Heterogeneity of cyclic nucleotide phosphodiesterases in liver endoplasmic reticulum.

A microsomal fraction from rat liver was subfractionated into three rough endoplasmic reticulum fractions RIII, RII and RI, together with a smooth endoplasmic reticulum plus Golgi fraction. Cyclic nucleotide phosphodiesterase activity was found in all fractions. Subsequently it was shown that Golgi fractions were essentially devoid of cyclic AMP phosphodiesterase activity and the activity resided in the smooth endoplasmic reticulum fraction. The activity of the endoplasmic reticulum constituted some 20% of the homogenate activity, with the major fraction of this being associated with the RII fraction and the least with the RI fraction. With the exception of the activity of the RI fraction, which was a peripheral enzyme, all of the other enzyme activities were integral, requiring detergent or repeated freeze-thawing to effect solubilization. All of the activities appeared to be exposed at the external surface of the endoplasmic reticulum, as they were inactivated by trypsin under conditions where glucose 6-phosphatase was not. All of these activities displayed distinct sensitivities to both thermal and trypsin inactivation, yielding activity decays consistent with a single enzyme species being present in each case. The freeze-thaw-solubilized enzymes yielded single symmetrical peaks on sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The sedimentation coefficients for the enzymes in the smooth-endoplasmic-reticulum-plus-Golgi, RIII, RII and RI fractions were 3.2S, 4.2S, 4.5S and 4.5S respectively. Whereas the activity in the smooth-endoplasmic-reticulum-plus-Golgi fraction exhibited normal Michaelis kinetics, those in the other fractions yielded kinetics indicative of apparent negative co-operativity. All of the enzymes exhibited low Km values towards cyclic AMP. The enzymes did not appear to be regulated by Ca2+ or calmodulin. ZnCl2 was found to be a potent non-competitive inhibitor of the enzyme in all fractions. NaF was a weak non-competitive inhibitor. The bilayer fluidizing agent benzyl alcohol exerted dissimilar effects on the enzyme activities. It is concluded that the endoplasmic reticulum displays lateral heterogeneity, with single, rather distinct, cyclic AMP phosphodiesterases being found in the different fractions.     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : I. Glucose-6-Phosphatase Distribution In Situ

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.     

Subcellular localization of cyclo-oxygenase-prostaglandin E2 synthetase complex in goat vesicular gland by catalytic activity analysis

The catalytic activity of the cyclo-oxygenase prostaglandin E2 synthetase complex in subcellular organelles of goat vesicular gland was determined. The enzyme activity was found to be located mostly in the rough endoplasmic reticulum and partly in the nuclear membrane; comparatively very little activity could be detected in the smooth endoplasmic reticulum. There was no detectable activity of the enzyme in the plasma membrane.     

Phosphatidylglycerol synthesis in castor bean endosperm: kinetics, requirements, and intracellular localization

The synthesis of phosphatidylglycerol in castor bean (Ricinus communis var. Hale) endosperm tissue was found to be located in both the endoplasmic reticulum and mitochondrial fractions separated on sucrose density gradients. The enzyme of both fractions attained maximum activity at 5 mm Mn(2+), 0.075% Triton X-100, and pH 7.3. The addition of dithiothreitol produced little effect, but sulfhydryl inhibitors reduced activity in both systems. Cytidine diphosphate-diglyceride exhibited an apparent Michaelis constant for the endoplasmic reticulum enzyme of 2.8 mum and for the mitochondrial enzyme of 2.0 mum; the maximum reaction rate was achieved at about 20 mum. For the second substrate, glycerol-phosphate, the apparent Michaelis constant for both fractions was about 50 mum and maximum velocity was reached at 400 mum. The specific activity of the mitochondrial enzyme was generally twice that of the endoplasmic reticulum.     

Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Delta6-desaturase activity

The Delta6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat Delta6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of Delta6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and Delta6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for Delta6-desaturase activity.     

Subcellular localization of cyclo-oxygenase-prostaglandin E2 synthetase complex in goat vesicular gland by catalytic activity analysis

The catalytic activity of he cyclo-oxygenase prostaglandin E₂ synthetase complex in subcellular organelles of goat vesicular gland was determined. The enzyme activity was found to be located mostly in the rough endoplasmic reticulum and partly in the nuclear membrane; comparatively very little activity could be detected in the smooth endoplasmic reticulum. There was no detectable activity of the enzyme in the plasma membrane.     

