphatase values were decreased by PCB exposure. Analysis of liver homogenate and microsomal fraction revealed an increase in lipid in PCB-exposed animals. Phospholipids, cholesterol and triglyceride were significantly increased after PCB exposure; however, the greatest percentage increase was seen in the triglyceride pool. The finding of an increase in microsomal triglyceride to phospholipid ratios with exposure to PCB is suggestive of an increase in membrane-enclosed lipid (liposomes). Studies with labelled glycerol indicated that the PCB-induced fatty liver resulted from increased half life but not increased synthesis of liver lipid moieties. The rate of incorporation of leucine into microsomal membrane and albumin was somewhat enhanced in rats exposed to PCB indicative of increased protein synthesis. Morphological studies showed increased occurrence of lipid material, both in cytoplasmic droplets and within rough and smooth-surfaced endoplasmic reticulum. Proliferation of smooth endoplasmic reticulum and flattened Golgi cisternae with no secretion granules containing lipoprotein particles characterized the liver from animals exposed for 6 days. The increase in lipid within membranes of the endoplasmic reticulum together with the flattened Golgi lacking typical secretory vesicles indicates a defect in transport of lipoproteins from the endoplasmic reticulum to the Golgi apparatus and may be the cause of the PCB-induced fatty liver.