ADP-ribosylation of nuclear proteins in normal lymphocytes and in low-grade malignant non-Hodgkin lymphoma cells

Normal lymphocytes and lymphocytes from patients with low-grade malignant non-Hodgkin lymphoma were isolated from blood by a Percoll gradient procedure. Absence of cell proliferation in both cell types was indicated by very low [3H]thymidine incorporation rates. Determination of endogenous protein-bound single ADP-ribose residues by a radioimmunoassay revealed that the leukemic cells had 2.5-times lower levels of the NH2OH-sensitive and a 4-fold lower amount of NH2OH-resistant ADP-ribose . protein conjugate subfractions, respectively, than normal lymphocytes. By contrast, "total" ADP-ribose transferase activity, as measured in homogenates or permeabilized cells in the presence of DNase, was two-times higher in leukemic cells, whereas activity determined in permeabilized cells in the absence of added DNase was practically identical in both cell types. The apparent discrepancy between ADP-ribose transferase activity and endogenous levels of protein-bound single ADP-ribose residues may be explained in part by an enzyme inhibitor present in normal human lymphocytes. NAD + NADH levels were decreased 2.5-fold in the leukemic cells. This decrease, however, does not explain the reduced levels of mono(ADP-ribose) . protein conjugates since the ratio of protein-bound single ADP-ribose residues to NAD is distinctly different in leukemic lymphocytes compared to normal lymphocytes.     

Enzyme encapsulation in permeabilized Saccharomyces cerevisiae cells

The Saccharomyces cerevisiae cell wall provides a semipermeable barrier that can retain intracellular proteins but still permits small molecules to pass through. When S. cerevisiae cells expressing E. coli lacZ are treated with detergent to extract the cell membrane, beta-galactosidase activity in the permeabilized cells is approximately 40% of the activity of the protein in cell extract. However, the permeabilized cells can easily be collected and reused over 15 times without appreciable loss in activity. Cell wall composition and thickness can be modified using different cell strains for enzyme expression or by mutating genes involved in cell wall biosynthesis or degradation. The Sigma1278b strain cell wall is less permeable than the walls of BY4742 and W303 cells, and deleting EXG1, which encodes a 1,3-beta-glucanase, can further reduce permeability. A short Zymolyase treatment can increase cell wall permeability without rupturing the cells. Encapsulating multiple enzymes in permeabilized cells can offer kinetic advantages over the same enzymes in solution. Regeneration of ATP from AMP by adenylate kinase and pyruvate kinase encapsulated in the same cell proceeded more rapidly than regeneration using a cell extract. Combining permeabilized cells containing adenylate kinase with permeabilized cells containing pyruvate kinase can also regenerate ATP from AMP, but the kinetics of this reaction are slower than regeneration using cell extract or permeabilized cells expressing both enzymes.     

Aspartokinase II from Bacillus subtilis is degraded in response to nutrient limitation

Aspartokinase II from Bacillus subtilis was shown by immunochemical methods to be regulated by degradation in response to starvation of cells for various nutrients. Ammonium starvation induced the fastest aspartokinase II decline (t1/2 = 65 min), followed by amino acid starvation (t1/2 = 80 min) and glucose limitation (t1/2 = 120 min). Loss of enzyme activity was closely correlated with the disappearance of the alpha subunit; degradation of the beta subunit was somewhat delayed or slower under some conditions. Pulse-chase experiments demonstrated that aspartokinase II was stable during exponential growth; the synthesis of the enzyme rapidly declined in response to nutrient exhaustion. The degradation of aspartokinase II was interrupted by inhibitors of energy production and protein synthesis but was not changed in a mutant lacking a major intracellular protease. Mutants lacking a normal stringent response displayed only a slight decrease in the rate of aspartokinase II degradation, even though aspartate transcarbamylase was degraded more slowly in the same mutant cells. These results indicate that although energy-dependent degradation of biosynthetic enzymes is a general phenomenon in nutrient-starved B. subtilis cells, the degradation of specific enzymes probably involves different pathways.     

Poly(ADP-ribose) metabolism appears normal in EM9, a mutagen-sensitive mutant of CHO cells

EM9 is a mutagen-sensitive CHO cell whose phenotype resembles that of normal CHO cells exposed to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis. This phenotype suggested that EM9 might be defective in poly(ADP-ribose) metabolism, but we now cannot find any abnormality in the synthesis or in the degradation of poly(ADP-ribose) in permeabilized EM9 cells. Thus the effects of 3-aminobenzamide on wild-type cells may be due to the inhibition of processes other than poly(ADP-ribose) synthesis. 3-Aminobenzamide enhances the cytotoxicity of EMS toward EM9 and control cells to the same degree.     

