Characterization of the NAD+ glycohydrolase associated with the rat liver nuclear envelope.

The localization of NAD+ glycohydrolase [EC 3.2.2.5] (NADase) in purified rat liver nuclei has been examined. Subnuclear fractionation revealed that at least 70% of the NADase in nuclei was associated with the nuclear envelope fraction. The nuclear envelope fraction was practically free of microsomal contamination as judged by electron microscopic morphometry and assays of microsomal marker enzymes. Therefore, NADase was found to be an integral component of the nuclear envelope. The enzymological properties of the nuclear envelope NADase were compared with those of the microsomal enzyme. The nuclear envelope NADase was identical to the microsomal enzyme in its Km for NAD+ (60 muM), pH optimum (pH 6.5), ratio of transglycosidase activity to NADase activity (about 0.5), thermal stability and sensitivity to various inhibitors. Thus, NADase is a common enzymic component of both the nuclear envelope and the endoplasmic reticulum.     

The association of lysosomal enzymes with nuclei during enzyme induction in rat liver.

Male rats of the Holtzman strain were fasted for 3 days and refed a diet high in carbohydrate (68.9%). The induction of liver glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was monitored for up to 48 h after refeeding. Induction occurred by 24 h, and by 48 h, 4.2- and 1.5-fold increases were observed for glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, compared with that of livers of pellet-fed rats. After refeeding, lysosomes increased in fragility as judged by an increased release of acid phosphatase activity during standard homogenization. Fragility was greatest 3 h after refeeding, but normal fragility was observed 24 h after refeeding. Nuclei were isolated from the liver samples before and after refeeding. Those isolated just before refeeding revealed small latent acid phosphatase activity (4–6%). However, after refeeding the carbohydrate-rich diet, a transient and significant (P < 0.01) increase in the latent activity occurred that was maximal (20%) at 1 h, returning to normal by 24 h. Cross-mixing the 800g nuclear pellet from livers of animals starved for 3 days with the 800g supernatant fraction from livers of animals refed the carbohydrate-containing diet did not alter the nuclear lysosomal-free (overt) or latent (detergent-released) enzyme activity. Similarly, mixing the 800g nuclear pellet from livers of animals refed for 1 h with the 800g supernatant fraction from livers of animals starved for 3 days, but not refed, did not change the nuclear lysosomalfree or latent enzyme activity. Purified nuclei, further washed in hypotonic buffer, lost acid phosphatase activity, but those isolated from livers of rats refed for 1 h retained 10% of the enzyme latency, whereas all latency was lost from those isolated from uninduced rats. A second lysosomal enzyme, β-galactosidase, became associated with the nuclei with the same temporal pattern as for acid phosphatase. However, no variation in nuclear content of cytosolic lactate dehydrogenase occurred as a result of feeding the high-carbohydrate diet to starved rats. When similarly starved rats were refed a diet high in lipid and carbohydrate-free, no induction of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was observed. Lysosomes were not temporarily fragile and purified nuclei did not exhibit increased latency of acid phosphatase activity. Though the evidence presented does not establish a direct correlation between lysosome migration to nuclei as a required function in enzyme induction, the temporal and specific nature of the phenomenon support the hypothesis that liver lysosomal enzymes participate in early signals in the induction of enzymes of lipogenesis.     

Marker enzymes in rat liver vesicles involved in transcellular transport

In order to label the vesicles involved in transcellular transfer (transcytosis) through hepatocytes, polymeric IgA (pIgA) was conjugated to horseradish peroxidase (HRP) and injected into rats. The endosomes containing this ligand at 10 or 20 min after injection were isolated by the diaminobenzidine-induced density-shift procedure and their content in various marker enzymes was measured. The endosomes carrying pIgA-HRP 10 min after injection contained only traces of 5'-nucleotidase and low amounts of alkaline phosphodiesterase I. The estimated marker enzyme content is similar to that observed for the particles containing galactosylated bovine serum albumin conjugated to HRP, a ligand degraded in lysosomes. However, 20 min after injection, the transcytotic endosomes showed a marked enrichment in 5'-nucleotidase and especially in alkaline phosphodiesterase I. The results confirm the heterogeneity of rat liver endosomes and substantiate the concept of distinct endosomal compartments.     

