Inosine Diphosphatase Research Articles

NUDT16 is a (deoxy)inosine diphosphatase, and its deficiency induces accumulation of single-strand breaks in nuclear DNA and growth arrest

Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest kcat/Km values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.

Purification and Characterization of Golgi Membrane-Bound Nucleoside Diphosphatase from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

Inosine diphosphatase (IDPase) isoforms associated with Golgi membranes were studied in sycamore cell culture. These enzyme isoforms were solubilized with Triton X-100 and purified by chromatography using DEAE-Toyopearl and SOURCE-S columns. The isoforms were separated into two distinguishable fractions (peak 1 and 2) by SOURCE-S column chromatography. Furthermore the peak 1 contained at least two isoform bands detected by native-PAGE analysis. The apparent molecular sizes of these three isoforms were estimated by both gel filtration and SDS-PAGE to be 50 kDa, indicating that the Golgi membrane-bound IDPase has a monomeric structure. These IDPase isoforms required divalent cations (Ca2+, Mg2+, Co2+, Mn2+) for their hydrolyzing activity, and were inhibited by ATP. IDP, UDP, and GDP were effective substrates for these enzymes. It is clearly indicated that the sycamore Golgi membrane-bound IDPase is a nucleoside diphosphatase.

Innervation and myoepithelial arrangements in the submandibular salivary gland of ferret investigated by enzyme, catecholamine and filament histochemistry

Although the submandibular gland of ferret is useful for studying salivary secretory processes which are regulated by nerves and involve myoepithelial activity, little attention has been paid to its parenchymal innervation and myoepithelial arrangements. Therefore, glands obtained postmortem from mature ferrets of both sexes were here examined with the use of light-microscopic histochemical techniques for cholinesterases, phosphatases and phosphorylase, histofluorescence for catecholamines, and milling dyes. Acetylcholinesterase staining was associated with nerve trunks in the interlobular stroma and an extensive intralobular network of nerve fibres, presumably of a cholinergic type, embracing acini and ducts. There were fewer fibres containing fluorescing catecholamines, presumably adrenergic. They were largely associated with acini. Numerous stellate cells with fine branching processes embracing acini, presumably myoepithelial cells, and a few spindle-shaped basal cells, investing striated ducts, were demonstrated on frozen tissue by alkaline phosphatase, but not by adenosine triphosphatase, inosine diphosphatase and phosphorylase. Cells of similar shape and distribution were also demonstrated by staining with milling dyes on fixed tissues, indicating possibly a filamentous constituent conferring mechanical stability and/or contractile ability. Together, these results suggest, firstly, that a cholinergic-type parenchymal innervation is prominent in the submandibular gland of ferret, although many adrenergic nerves are also present, and, secondly that the gland has a very extensive myoepithelial network which is possibly involved in membrane transport, and the support and/or contraction of the secretory parenchyma.

Ca2+‐dependent ATPase associated with plasma membrane from a calcareous alga, Serraticardia maxima (Corallinaceae, Rhodophyta)

SUMMARYThe plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two‐phase partitioning. Its purity was examined with marker enzymes, Mg2+‐dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH‐cytochrome c reductase, as well as the sensitivity of Mg2+‐dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid‐chromic acid. Mg2+‐ or Ca2+‐dependent ATPases were associated with the plasma membrane. Ca2+‐dependent ATPase was activated at physiological cytoplasmic concentrations of Ca2+ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg2+‐dependent ATPase. The isolated plasma membrane vesicles were mostly right side‐out. To test for H+‐translocation, right side‐out vesicles were inverted; 27% of vesicles were inside‐out after treatment with Triton X‐100. The inside‐out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca2+. The reduced fluorescence was recovered with the addition of 10 mmol/L NH4Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca2+‐dependent ATPase might pump H+ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification.