Differential poly(ADP-ribose) metabolism in repair-proficient and repair-deficient murine lymphoma cells

The UV254nm-sensitive, tumorigenic murine lymphoma cell line LY-R undergoes spontaneous conversion into a UV254nm-resistant, non-tumorigenic LY-S subline after prolonged culture in vitro. Here we describe that this conversion leads to distinct changes in several features of intranuclear ADP-ribose polymer metabolism, which may contribute to the altered processing of DNA damage in these cells. The UV254nm-resistant LY-S cells contain 3-fold higher levels of ADP-ribose polymers than LY-R cells. The initial catabolic rate of degradation of these polymers is more than 6-fold higher in LY-S cells. UV254nm irradiation raises the catabolic rates of ADP-ribose polymers in both cell lines. As a consequence, the polymer half-lives decrease from 15 min to 4 min in LY-S cells, and from 96 min to 19 min in LY-R cells. In addition, the rapidly turning over fraction of polymers is much larger in the UV254nm-resistant LY-S cells. These data suggest that the catabolism of poly(ADP-ribose) may be an important factor in the biological expression of DNA damage.     

Cytosolic components are required for proteasomal degradation of newly synthesized apolipoprotein B in permeabilized HepG2 cells.

Recent studies have proposed that post-translational degradation of apolipoprotein B100 (apoB) involves the cytosolic ubiquitin-proteasome pathway. In this study, immunocytochemistry indicated that endoplasmic reticulum (ER)-associated proteasome molecules were concentrated in perinuclear regions of digitonin-permeabilized HepG2 cells. Signals produced by antibodies that recognize both alpha- and beta-subunits of the proteasome co-localized in the ER with specific domains of apoB. The mechanism of apoB degradation in the ER by the ubiquitin-proteasome pathway was studied using pulse-chase labeling and digitonin-permeabilized cells. ApoB in permeabilized cells incubated at 37 degrees C in buffer alone was relatively stable. When permeabilized cells were incubated with both exogenous ATP and rabbit reticulocyte lysate (RRL) as a source of ubiquitin-proteasome factors, >50% of [3H]apoB was degraded in 30 min. The degradation of apoB in the intact ER of permeabilized cells was much more rapid than that of extracted [3H]apoB incubated with RRL and ATP in vitro. The degradation of apoB was reduced by clasto-lactacystin beta-lactone, a potent proteasome inhibitor, and by ubiquitin K48R mutant protein, an inhibitor of polyubiquitination. ApoB in HepG2 cells was ubiquitinated, and polyubiquitination of apoB was stimulated by incubation of permeabilized cells with RRL. These results suggest that newly synthesized apoB in the ER is accessible to the cytoplasmic ubiquitin-proteasome pathway and that factors in RRL stimulate polyubiquitination of apoB, leading to rapid degradation of apoB in permeabilized cells.     

Production of trehalose by permeabilized Micrococcus QS412 cells

Permeabilized Micrococcus QS412 cells were used to produce trehalose from starch through catalysis of maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase in the cells. The permeabilized cells could omit the enzyme purification and simplify the immobilization of intracellular enzymes. The reagent, reagent dosage and time of cell permeabilization treatment were determined. The maximum trehalose biosynthesis activity was obtained after the cells were treated with 5% (w/v) of toluene at 30 °C for 40 min. Reaction conditions of trehalose synthesis of permeabilized cells were optimized. The yield of trehalose was up to 188 mg/g wet permeabilized cells in pH 8.0, 100 mmol/l phosphate buffer at 30 °C after 12 h reaction. Batch reactions showed that the permeabilized cells could be reused for 16 cycles in the biosynthesis reaction. The total trehalose yield was up to 2.5 g/g wet permeabilized cells. Development of permeabilized cells provide a new cheaply alternative technology for trehalose production.     

In situ assay of intracellular enzymes of yeast (Kluyveromyces fragilis) by digitonin permeabilization of cell membrane

The yeast, Kluyveromyces fragilis was permeabilized to a number of low-molecular-weight substrates using digitonin. The activities of intracellular yeast enzymes, viz., alcohol dehydrogenase (ADH), beta-galactosidase, glucose-6-phosphate dehydrogenase, aspartase, and hexokinase were found to be much higher in the permeabilized cells than the untreated cells. The optimum conditions for permeabilization with reference to ADH were 0.1% digitonin at 37 degrees C for 15 min. The ADH activity in permeabilized cells was several-fold higher than that in cell free extracts prepared by either physical or chemical methods.     

A novel technique for the preparation of osmotically stabilized and permeabilized cells of extremely halophilic bacteria.

The cells of Haloferax mediterannei were stabilized by cross-linking with 0.5% glutaraldehyde for 10 min. Such cells were found to be osmotically stable even when suspended in water. The stabilized cells could be permeabilized by treatment with chloroform without leakage of intracellular components. No significant difference in the properties of an intracellular enzyme aldolase was observed, using either cell-free extract or the osmotically stabilized and permeabilized cells. This novel technique can serve as a useful tool for studying in situ regulatory characteristics of intracellular functions in halobacteria and can also help in their re-use under more stabilized conditions for biotechnological applications.     

Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells

Permeabilization was evaluated as a rapid method to prepare mammalian cells for subcellular enzyme activity measurement. It was observed that enzymes can be measured directly in cell suspensions permeabilized by Triton X-100 and digitonin with various concentrations. Total enzyme activities measured in permeabilized cells were identical to those measured in sonicated cells showing that permeabilization can replace the more complicated sonication method. Tuning of digitonin concentration allowed selective permeabilization of plasma and mitochondrial membranes. This was studied by analyzing the release of extramitochondrial and mitochondrial marker enzymes on treatment with different concentrations of the agent. Solely the plasma membrane was permeabilized by using 0.01–0.02% (w/v) digitonin. Access to all cellular enzymes was achieved by using 0.05% (v/v) Triton X-100. This selective permeabilization was further evaluated in a 96-well plate format by testing additional marker enzymes and additional cell lines, Hep G2 and CHO-K1, applying the developed protocol. The presented method is well suited for the high-throughput analysis of subcellular localization and activity of enzymes. The method is simple and enables one to distinguish between mitochondrial and extramitochondrial activities, which is usually achieved only by much more complicated and time-consuming cell preparation.     

Cell cycle effects on the basal and DNA-damaging-agent-stimulated ADPRT activity in cultured mammalian cells

ADP-ribosyl transferase (ADPRT) is a DNA-dependent chromatin-associated enzyme which covalently attaches ADP-ribose moieties derived from NAD+ to protein acceptors to form poly(ADP-ribose). ADPRT activity is strongly stimulated by breaks in DNA, and it is suggested that its activity is required for efficient DNA excision repair. In this paper, a cell-cycle-dependent fluctuation of basal ADPRT activity was demonstrated by measuring it in permeabilized FL cells. The cell used was subjected to arginine starvation for 48 h before being released from the block by replacement of deficient medium with complete medium and cells in different proliferating stages were traced by [3H]TdR pulse labelling and obtained at different intervals after block release. The peak basal ADPRT activity appeared 4-6 h after the appearance of the peak of DNA synthesis. After treating the cells with MNNG (10(-4) M), MMS (10(-3)-10(-4) M) and 4NQO (10(-5) M) for 90 min just after release of the block, the ADPRT activity was markedly stimulated. It was further demonstrated that the effects of MNNG/4NQO and cell cycle influence on the level of poly(ADP-ribose) synthesis appear to be additive. While concerning MMS, quite a different pattern of ADPRT stimulation in the cell cycle was demonstrated, i.e., the activity of ADPRT stimulation of 10(-3) M MMS was found to be completely dependent on the basal ADPRT activity. In the cells with the highest basal ADPRT activity 12 h after block release, the MMS-induced ADPRT stimulation could not be observed. It was suggested that more than one pathway might be present in ADPRT stimulation induced by DNA-damaging chemicals, and the cells synchronized in late G1 stage might be the most suitable for demonstrating poly(ADP-ribose) synthesis after DNA damage.     

Resistance of ADP-ribosylated histones and HMG proteins to proteases.

Calf thymus histones (individually isolated or mixtures) and high mobility group proteins were ADP-ribosylated in vitro using [32P]NAD+ and immobilized purified poly(ADP-ribose) polymerase. The modified histones were then subjected to V8 protease or alpha-chymotrypsin digestion and the resulting peptides were separated by electrophoresis on acetic acid-urea-Triton gels. It was found that in vitro ADP-ribosylated histones were much more resistant to proteases than unmodified histones. A similar approach was applied to histones modified by the endogenous poly(ADP-ribose) polymerase in permeabilized NS-1 mouse myeloma cells in culture. In this case, the proteases could not discriminate between modified and unmodified histones and putative mono(ADP-ribosyl)ated peptides appeared in a digestion frame corresponding to that of bulk peptides. These differences are most probably due to the specificity or number of ADP-ribose groups added to the histones by the endogenous or exogenous poly(ADP-ribose) polymerase. Thus, depending on the size of poly(ADP-ribose) attached to nuclear proteins, these modified proteins might display different degrees of resistance to proteolysis.     

Poly(ADP-ribose) polymerase 1 accelerates single-strand break repair in concert with poly(ADP-ribose) glycohydrolase

Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.     

