Eva Kirsten

[14] Stabilization of mitochondrial functions with digitonin

Publisher Summary The great diversity of intrinsic stabilities of various mitochondrial systems, especially those contributing to the intactness of the transfer capabilities of the inner mitochondrial membrane, poses special difficulties to experimentalists. The acceptor control index measured within short periods after isolation of mitochondria by conventional methods is a correct test of mitochondrial intactness, yet the specific activity of the adenosine triphosphate (ATP) synthetase system may vary greatly in various preparations exhibiting the same acceptor control index. Therefore, it is evident that remaining within the realm of oxidative phosphorylation, two diverse criteria of apparent functional intactness of the inner mitochondrial membrane can be obtained. This phenomenon is readily demonstrable if conventionally prepared mitochondria are incubated without substrates or cofactors at 30 ° C for 10–20 min and both acceptor control index and ATP synthetase are simultaneously monitored. The relatively slow mitochondrial processes play a significant role in experimental work when intracellular regulation of mitochondria is to be investigated with isolated mitochondria because functional intactness of these particles must be maintained for prolonged periods in vitro .

Coenzymatic activity of randomly broken or intact double-stranded DNAs in auto and histone H1 trans-poly(ADP-ribosylation), catalyzed by poly(ADP-ribose) polymerase (PARP I).

The enzymatic transfer of ADP-ribose from NAD to histone H1 (defined as trans-poly(ADP-ribosylation)) or to PARP I (defined as auto-poly(ADP-ribosylation)) was studied with respect to the nature of the DNA required as a coenzyme. Linear double-stranded DNA (dsDNA) containing the MCAT core motif was compared with DNA containing random nicks (discontinuous or dcDNA). The dsDNAs activated trans-poly(ADP-ribosylation) about 5 times more effectively than dcDNA as measured by V max. Activation of auto-poly(ADP-ribosylation) by dcDNA was 10 times greater than by dsDNA. The affinity of PARP I toward dcDNA or dsDNA in the auto-poly(ADP-ribosylation) was at least 100-fold lower than in trans-poly(ADP-ribosylation) (K a = 1400versus 3–15, respectively). Mg2+ inhibited trans-poly(ADP-ribosylation) and so did dcDNA at concentrations required to maximally activate auto-poly(ADP-ribosylation). Mg2+ activated auto-poly(ADP-ribosylation) of PARP I. These results for the first time demonstrate that physiologically occurring dsDNAs can serve as coenzymes for PARP I and catalyze preferentially trans-poly(ADP- ribosylation), thereby opening the possibility to study the physiologic function of PARP I.

Molecular interactions between poly(ADP‐ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding

The molecular interactions of poly(ADP‐ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the ‘global’ Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP‐ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the ‘global’ Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I–Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S‐transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the ‘C clamp’ model derived from X‐ray crystallography.

Cellular regulation of ADP-ribosylation of proteins: IV. Conversion of poly(ADP-ribose) polymerase activity to NAD-glycohydrolase during retinoic acid-induced differentiation of HL60 cells

Two enzymatic activities of the nuclear enzyme poly(ADP-ribose) polymerase or transferase (ADPRT, EC 2.4.2.30), a DNA-associating abundant nuclear protein with multiple molecular activities, have been determined in HL60 cells prior to and after their exposure to 1 microM retinoic acid, which results in the induction of differentiation to mature granulocytes in 4-5 days. The cellular concentration of immunoreactive ADPRT protein molecules in differentiated granulocytes remained unchanged compared to that in HL60 cells prior to retinoic acid addition (3.17 +/- 1.05 ng/10(5) cells), as did the apparent activity of poly(ADP-ribose) glycohydrolase of nuclei. On the other hand, the poly(ADP-ribose) synthesizing capacity of permeabilized cells or isolated nuclei decreased precipitously upon retinoic acid-induced differentiation, whereas the NAD glycohydrolase activity of nuclei significantly increased. The nuclear NAD glycohydrolase activity was identified as an ADPRT-catalyzed enzymatic activity by its unreactivity toward ethenoadenine NAD as a substrate added to nuclei or to purified ADPRT. During the decrease in in vitro poly(ADP-ribose) polymerase activity of nuclei following retinoic acid treatment, the quantity of endogenously poly(ADP-ribosylated) ADPRT significantly increased, as determined by chromatographic isolation of this modified protein by the boronate affinity technique, followed by gel electrophoresis and immunotransblot. When homogenous isolated ADPRT was first ADP-ribosylated in vitro, it lost its capacity to catalyze further polymer synthesis, whereas the NAD glycohydrolase function of the automodified enzyme was greatly augmented. Since results of in vivo and in vitro experiments coincide, it appears that in retinoic acid-induced differentiated cells (granulocytes) the autopoly(ADP-ribosylated) ADPRT performs a predominantly, if not exclusively, NAD glycohydrolase function.