Purification of plasma membranes from leaves of conifer and deciduous tree species by phase partitioning and free‐flow electrophoresis

Purified plasma membrane fractions were obtained from leaves of Picea abies L., Pinus sylvestris L., Fagus sylvatica L. and Quercus robur L., whereas plasma membranes from Pinus halepensis Mill, proved to be more difficult to obtain, perhaps due to the higher content of volatile substances in this plant species. Plasma membranes were purified by both phase partitioning and free‐flow electrophoresis from microsomal fractions and identified on the basis of biochemical and in some cases morphological and cytochemical markers. Electron micrographs revealed that membrane vesicles from Pinus sylvestris exhibited a very clear dark‐light‐dark pattern and measurements of membrane thickness showed that it ranged from 6 to 10 nm. Most membranes were 8 nm thick and stained with phosphotungstic acid at low pH, both typical characteristics of the plasma membrane. Enzymatic identification of plasma membranes consisted in the determination of the vanadate‐sensitive ATPase (EC 3.6.1.3) activity. The specific activity in the upper phase (U2) fraction was 10–25 times higher than those in the lower phase and microsomal fractions, depending on plant species. 1,3‐β‐glucan synthase II (EC 2.4.1.3), another putative plasma membrane marker, was not detected in the plasma membrane‐enriched fractions of conifer needles and showed a very low specific activity in membranes of deciduous trees. Contamination by membranes of other origin was determined by analysis of membrane markers: cytochrome c oxidase (EC 1.9.3.1) for mitochondria, inosine diphosphatase (EC 3.6.1.6) for Golgi apparatus, cytochrome c reductase (EC 1.6.2.4) for endoplasmic reticulum, and pyrophosphatase (EC 3.6.1.1) for tonoplasts. The main, but relatively low contamination, was due to tonoplasts, as determined by the activity of pyrophosphatase. Plasma membrane characteristics were quite different depending on the season during which needles were taken. Membrane preparations of better quality were more easily obtained from samples taken during winter.

Structure and Function of the Golgi Complex in Rice Cells (II. Purification and Characterization of Golgi Membrane-Bound Nucleoside Diphosphatase).

Inosine diphosphatase bound to Golgi membranes was studied in rice (Oryza sativa L. cv Nipponkai) cells. The enzyme was solubilized with Triton X-100 from isolated rice Golgi membranes and was highly purified employing a series of chromatography steps in the presence of 20% glycerol and 0.1% Triton X-100. The apparent molecular mass of the enzyme was estimated by gel filtration column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 200 and 55 kD, respectively. The isoelectric point of the enzyme was determined to be 7.5. The optimal pH for the enzyme activity was around 7 and the enzyme required Mg2+ for hydrolyzing activity. IDP, UDP, and GDP were effective substrate for the purified rice Golgi membrane-bound inosine diphosphatase, whereas activity with ADP, CDP, and thymidine 5[prime]-diphosphate was 10 to 20% of IDP. The Km values for IDP, UDP, and GDP were 0.48, 0.50, and 0.67 mM, respectively, and Vmax values were 1.85, 1.54, and 1.67 [mu]mol min-1 mg-1, respectively. These results indicate that the rice Golgi enzyme is a nucleoside diphosphatase that is specific for IDP, UDP, and GDP. Furthermore, this rice Golgi nucleoside diphosphatase stimulated the activity of glucan synthase I also localized in rice Golgi membranes. The results strongly support the view that this nucleoside diphosphatase is involved in regulation of [beta]-glucan synthesis in the plant Golgi complex.

Filamentous Fungi with High Cytosolic Phospholipid Transfer Activity in the Presence of Exogenous Phospholipid

The phospholipid transfer activity of cell extracts from 15 filamentous fungus strains grown on a medium containing phospholipids as the carbon source was measured by a fluorescence assay. This assay was based on the transfer of pyrene-labeled phosphatidylcholines forming the donor vesicles to acceptor vesicles composed of egg phosphatidylcholines. The highest phosphatidylcholine transfer activity was obtained with cell extracts from Aspergillus oryzae. The presence of exogenous phospholipids in the culture medium of A. oryzae was shown to increase markedly the activity of phospholipid transfer as well as the pool of exocellular proteins during the primary phase of growth. Modifications in the biochemical marker activities of cellular organelles were observed: succinate dehydrogenase, a mitochondrial marker; inosine diphosphatase, a Golgi system marker; and cytochrome c oxidoreductase, an endoplasmic reticulum marker, were increased 7.3-, 2-, and 22-fold, respectively, when A. oryzae was grown in the presence of phospholipids.