Tert-butyl hydroperoxide selectively inhibits mitochondrial respiratory-chain enzymes in isolated rat hepatocytes

Sensitivity of various mitochondrial enzymes to oxidative damage was tested on isolated rat liver hepatocytes permeabilized by digitonin. In permeabilized hepatocytes normal respiratory control values were obtained and mitochondrial membranes remained intact. Respiratory rates of NADH-dependent (glutamate + malate, palmitylcarnitine + malate) and flavoprotein-dependent (succinate) substrates were determined in hepatocytes exposed for 5 min to 0.5-3 mM tert-butyl hydroperoxide before addition of digitonin. Our data showed that oxidation of NADH-dependent substrates is much more sensitive to oxidative stress than oxidation of flavoprotein-dependent ones, evidently due to the modification of iron-sulfur clusters or SH groups in the NADH dehydrogenase enzyme complex (Complex I).     

Improvement of oxidoreductase stability of cethyltrimethylammoniumbromide permeabilized cells ofZymomonas mobilis through glutaraldehyde crosslinking

Summary Permeabilization ofZymomonas mobilis with CTAB(Cetyltrimethylammoniumbromide) was investigated in order to obtain a stable process for sorbitol production in the immobilized system. The optimum conditions for sorbitol formation were treating cells with 0.2% CTAB at 4°C for 10 min. For the immobilized system permeabilized cells were treated with glutaraldehyde to improve the system with cross-linking of enzymes. In this way, no significant loss of enzyme activity was apparent during 30 day operation in a continuous process. The productivity of the continuous process at a dilution rate 0.2 h^–1 was 6.51g/L-h for sorbitol. The CTAB-permeabilized cells could be used to produce sorbitol and gluconic acid simultaneously in the long term continuous process.     

Over-expression of DAAO and catalase in Kluyveromyces marxianus through media optimization, permeabilization and GA stabilization techniques

The selected thermotolerant, lactose-utilizing yeast strain Kluyveromyces marxianus NBIMCC 8362 possesses high specific d-amino acid oxidase activity (60Ug(-1)), which was increased nine-fold (545Ug(-1)) by design of the growth medium and conditions for d-amino oxidase induction. Applying an optimized simple and rapid procedure for chemical permeabilization of K. marxianus cells with the cationic detergent cetyltrimethylammonium bromide, the enzyme activities (d-amino acid oxidase and catalase) of the cells have been further increased for up to 43- and 58-fold, respectively. However, the enzyme activities of the permeabilized cells decreased rapidly due to the leakage of the enzymes. Treating the permeabilized cells with 0.1% glutaraldehyde at 4°C for 10min stabilized the enzyme in the cells and prevented their outflow. The process is stable for 10 cycles and the productivity measured was 16.6mmmoll(-1)h(-1). The d-alanine transformation efficiency of K. marxianus permeabilized and GA entrapted cells was 98%.     

Glycolysis compared in intact, permeabilized and sonicated L-929 cells.

The effects of incubation time and cell density on glycolytic rate were examined in suspensions of intact, permeabilized and sonicated L-929 cells. Sonicates exhibited strong dependence on cell density and a distinct lag in glycolytic rate, while intact cells showed no cell density dependence and linear glycolytic rates. Permeabilized cells exhibited linear glycolytic rates, but sometimes showed dependence on cell density. Rates of lactate production (nmol at 30 min/10(6) cells) were highest in sonicates and lowest in intact cells. These results are interpreted as support for the previously proposed hypothesis that enzymes of the glycolytic pathway are highly organized in intact L-929 cells.     

Poly(ADP-ribose) polymerase activity in intact or permeabilized leukocytes from mammalian species of different longevity

Poly(ADP-ribosyl)ation is a eukaryotic posttranslational protein modification catalyzed by poly(ADP-ribose) polymerase (PARP), a highly conserved nuclear enzyme which uses NAD as substrate. We have previously tested PARP activity in permeabilized mononuclear blood cells (MNC) from 13 mammalian species as a function of the species-specific life span. A direct and maximal stimulus of PARP activation was provided by including saturating amounts of a double-stranded ollgonucleotide in the PARP-reaction buffer. The data yielded a strong positive correlation between PARP activities and the species' maximal life spans (r=0.84; p0.001). Here, we investigated the formation of poly(ADP-ribose) inliving MNC from two mammalian species with widely differing longevity (rat and man) by immunofluorescence detection of poly(ADP-ribose). The fraction of positive cells was recorded, following -irradiation of intact MNC, as a semiquantitative estimation of poly(ADP-ribose) formation. Human samples displayed a significantly higher percentage of positivity than did those from rats, consistent with our previous results on permeabilized cells. While rat MNC had a higher NAD content than human MNC, the number of radiation-induced DNA strand breaks was not significantly different in the two species. Since poly(ADP-ribosyl)ation is apparently involved in DNA repair and the cellular recovery from DNA damage, we speculate that the higher poly(ADP-ribosyl)ation capacity of long-lived species might more efficiently help to slow down the accumulation of unrepaired DNA damage and of genetic alterations, as compared with short-lived species. (Mol Cell Biochem138: 85–90, 1994)