Cytochemical evidence of the origin of the dense tubular system in the mouse platelet

Summary Glucose-6-phosphatase (G6Pase) was used as a marker enzyme for the endoplasmic reticulum in mouse megakaryocytes and platelets. G6Pase activity was localized in the dense tubular system of the platelets. Enzyme activity was also observed in the nuclear envelope, and in the rough endoplasmic reticulum of the megakaryocytes. However, the Golgi apparatus of the megakaryocyte was never involved. The present study has added new cytochemical evidence for the hypothesis that the dense tubular system of the platelet originates from the endoplasmic reticulum of the megakaryocyte.     

The localization of cholinephosphotransferase in the outer membrane of guinea-pig lung mitochondria

The activity of cholinephosphotransferase was measured in the subcellular fractions of guinea-pig lung. The specific activity of the enzyme was highest in a fraction, intermediate in density between mitochondria and microsomes. Similar subcellular distribution patterns were observed for both cholinephosphotransferase and rotenone-insensitive NADH-cytochrome c reductase, an enzyme associated with the outer membrane of mitochondria and endoplasmic reticulum, suggesting that cholinephosphotransferase may be localized in both of these organelles. The distribution of cholinephosphotransferase activity in the subfractions of mitochondria and the intermediate fractions recovered by linear density gradient paralleled that of the mitochondrial outer membrane marker enzyme, monoamine oxidase. RNA content of a subfraction enriched in cholinephosphotransferase and monoamine oxidase was not typical to that of either rough or smooth endoplasmic reticulum. The results of this study suggest that in guinea-pig lung, cholinephosphotransferase is localized in both the outer membrane of mitochondria, and the endoplasmic reticulum.     

Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol- dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.     

Gonadotropin and prostaglandins binding sites in rough endoplasmic reticulum and Golgi fractions of bovine corpora lutea.

Highly purified rough endoplasmic reticulum and three subfractions of golgi were prepared from 105,000g pellet of the homogenate by centrifugation in floatation and sedimentation discontinuous sucrose gradients. Highly purified plasma membranes were also prepared from 9,000g pellet of the same homogenates for assessment under the same experimental conditions. Although 5′-nucleotidase, a marker for plasma membranes, was markedly enriched in plasma membranes, very little or none of this enzyme activity was found in other fractions. Very little or no NADH cytochrome c reductase activity, a marker for rough endoplasmic reticulum, was found in fractions other than rough endoplasmic reticulum. Galactosyl transferase, a marker for golgi, was found and enriched in all the fractions; however, enrichment in golgi fractions was higher than in other fractions. Very little or no lysosomal marker activity, i.e., acid phosphatase, was found in rough endoplasmic reticulum or golgi fractions as compared to lysosomes. These marker enzyme data suggested that rough endoplasmic reticulum and golgi fractions were relatively pure with little or no cross contamination with other organelles. The [¹²⁵I]human choriogonadotropin ([¹²⁵I]hCG), [³H]prostaglandin (PG)E₁, and [³H]PGF_2a specifically bound to rough endoplasmic reticulum and golgi fractions in addition to plasma membranes. The enrichments of binding in the former two fractions, in some cases, were as high as plasma membranes itself. The specific binding of some of the ligands was found to be partially latent in rough endoplasmic reticulum and golgi fractions but not in plasma membranes. Marker enzyme data, ratio between bindings and marker enzyme activities (an index of organelle contamination), and partial latency of binding suggest that rough endoplasmic reticulum and golgi fractions intrinsically contain gonadotropin and PGs binding sites.     

Ultrastructural demonstration of glucose 6-phosphatase in cerebral cortex

Summary A two-stage fixation technique has been developed to obtain morphological preservation and retention of glucose 6-phosphatase (G6-Pase) activity for its demonstration in rat cerebral cortex. The technique was then employed to localize the enzyme in the cortex where it produced a dense reaction over the well developed granular endoplasmic reticulum cisternae in nerve cells and oligodendrocytes which contrasted with a thin reaction in astrocytes. Other membranous organelles showed no reaction.     

Ultrastructural demonstration of glucose 6-phosphatase in cerebral cortex

A two-stage fixation technique has been developed to obtain morphological preservation and retention of glucose 6-phosphatase (G6-Pase) activity for its demonstration in rat cerebral cortex. The technique was then employed to localize the enzyme in the cortex where it produced a dense reaction over the well developed granular endoplasmic reticulum cisternae in nerve cells and oligodendrocytes which contrasted with a thin reaction in astrocytes. Other membranous organelles showed no reaction.     