Allosteric regulation of the Golgi inosine diphosphatase from corn roots

Light microsomes of corn roots, enriched in endoplasmic reticulum and Golgi membranes, have an IDPase activity which is stimulated by Triton X-100 and by cold storage. In the native state, the enzyme activity does not follow Michaelis-Menten kinetics. It hydrolyses IDP with K0.5 of about 900 μM and Vmax of 300–400 nmol Pi/mg protein/min. In the presence of Triton X-100, the enzyme is maximally stimulated and it renders to a Michaelis-Menten behavior with a Km of about 500 μM and a Vmax of 800–1200 nmol Pi/mg protein/min. The maximal effect of the detergent occurs at about 1 mM IDP (270%), being reduced (190%) at high IDP concentrations (>2 mM) which, per se, have a slight stimulatory effect on the enzyme. On the other hand, we observed that ATP (>2 mM) and adenosine inhibit the IDPase. The effects of the nucleotides and of the adenosine are abolished in the presence of Triton X-100, which makes the enzyme fully active. Furthermore, we observed that detergent treatment of the membranes reduces the change in the activation energy which occurs at 10 °C and eliminates cooperative effects, as revealed by the Arrhenius analysis and the Hill analysis, respectively. We also observed that IDPase inhibition by ATP is maximal at low IDP concentrations (1 mM), whereas it decreases at high concentrations of IDP (4 mM), which promote maximal velocities in the native enzyme. Conversely, the inhibitory effect of adenosine is not reduced at high IDP concentrations. Pyrophosphate also inhibits the IDPase, but the effect is non-competitive and it is cumulative with that of ATP. We also observed that the latent activity of the IDPase (Triton-stimulated IDPase) is reduced by pre-treatment of the membranes with glutaraldehyde. The results indicate that Golgi IDPase is an allosteric enzyme which is positively modulated by IDP and negatively modulated by ATP and adenosine. Pyrophosphate inhibits the IDPase, but it seems to act at the catalytic site, whereas the other modulators appear to interact with a distinct regulatory site.

Differentiation between Several Types of Phosphohydrolases in Light Microsomes of Corn Roots

The phosphohydrolase activity of a light microsomal fraction isolated from corn roots (Zea mays L. cv LG 55) was investigated. The fraction, which appears to be enriched in endoplasmic reticulum and Golgi membranes, has ATPase and pyrophosphatase activities that hydrolyze ATP and pyrophosphate at an optimum pH of 7.0, with K(m) values of about 160 and 240 micromolar and with V(max) values of about 200 and 50 nanomoles substrate hydrolyzed per milligram protein per minute, respectively. These enzymes differ in their sensitivity to anions and inhibitors. The ATPase is stimulated by sulfate anions, whereas pyrophosphatase is inhibited by molybdate. Furthermore, the simultaneous addition of ATP and pyrophosphate to the reaction medium increases phosphohydrolysis, suggesting that separate enzymes are operating in the membranes. We also observed that pyrophosphate competitively inhibits the ATPase, whereas ATP has no significant effect on the pyrophosphatase. On the other hand, we observed a detergent-stimulated, molybdate-insensitive inosine diphosphatase activity which, in the native state, hydrolyzes inosine diphosphate with a K(m) of about 700 micromolar and a V(max) of about 450 nanomoles inosine diphosphate hydrolyzed per milligram protein per minute. In the solubilized form, the enzyme appears to be fully active, exhibiting lower K(m) values to hydrolyze inosine diphosphate. Furthermore, we found that native inosine diphosphatase is inhibited either by ATP or pyrophosphate, whereas inosine diphosphate inhibits the ATPase, but has no significant effect on the pyrophosphatase. It appears that inosine diphosphate is a positive modulator of the inosine diphosphatase, whereas ATP and pyrophosphate act as negative modulators of this enzyme.

Distribution of thiamine, thiamine phosphates, and thiamine metabolizing enzymes in neuronal and glial cell enriched fractions of rat brain.