Characterization of the nuclear envelope, pore complexes, and dense lamina of mouse liver nuclei by high resolution scanning electron microscopy

We have used high resolution scanning electron microscopy (SEM) to study the nuclear envelope components of isolated mouse liver nuclei. The surfaces of intact nuclei are covered by closely packed ribosomes which are distinguishable by SEM from nuclear pore complexes. After removal of nuclear membranes with the nonionic detergent Triton X-100, the pore complexes remain attached to an underlying, peripheral nuclear lamina, as described by others. The surface of this dense lamina is composed of particulate granules, 75-150 A in diameter, which are contiguous over the entire periphery. We did not observe the pore-to-pore fibril network suggested by other investigators, but such a structure might be the framework upon which the dense lamina is formed. Morphometric analysis of pores and pore complexes shows their size, structure, and density to be similar to that of other mammalian cells. In addition, several types of pore complex-associated structures, not previously reported by other electron microscope (EM) techniques, are observed by SEM. Our studies suggest that the major role of the dense lamina is associated with the distribution, stability, and perhaps, biogenesis of nuclear pore complexes. Treatment of isolated nuclei with a combination of Triton X-100 and sodium deoxycholate removes membranes, dense lamina, and nuclear pore complexes. The resulting "chromatin nuclei" retain their integrity despite the absence of any limiting peripheral structures.     

Correction of the activity of certain enzymes in the rat liver mitochondrial electron transport chain by derivatives of alpha-tocopheryl acetate in toxic damage to the liver

Correcting action of vitamin E and it's short chain derivative on the activity of some mitochondria electron transport chain enzymes were investigated on models of acute and chronic toxic hepatitis. Inhibition of NADH- and succinate-cytochrome c oxidoreductase complexes activity was established in short term action of xenobiotics. Treatment of rats with CCl4 during 60 days lowered activity of NADH-cytochrome c oxidoreductase complex and significantly increased activity of succinate-cytochrome c oxidoreductase complex and succinate dehydrogenase. Obviously, as a result of long term influence of hepatotoxic agents switching over in rat mitochondria electron transport from NAD-dependent way of substrate oxidation to succinate-dependent way took place. This event could be a part of the body adaptation mechanisms. Vitamin E and its short chain analogue corrected activities of investigated enzymes of mitochondria liver in the animals with acute and chronic hepatitis.     

Characterization of the electron transport system in Brucella abortus.

The electron transport system in Brucella abortus has been characterized. Spectral studies of membrane preparations have indicated the presence of cytochromes a + a3 (maxima at 612 nm), cytochrome b (maxima at 560, 530, and 428 nm), cytochrome c (maxima at 552 and 522 nm), cytochrome o (maxima of carbon monoxide complex at 418 nm), and flavoproteins (minimum at 582 and 450 nm). Cytochromes a + a3 appeared only after cells had reached late log phase, possibly due to lowered oxygen tension in the medium. Dehydrogenases were shown to be present for D-erythritol 1-phosphate, L-lactate, reduced nicotinamide adenine dinucleotide, and succinate. All of the above substrates reduced the electron transport chain and at least some of the flavoproteins, indicating similar pathways of electron transport. N-ethylmaleimide, p-chloromercuribenzoate, and KCN were the only electron transport inhibitors that blocked electron transport by 100%. The system seemed to be uniquely resistant to other electron transport inhibitors.     

Characterization of mevalonate-activating enzymes in the neonatal chick liver.