Pulmonary surfactant synthesis. A highly active microsomal phosphatidate phosphohydrolase in the lung.

Lung cell-free homogenate, which contains about twice the units of phosphatidate phosphohydrolase per mg of protein compared to liver, was fractionated by differential centrifugation and the fractions were assayed for phosphatidate phosphohydrolase and marker enzymes of endoplasmic reticulum, mitochondria, and lysosomes. Over 60% of the lung phosphatidate phosphohydrolase was associated with the endoplasmic reticulum, compared to 50% of the total liver enzyme. Thus a major portion of the more active lung enzyme is potentially involved in lipid biosynthesis by the endoplasmic reticulum. Less than 0.2% of the total lung enzyme was found in a lamellar body fraction, consistent with previous findings. The lung microsomal phosphohydrolase was specific for lipid substrates, showing equal activity towards phosphatidic acid or lysophosphatidic acid and relatively low activities towards glycerophosphates. It had a neutral pH optimum, similar to the liver enzyme, but differed somewhat in its relative activity at extremes of pH. Stability at 65 degrees C was greater for the lung enzyme. Fluroide inhibited lung (or liver) microsomal phosphatidate phosphohydrolase, while tartrate, MgCl2, or EDTA had no effect. The presence of a high activity of phosphatidate phosphohydrolase in lung endoplasmic reticulum is consistent with the rapid synthesis of pulmonary surfactant phosphatidylcholine.     

Ultrastructural localization of acetylcholinesterase activity in primary cultures of rat substantia nigra

Summary Ultrastructural localization of acetylcholinesterase activity was studied in primary cultures of the substantia nigra microdissected from newborn rat brains. Light microscopic observations were also made on the characteristics of dopamine neurones and acetylcholinesterase containing cells in these cultures. Ultrastructurally acetylcholinesterase activity was localized in the nuclear envelope and rough endoplasmic reticulum of neurones, which had deeply infolded, round or oval nucleus, a prominent Golgi apparatus and varying amounts of rough endoplasmic reticulum. In the neuropil acetylcholinesterase activity was seen within microtubules of neuronal processes and in the rough endoplasmic reticulum of dendrites. The enzyme activity was also demonstrated within the nuclear envelope and rough endoplasmic reticulum of probably capillary endothelial cells. Dopaminergic neurones were identified on the basis of the green catecholamine fluorescence they exhibited. Small dopaminergic neurones could be observed and there was indirect evidence that these cells did not stain for acetylcholinesterase.     

Effect of glutamine or glucose deprivation on the expression of cyclin and cyclin-dependent kinase genes in glioma cell line U87 and its subline with suppressed activity of signaling enzyme of endoplasmic reticulum-nuclei-1

Ischemia has been shown to induce a set of complex intracellular signaling events known as the unfolded protein response, which is mediated by endoplasmic reticulum-nuclei-1 sensing enzyme. We have studied the expression of several cyclin and cyclin-dependent kinase genes which participate in the control of cell cycle and proliferation under ischemic conditions (glucose or glutamine deprivation) in endoplasmic reticulum-nuclei-1-deficient glioma cells. It was shown that blockade of endoplasmic reticulum-nuclei signaling enzyme-1, the key endoplasmic reticulum stress sensor, leads to an increase of the expression levels of cyclin-dependent kinase-2 and cyclin A2, D3, E2 and G2 genes but suppresses cyclin D1. Moreover, the expression level of cyclin-dependent kinase-2 as well as cyclin A2, D3 and E2 mRNAs is significantly decreased under glucose or glutamine deprivation conditions both in control and endoplasmic reticulum-nuclei-1-deficient glioma cells. However, cyclin-dependent kinase-4 and -5 mRNA expressions is increased, but in glucose deprivation conditions only. Results of this study have shown that the expression of most tested genes of encoded cyclins and cyclin-dependent kinases is dependent on endoplasmic reticulum-nuclei-1 signaling enzyme function both in normal and glutamine and glucose deprivation conditions and possibly participates in cell adaptive response to endoplasmic reticulum stress associated with ischemia.     

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.     