The distribution of thiamine, thiamine phosphoesters, and the thiamine pyrophosphate synthetizing [thiamine-pyrophosphokinase (TPKase)] as well as hydrolyzing [thiamine pyrophosphatase (TPPase) and thiamine monophosphatase (TMPase)] enzymes was determined in neuronal and glial enriched fractions prepared from rat brain. Nucleoside diphosphatases [inosine diphosphatase (IDPase) and uridine diphosphatase (UDPase)] and nucleoside monophosphatases [uridine monophosphatase (UMPase) and inosine monophosphatase (IMPase)] were also determined. Thiamine and thiamine mono- and pyrophosphate were present in neuronal enriched fractions at concentrations 2.8, 3.6, and 4.6 times higher than in glial fractions. TMPase was found only in glial enriched fractions, whereas the levels of TPKase, UMPase, IMPase, IDPase, UDPase, and TPPase were 2.0-, 2.2-, 1.3-, 2.8-, 3.7-, and 20.8-fold higher in neuronal than in glial fractions.

Sugar nucleotides dissipate ATP-generated transmembrane pH gradient in Golgi vesicles from suspension-cell protoplasts ofChenopodium rubrum L.

A microsomal vesicle fraction (GV) markedly enriched by the Golgi marker enzyme latent inosine diphosphatase (IDPase) has been isolated from photoautotrophic suspension-cell protoplasts ofChenopodium rubrum L. Addition of ATP creates a substantial pH gradient across the GV membrane as measured by accumulation of acridine orange. The GV showed a density of 1.14 g·cm(-3) by equilibrium density centrifugation on sucrose gradients. Coincidence of acridine-orange accumulation and IDPase activity was confirmed on Percoll gradients. Formation of the pH gradient half-saturates at 0.3 mM MgATP, peaks at pH 7, and is competitively inhibited by ADP (k i≤0.1 mM), but not by Pi; it is hardly inhibited by orthovanadate, quickly dissipated by monensink 2=18 nM), nigericin (k 1/2=25 nM), and sluggishly by N-ethylmaleimide (k 1/2≈35 μM). Inhibition by KNO3 (k 1/2≈6.7 mM) is incomplete (60%). Uridine 5'-diphosphate (UDP)-glucose, UDP-galactose, but not UDP-mannose and the pertinent sugars, dissipate the ATP-generated pH gradient (k 1/2≈10-20 mM UDP-glucose; optimum pH at 7.8). This UDP-glucose activity is accompanied by release of Pi, but not of glucose or sucrose. UDP-glucoseinduced Pi release from the GV saturates (k 1/2=1 mM UDP-glucose; optimum pH at 7) and is completely inhibited by the anion-channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS;k 1/2=140 μM). The GV incorporates UDP-[U-(14)C]glucose into an acid-labile, alkaline-stable macromolecular compound; this process is like-wise inhibited by DIDS. We propose a model including, inter alia, a UDP-glucose/uridine-5'-monophosphate translocator and a phosphate-permeable anion channel to operate in Golgi vesicles ofChenopodium rubrum.

Cytochemical studies on induced autophagocytosis in mouse exocrine pancreas

1. 1. The origin of the limiting membranes of autophagic vacuoles (AVs) in mouse pancreatic acinar cells was studied in vinblastine-induced autophagocytosis. 2. 2. The marker enzymes used were adenosine triphosphatase, lipase, inosine diphosphatase and thiamine pyrophosphatase. The following impregnation techniques were used: unbuffered osmium tetroxide impregnation, imidazole-buflered osmium tetroxide impregnation and uranyl-lead-copper impregnation. 3. 3. Only a weak lipase activity was observed between the limiting membranes of a few AVs. The AV membranes were stained heavily with all impregnation techniques used. 4. 4. The origin of AV membranes seems to be same in mouse liver and exocrine pancreas in vinblastine-induced autophagocytosis.

Subcellular Localization of Glycosidases and Glycosyltransferases Involved in the Processing of N-Linked Oligosaccharides

Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc(3)Man(9)(GlcNAc)(2), was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man(6-9)[GlcNAc](2)), mannosidase II (removes mannose residues from GlcNAcMan(5)[GlcNAc](2)), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.

Prenatal exposure to alcohol alters the Golgi apparatus of newborn rat hepatocytes: a cytochemical study.