1. The 5-phosphomevalonate (MVAP) and 5-pyrophosphomevalonate (MVAPP) formation by cell-free extracts from 7-10 days chick liver shows an absolute nucleotide requirement, ATP being the most effective phosphate donor, though ITP and UTP can be used less effectively. 2. Mn2+ is a better activator than Mg2+ at low concentrations (0.1-5.0 mM). At higher concentrations (10.0 mM) Mn2+ produces a clear decrease in the MVAP formation, whereas the maximum MVAPP formation occurs in the presence of 10.0 mM Mg2+. 3. Mevalonate-activating enzymes maintain their activities for 48 hr at 4 degrees C and 24 hr at 37 degrees C. No MVAP is formed when the extracts are heated to 65 degrees C for 10 min. 4. Unlike other vertebrate mevalonate and phosphomevalonate kinases, these enzymes from chick liver are not activated by -SH group protectors as dithiothreitol, reduced glutathione, cysteine or beta-mercaptoethanol. However, the enzymes are found to be sensitive to thiol binding reagents p-hydroxymercuribenzoate and 5,5'-dithiobis-(2-nitrobenzoic acid).     

Characterization of membrane-bound electron transport enzymes from castor bean glyoxysomes and endoplasmic reticulum

Membranes purified from castor bean endosperm glyoxysomes by washing with sodium carbonate exhibited integral NADH:ferricyanide and NADH:cytochrome c reductase activities. The enzyme activities could not be attributed to contamination by other endomembranes. Purified endoplasmic reticulum membranes also contained the redox activities; and marker enzyme analysis indicated minimum cross contamination between glyoxysomal and endoplasmic reticulum fractions. The glyoxysomal redox activities were optimally solubilized at detergent to protein ratios (weight to weight) of 10 (Triton X-100), 50 (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), and 100 (octylglucoside). Detergent in excess of the solubilization optimum was stimulatory to NADH:ferricyanide reductase and inhibitory to NADH:cytochrome c reductase. Endoplasmic reticulum redox activity solubilization profiles were similar to those obtained for glyoxysomal enzymes using Triton X-100. Purification of the glyoxysomal and endoplasmic reticulum NADH:ferricyanide reductases was accomplished using dye-ligand affinity chromatography on Cibacron blue 3GA agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of NADH:ferricyanide reductase preparations purified by rate-zonal density gradient centrifugation, affinity chromatography, and nondenaturing electrophoresis of detergent-solubilized glyoxysomal and endoplasmic reticulum membranes consistently displayed 32- and 33-kDa silver-stained polypeptide bands, respectively.     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

The role of ATP in the transport of rapidly-labeled RNA from isolated nuclei of rat liver in vitro.

To understand the mechanism of the action of ATP on the in vitro transport of the rapidly-labeled RNA from isolated nuclei, the fate of ATP during the incubation as well as the effect of ATP, its analogues and other ribonucleoside triphosphates on the transport was examined and the following results were obtained. (1) More than 97% of added ATP remained acid soluble. No polyadenylation of the rapidly-labeled RNA in the released fraction by added ATP occurred although new polyadenylate segments smaller than 10 S were synthesized. (2) The addition of an ATP-generating system to the reaction mixture restored the initial rate of the release of the rapidly-labeled RNA from isolated nuclei. (3) Among the ribonucleoside triphosphates tested, ATP was most effective in stimulating the release. GTP was about 2/3 as effective as ATP. UTP showed some effect, but CTP showed no effect. EDTA was also non-effective. (4) When no ATP-generating system was added to the reaction mixture, AMP failed to mimic the effect of ATP. However, the combination of AMP and pyrophosphate could take the place of ATP. (5) Both AMP-CPP and AMP-PCP, the ATP analogues, showed the equal degree of their effect on the release, regardless of the position of the methylene bond. From these results, the principal role of ATP in the in vitro transport systems seemed to be its interaction with isolated nuclei to dissociate a structure which retains the rapidly-labeled RNA in the nucleus.