西瓜柱头乳突细胞分泌活动期间ATP酶活性超微结构定位

研究了西瓜柱头乳突细胞ＡＴＰ酶活性的超微结构定位。分泌活动旺盛的细胞中,质膜、内质网、质体的内部片层、胞间连丝以及多数大液泡的膜上面都有大量ＡＴＰ酶活性反应产物,线粒体和小泡上只有少量酶活性反应产物。分泌活动停止后处于解体状态的细胞内,反应产物主要定位于液泡膜上。分泌旺盛的乳突细胞质膜具有高的ＡＴＰ酶活性表明分泌物运出需要大量能量,内质网ＡＴＰ酶活性强可能意味着该细胞参与分泌物合成。     

Stability of alpha-fetoprotein messenger RNA in mouse yolk sac

Changes in the activity of DNA polymerase-α and in subcellular distribution were studied during gastrulation of the sea urchin, Hemicentrotus pulcherrimus. Although the activity of DNA polymerase-α for each embryo was constant up to the blastula stage as reported previously, the enzyme activity increased during gastrulation by about twofold prior to an increase in its DNA content. Thereafter the enzyme activity remained constant at a high level until the early pluteus stage. During gastrulation, an increase in the fraction of DNA polymerase-α was associated with the rough endoplasmic reticulum. During the period between the gastrula and pluteus stages, the cytoplasmic DNA polymerase-α activity decreased gradually with a concomitant increase of activity in the nucleus fraction. The timing of this increase in the nucleus coincided with the increase of DNA content per embryo. These results suggest that DNA polymerase-α accumulates on the rough endoplasmic reticulum during gastrulation and then translocates to the nucleus for DNA synthesis as seen before the blastula stage. DNA polymerase-α obtained from gastrula nuclei did not associate with the endoplasmic reticulum from gastrulae. DNA polymerase-α obtained from the gastrula endoplasmic reticulum membranes became bound to the salt-washed membranes from gastrulae but not to those from unfertilized eggs. Likewise, DNA polymerase-α from the rough endoplasmic reticulum of unfertilized eggs became attached to salt-washed membranes from unfertilized eggs, but not to those from gastrulae. This suggests that DNA polymerase-α is synthesized anew, and a transition of both DNA polymerase-α and endoplasmic reticulum occurs at the gastrula stage.     

The effects of triton X-100 on the transfer of mannose, glucose and n-acetylglusomine phosphate to dolichol monophosphate by preparations of rough and smooth endoplasmic reticulum and of mitochondria of rat liver.

Triton X-100 and exogenous dolichol monophosphate have been used to investigate the nature of enzymes responsible for the transfer of mannose, glucose and N-acetylglucosamine phosphate from nucleotide donors to dolichol monophosphate in vesicles derived from rough and smooth endoplasmic reticulum and mitochondria. Mitochondria were shown to contain the highest specific activities of these enzymes. The responses of the glycosyltransferases to increasing concentrations of Triton X-100 and the effect on these responses of exogenous dolichol monophosphate suggest that the enzymes for mannose and glucose transfer are less hydrophobic, and therefore less intrinsic, in the membrane than the enzyme for N-acetylglucosamine phosphate transfer. In smooth vesicles the results are consistent with mannosyl- and glucosyl-transferases being located at both inner and outer faces of the membrane. In rough vesicles and in mitochondria mannosyl- and glucosyl-transferases were confirmed at the outer face. There is, however, only one site of N-acetylglucosamine phosphate transfer, this being more hydrophobically located in the membrane than the other sites of glycosyl transfer. Mitochondrial enzyme activity closely resembled that of rough endoplasmic reticulum in response to Triton X-100 and exogenous dolichol monophosphate, and is probably associated with the outer membrane.     

西瓜柱头乳突细胞分泌活动期间ATP酶活性超微结构定位

研究了西瓜柱头乳突细胞ATP酶活性的超微结构定位。分泌活动旺盛的细胞中,质膜、内质网、质体的内部片层、胞间连丝以及多数大液泡的膜上面都有大量ATP 酶活性反应产物,线粒体和小泡上只有少量酶活性反应产物。分泌活动停止后处于解体状态的细胞内,反应产物主要定位于液泡膜上。分泌旺盛的乳突细胞质膜具有高的ATP酶活性表明分泌物运出需要大量能量,内质网 ATP 酶活性强可能意味着该细胞器参与分泌物合成。