The effect of prenatal exposure to ethanol on the Golgi apparatus of newborn rat hepatocytes has been studied cytochemically using several trans-Golgi markers (thiamine pyrophosphatase, uridine diphosphatase, inosine diphosphatase, acid phosphatase, and 5'-nucleotidase) as well as a cis-side marker (osmium impregnation). The amount of cerium phosphate formed in the cytochemical reactions was roughly quantitated by stereologic methods. The Golgi apparatus of about 40% of the hepatocytes appeared disorganized after alcohol treatment, and in the other 60%, the electron density of reaction product deposits for all phosphatases investigated was decreased. 5'-Nucleotidase was completely absent in cisternae of Golgi apparatus of treated cells. In control cells impregnated with osmium tetroxide, reduced osmium compounds were observed in most Golgi cisternae and in nearby vesicles. In contrast, only small vesicles appeared positive in treated hepatocytes. These results suggest that prenatal alcohol exposure alters some Golgi functions. Thus, the decrease in nucleoside diphosphatase and 5'-nucleotidase cytochemical activities after ethanol exposure strongly suggests that this treatment could affect glycosylation in the Golgi apparatus of newborn rat hepatocytes.

Identification of adipose tissue primordia in perirenal tissues of pig fetuses: utility of phosphatase histochemistry.

Perirenal adipose tissue samples were obtained from fetuses removed from pregnant (crossbred) sows at 3 stages of gestation (70, 90 and 110 days). Phosphatase histochemistry, succinate dehydrogenase (SDH) histochemistry and factor VIII antigen immunocytochemistry were conducted on fresh-frozen cryostat sections. Age-associated changes in nucleosidediphosphatase (NDPase) reactions in the arteriolar system were correlated with the morphological development of the medial layer of arterioles and arteries. For instance, a strong NDPase reaction in small arterioles was associated temporally with the assumption of a normal smooth-muscle cell morphology and arrangement in the medial layer. Age-associated changes in blood vessel reactions for factor VIII antigen and alkaline phosphatase activity were not correlated with morphological development. In the youngest fetuses, alkaline phosphatase activity was evident in large and small arterioles, but in the oldest fetuses, alkaline phosphatase activity was restricted to the smallest arterioles and vessels associated with them. Arteriolar differentiation was demonstrable with either adenosine triphosphatase (ATPase) or inosine diphosphatase (IDPase) reactions. Primordial stromal cells around differentiated arterioles were reactive for ATPase but not for IDPase activities. In older fetuses, there were large areas that contained ATPase-reactive stromal cells, no adipocytes, differentiated (ATPase and IDPase) arterioles and few capillaries. Positive reactions for SDH were evident in the ATPase-reactive stromal areas that contained no adipocytes. Differentiated adipocytes were SDH- and ATPase-reactive. These data illustrate the utility of differential phosphatase histochemistry to identify adipose tissue primordia.

Adipocyte development in primary rat cell cultures: a scanning electron microscopy study.

This study utilized scanning electron microscopy (SEM) techniques to observe primary cultures of stromal-vascular (SV) cells derived from postnatal rat inguinal adipose tissue. Cells were grown on collagen-coated, fibronectin-coated, or uncoated glass coverslips. Coverslips were normally fixed in glutaraldehyde, osmium tetroxide, dehydrated, and critical-point-dried. Other coverslips were frozen in isopentane (cooled in LN2) and dried or fixed in Baker's formalin for demonstration of inosine diphosphatase (IDPase) by X-ray microprobe analysis (XRMA). Adipocyte morphologies were similar on all substrates. At 2 days of culture, actin cables were detected extending from developing adipocytes. No difference in actin cable structure, cellular shape, or lipid accumulation was observed among the different substrates. Some stromal cells did not accumulate lipid but proliferated into a multilayer by 9 days in culture. Inosine diphosphatase was detected in the Golgi apparatus of developing adipocytes utilizing the technique of XRMA. This study demonstrates the potential for using SEM and XRMA techniques to define morphological features and cytochemical markers of adipocytes in vitro and the response of primary cultured rat SV cells to other attachment substrates.

Loss of endoplasmic reticulum-associated enzymes in affected brain regions in Huntington's disease and Alzheimer-type dementia

Post-mortem brain samples from patients with either Huntington's disease, Alzheimer-type dementia or appropriate controls were assayed for endoplasmic reticulum enzymes, NADPH-cytochrome c reductase, neutral α-glucosidase, inosine diphosphatase, α-mannosidase and glucose-6-phosphatase and for Golgi enzymes, fucosyl- and galactosyl-transferases. In Alzheimer-type dementia there was a selective decrease in α-glucosidase activity in the temporal cortex. In Huntington's disease there was a selective decrease of putamen α-glucosidase and fucosyl-transferase activities. It is suggested that these changes reflect highly specific alterations in glycoprotein synthesis and processing and may contribute to the underlying pathology of these disorders.