Inhibition of carcinogen-induced cellular transformation of human fibroblasts by drugs that interact with the poly(ADP-ribose) polymerase system: Initial evidence for the development of transformation resistance

Two types of interactions of 13 drugs with human fibroblasts were determined: (a) I 50 of nuclear poly(ADP‐ribose) polymerase, as assayed with isolated nuclei in vitro, and (b) the non‐toxic concentration of drugs that prevented carcinogen‐induced cell transformation of intact fibroblasts (RCF1). In general, RCF1 was much lower than I 50, and one antitransformer did not inhibit the enzyme in vitro, indicating that low‐affinity enzyme inhibitory sites appear to play no role in the mechanism of prevention of cell transformation. Two enzyme inhibitors, caffeine and 1‐methylnicotinamide, exhibited no antitransforming activity. Benzamide when applied in population doubling 1 induced resistance to cell transformation in population doubling 6 by carcinogens added at this stage.

Isolation of adenosine diphosphoribosyltransferase by precipitation with reactive red 120 combined with affinity chromatography

The DNA-associating enzyme, adenosine diphosphoribosyltransferase, has been isolated from calf thymus by selective precipitation with a solution of dihydroxy Reactive Red 120, followed by extraction of the enzyme from the precipitate with 2 M KCl and an on-line train of three successive column chromatographic steps, including a final 3-aminobenzamide-Sepharose 4B affinity chromatography. The method yields 8-9 mg of more than 95% homogeneous enzyme protein per kilogram starting material and requires about 3 working days. This dye precipitation method is distinct from affinity precipitation, since it involves the binding of the dye to both nonspecific sites and the substrate and DNA sites of the transferase as indicated by enzyme inhibition by dihydroxy Reactive Red 120 at both enzyme sites.

Cellular regulation of poly(ADP) ribosylation of proteins. I. Comparison of hepatocytes, cultured cells and liver nuclei and the influence of varying concentrations of NAD.

The in vitro rates (vinit) of poly(ADP-ribose) polymerase of permeabilized rat hepatocytes and of nuclei, isolated from hepatocytes, did not differ significantly. Incubation beyond 3 min resulted in diminished poly(ADP) ribosylation in hepatocytes compared with nuclei, coinciding with high rates of plasma membrane-associated NAD-glycohydrolase. Cultured cells (Drosophila Kc cells, gliosarcoma 9L, human fibroblasts and mouse spleen lymphocytes) exhibit variations of NAD-glycohydrolase and poly(ADP-ribose) polymerase activities and the assessment of poly(ADP-ribose) polymerase activity in permeabilized cells requires simultaneous assay of NAD-glycohydrolase. In rat liver nuclei during 10 min incubation with 500 microM NAD, 40% of NAD is consumed, 10% ADP-ribose is bound to proteins, and 20% ADP-ribose, 5% AMP and 2.7% adenosine are liberated. As determined by solvent partitioning (Jackowski, G & Kun, E, J biol chem 258 (1983) 12587) [1], the phenol-soluble protein-ADP-ribose fraction represents largely mono(ADP)-ribose protein adducts, whereas the H2O-soluble phase contains poly(ADP)-ribosylated proteins. The quantity of ADP-ribose protein adducts, the chain length of oligomers and the nature of apparent acceptor proteins in liver nuclei vary significantly with the concentration of NAD as substrate. At 500 microM NAD concentration the quantity of ADP-ribose containing adducts was in the nmol per mg DNA range, the polymers are long chains and the acceptor proteins predominantly non-histone proteins. At 0.1 microM NAD as substrate pmol quantities of monomeric ADP-ribose adducts per mg DNA were formed and the main acceptors were sharply discernable on the basis of molecular mass as histones, high mobility non-histone proteins, two protein groups of a mass of 66 and 44 kD respectively, and the poly(ADP-ribose) polymerase enzyme protein of 119 kD mass. Whereas products in the presence of 0.1 microM NAD may indicate acceptors of highest reactivity, protein adducts formed in the presence of 500 microM NAD resemble a pattern found in vivo.