Alterations in the cytochemical activity of several phosphatases in hepatocytes from rats exposed prenatally to ethanol.

The activities of acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, uridine diphosphatase, inosine diphosphatase, thiamine pyrophosphatase and 5'-nucleotidase have been investigated cytochemically in hepatocytes of the offspring of alcohol-fed rats, using cerium ions as a capturing agent and qualitative and quantitative electron microscopy. All these enzyme activities were decreased in the experimental animals compared with controls not exposed to ethanol. The pattern of deposition of the product of glucose-6-phosphatase activity in the cisternae of the endoplasmic reticulum was also different in the two groups. The phosphatases analyzed are functional markers of different cell components, and the results suggest that prenatal exposure of rats to ethanol causes functional alterations in the endoplasmic reticulum, Golgi apparatus, lysosomes and plasma membrane of hepatocytes.

Subcellular Fractionation of Candida boidinii after Growth on Glucose or Methanol

Extracts of glucose-grown and methanol-grown Candida boidinii were fractionated on sucrose gradients. Mitochondria and microbodies from methanol-grown cells were more fragile than those from glucose-grown cells. Formaldehyde and formate dehydrogenases and antimycin A-insensitive NADPH: cytochrome c oxidoreductase were non-sedimentable enzymes. The mitochondrial zone defined by cytochrome c oxidase (ρ = 1·19 to 1·20 g ml–1) contained sedimentable activities of malate dehydrogenase and citrate synthase. The vacuolar enzyme acid phosphatase was also recovered from this region. The microbody enzyme catalase sedimented to a lower density (ρ = 1·17 g ml–1) than mitochondria after growth on glucose but, together with methanol oxidase activity, to higher densities (ρ = 1·23 and 1·27 g ml–1) after growth on methanol. Activity of the methanol assimilation enzyme dihydroxyacetone synthase was partially sedimentable and was recovered in the fraction containing mitochondria and membraneous vesicles. Inosine diphosphatase was probably multilocational in the cells.

Effect of colchicine on the Golgi complex of rat pancreatic acinar cells.

Colchicine administered to adult rats at a dosage of 0.5 mg/100 g of body weight effected a disorganization of the Golgi apparatus in pancreatic acinar cells. The results obtained after various periods of treatment (10 min to 6 h) showed (a) changes in all components of the Golgi complex, and (b) occurrence of large vacuoles that predominated in cytoplasmic areas outside the Golgi region. The alterations in Golgi stacks concerned elements of the proximal and distal side: (a) accumulation of transport vesicles, (b) formation of small, polymorphic secretion granules, and (c) alterations in the cytochemical localization of enzymes and reaction product after osmification. Transport vesicles accumulated and accompanied short, dilated cisternae, which lack mostly the reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase, and osmium deposits after prolonged osmification. After 4 to 6 h of treatment, accumulated transport vesicles occupied extensive cellular areas; stacked cisternae were not demonstrable in these regions. The changes on the distal Golgi side included GERL elements: condensing vacuoles were diminished; they were substituted by small, polymorphic zymogen granules, which appeared to be formed by distal Golgi cisternae and by rigid lamellae. Unusually extended coated regions covered condensing vacuoles, rigid lamellae, and polymorphic secretion granules. A cytochemical distinction between Golgi components and GERL was possible neither in controls nor after colchicine treatment. The cytochemical alterations in Golgi components were demonstrable 20-30 min following administration of colchicine; at 45 min, initial morphological changes--augmentation of transport vesicles and formation of polymorphic zymogen granules--became apparent. 20 min after administration of colchicine, conspicuous groups of large vacuoles occurred. They were located mostly in distinct fields between cisternae of the endoplasmic reticulum, and were accompanied by small osmium--reactive vesicles. Stacked cisternae were not demonstrable in these fields. Vacuoles and vesicles were devoid of reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase. The results provide evidence that formation of stacked Golgi cisternae is impaired after colchicine treatment. The colchicine--induced disintegration of the Golgi complex suggests a regulatory function of microtubules in the organization of the Golgi apparatus.