The invivo effect of benzamide and phenobarbital on liver enzymes: Poly(ADP-ribose) polymerase, cytochrome P-450, styrene oxide hydrolase, cholesterol oxide hydrolase, glutathione S-transferase and UDP-glucuronyl transferase

Rats fed a synthetic diet containing 0.25% benzamide, 0.1% phenobarbital, separately or in combination, for two weeks showed a significant augmentation in the activity of nuclear poly(ADP-ribose) polymerase as well as changes in various nuclear, microsomal and cytosolic liver enzymes involved in the metabolism of xenobiotics. A selective depression of microsomal styrene oxide hydrolase activity by benzamide feeding, and a contrasting augmentation by phenobarbital, were confirmed by immunological titration of the enzyme-protein content suggesting actual enzyme repression and induction. The NAD content of these livers is not altered significantly as a result of benzamide and phenobarbital feeding, indicating that the changes in enzymes are not a result of non-specific toxic effects.

Cellular regulation of ADP-ribosylation of proteins: III. Selective augmentation of in vitro ADP-ribosylation of histone H3 in murine thymic cells after in vivo emetine treatment☆

Thymic cells were isolated at intervals of between 0 and 144 h from mice that received one intraperitoneal injection of emetine (33 mg/kg), and thymus weight, incorporation of [14C]leucine into proteins and [3H]thymidine into DNA in intact thymic cells, as well as initial rates of protein ADP-ribosylation in permeabilized cells [A. Sóoki-Tóth, F. Asghari, E. Kirsten, and E. Kun (1987) Exp. Cell Res. 170, 93] were simultaneously monitored. The effect of emetine as an inhibitor of protein synthesis [F. Antoni, N. G. Luat, I. Csuka, I. Oláh, A. Sóoki-Tóth, and G. Bánfalvi (1987) Int. J. Immunopharmacol. 9, 333] corresponds to the induction of sequential cellular events, such as cell exit and remigration, by other antimitotic agents [C. Penit and F. Vasseur (1988) J. Immunol. 140, 3315] and produces an activation of proliferation of cells reentering into this organ. Proliferation, as demonstrated by a large increase in DNA synthesis and entrance into S phase, was kinetically related to an apparent increase in poly(ADP-ribose) polymerase activity in thymic cells and a highly significant in vitro ADP-ribosylation of histone H3. Since no DNA fragmentation occurred in thymic cells, as tested by a fluorometric technique [C. Birnboim and J. J. Jevac (1981) Cancer Res. 41, 1889], it is probable that a selective activation of poly(ADP-ribose) polymerase may have been induced in cells that undergo differentiation and proliferation while repopulating the thymus.

Coincidence of subnuclear distribution of poly(ADP-ribose) synthetase and DNA polymerase β in nuclei of normal and regenerating liver

An increase of hepatic poly(ADP)-ribosylation of predominantly non-histone chromatin proteins occurs at an early pre-cancerous state following treatment with dimethyl nitrosamine [ 11. This increase in poly(ADP)-Gbosylation is specific for the precancerous state and an opposite effect is induced by growth hormone [2]. In search of the mechanism of the dimethyl nitrosamine-induced increase in poly(ADP)ribosylation it was apparent that no measurable DNA fragmentation was detectable in vivo [l] therefore, the putative stimulatory effect of this process on poly(ADP-ribose) synthetase activity [3] seemed unlikely. Two distinct types of mechanisms are recognized that are germane to the carcinogenicity of dimethyl nitrosamine: (1) Covalent modification of DNA and of other macromolecules [4-71. A subsequent excision repair of modified DNA is also generally known but the cellular physiology of this process is poorly understood. (2) Cancer promotion, which is the second broadly defined stage of the 2step processes of carcinogenesis [ 81. Partial hepatectomy or cell death-induced by CCL, and subsequent regeneration [9,10] are powerful promotors and can initiate carcinogenesis following an otherwise ineffective dose of carcinogen. Dimethyl nitrosamine alone at a certain dose produces cell death and induces regeneration, thus requires no promotor [ 1 l] like partial hepatectomy. The dose of dimethyl nitrosamine used in [l] is therefore likely to have produced both stages of carcinogenesis. To determine whether or not the promotor process was responsible for the increase in protein poly(ADP)ribosylation [l] we determined the effect of surgicallyinduced liver regeneration [ 121 on, both poly(ADPribose) synthetase activity and on DNA synthesis. Instead of assaying whole nuclei, we have chosen to determine the membrane association of these 2 systems in normal and regenerating liver, because in both prokaryotes [13-151 and eukaryotes [16,17], ‘Mband’associated DNA and RNA synthetase activities are directly relevant to cell division. In mitochondria, the protein ADP-ribosylating system is significantly associated with the mitochondrial ‘M-band’ fraction and ADP-ribosylation in mitochondria inhibits DNApolymerase +y [ 18 ,191. The subnuclear association of the poly(ADP)-ribosylating system with the ‘M-band’ fraction had not been studied.