Development of a method to measure sensory perception in children at the European level.

BACKGROUND: The 5-year multilevel epidemiological IDEFICS (Identification and prevention of dietary- and lifestyle-induced health effects in children and infants) study, launched under the Sixth Framework Programme of the European Commission, aims at counteracting the epidemic of dietary- and lifestyle-induced adverse health effects in children. To reveal possible links between overweight/obesity in childhood with taste sensitivity and taste preferences, special procedures were developed for application at the European level. This paper presents these newly developed procedures. METHODS: Testing procedures to assess taste sensitivity for sucrose, sodium chloride, caffeine and monosodium glutamate and taste preferences for sweet, flavour, salty, fatty and umami tastes were developed with 191 children from nursery schools and preschools in northern Germany. To assess test-retest reliability, Cohen's kappa was calculated. RESULTS: The study shows that it is possible to assess taste sensitivity and taste preferences even in young children, provided the framework of the procedures applied is adapted to this scenario. Test-retest reliability was calculated for the procedures applied and the results show that they are very reliable for assessing taste preferences and taste sensitivity in young children. CONCLUSION: It is possible to assess taste sensitivity and taste preferences even in young children, provided the methods applied are adapted to the special requirements that working with young children entail.

Advanced prostate cancer is associated with a decrease in serum luteinizing hormone.

OBJECTIVE: Depletion of serum LH by LHRH agonists is used as a therapeutic treatment in hormone-sensitive prostate cancer (PCa). However, little information on serum LH in different patient groups is available. METHODS: Patients with biopsy-proven PCa, men with BPH (biopsy-proven absence of PCa), two subgroups (serum PSA <4 ng/ml; PCa and BPH), and a PCa cohort before and after radical prostatectomy were analyzed for serum LH, testosterone (T), dihydrotestosterone (DHT), total and free PSA by immunological procedures. RESULTS: PCa patients with cancer volumes >10 cm(3), or with advanced Gleason scores, had significantly lower LH values than men in a cancer-free control group (PSA <4 ng/ml). Eight weeks after radical prostatectomy, LH levels had returned to the level of the control group (p<0.0001). These alterations were not accompanied by corresponding changes of serum androgens. Introduction of a PSA/LH ratio appeared to increase the differences between BPH and PCA groups ranked according to Gleason scores, versus PSA or LH alone. However, the calculation of ROC curves indicated that PSA/LH ratios may not improve the discrimination of malignant and benign forms of the disease, compared to presently used parameters. CONCLUSIONS: A significant reduction of circulating LH is observed in the most advanced forms of PCa. The effect does not come about by T- or DHT-mediated feedback inhibition. Since LH values after prostatectomy returned to practically the same levels as seen in the control group (BPH with <4 ng/ml PSA), it appears that the healthy prostate has no marked influence on serum LH while advanced PCa induces a decrease in serum LH.

A novel covalent modification of nitrogenase in a cyanobacterium.

In extracts of the unicellular cyanobacterium Gloeothece, the Fe-protein of nitrogenase can be separated by SDS-PAGE into two antigenically identifiable components. Unlike the situation in photosynthetic bacteria such as Rhodospirillum rubrum, these two forms do not arise from covalent modification of the protein by ADP-ribosylation. Rather, the Fe-protein of Gloeothece nitrogenase is subjected to modification by palmitoylation.

Molecular heterogeneity of free PSA in sera of patients with benign and malignant prostate tumors.

OBJECTIVE: To analyze free prostate-specific antigen (f-PSA) in sera from patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to detect possible differences in subtypes as potential diagnostic parameters. MATERIALS AND METHODS: PSA was purified from sera by an immunoaffinity procedure developed on the basis of oriented antibody immobilization, and subjected to size exclusion chromatography (SEC), Western blotting, and N-terminal amino acid sequencing. RESULTS: The novel procedure allowed the purification of PSA with high yield from sera containing PSA <10 ng/ml. SEC under nonreducing conditions as well as Western blots demonstrated the presence of several molecular forms of f-PSA. Three of the smaller polypeptides exhibited the N-terminal sequence of PSA while one represented the C-terminal fragment Lys(146)-Pro(237). Shortening of some polypeptides by the N-terminal amino acid Ile(1) suggestive of aminopeptidase action was also observed. No propeptide sequence could be detected, and none of the bands from patient sera reacted with antibodies raised against propeptide antigens. BPH sera expressed higher proportions of smaller PSA fragments per unit p33, and contained significant amounts of fragments <14,000 which appeared to be very low or absent from most PCa sera. CONCLUSIONS: f-PSA as obtained from BPH and PCa sera represents a heterogeneous fraction. The major component (p33) is not in the nicked form and does not contain proPSA. Diagnostic potential could arise from the quantitative differences of the smaller PSA derivatives seen between PCa and BPH sera.

An enzymatic activity in the yeast Sir2 protein that is essential for gene silencing.

Despite its conservation in organisms from bacteria to human and its general requirement for transcriptional silencing in yeast, the function of the Sir2 protein is unknown. Here we show that Sir2 can transfer labeled phosphate from nicotinamide adenine dinucleotide to itself and histones in vitro. A modified form of Sir2, which results from its automodification activity, is specifically recognized by anti-mono-ADP-ribose antibodies, suggesting that Sir2 is an ADP-ribosyltransferase. Mutation of a phylogenetically invariant histidine residue in Sir2 abolishes both its enzymatic activity in vitro and its silencing functions in vivo. However, the mutant protein is associated with chromatin and other silencing factors in a manner similar to wild-type Sir2. These findings suggest that Sir2 contains an ADP-ribosyltransferase activity that is essential for its silencing function.

1-(5-phospho-beta-D-ribosyl)2'-phosphoadenosine 5'-phosphate cyclic anhydride induced Ca2+ release in human T-cell lines.

1-(5-Phospho-beta-D-ribosyl)2'-phosphoadenosine 5'-phosphate cyclic anhydride [2'-phospho-cyclic ADP-ribose, cAdo(2')P(5')PP-Rib] was prepared enzymatically from NADP+ using ADP-ribosyl-cyclase from Aplysia californica. The product was purified by HPLC and characterized by NMR and mass spectroscopy, by conversion to 1-(5-phospho-beta-D-ribosyl)adenosine 5'-phosphate cyclic anhydride (cADP-Rib) by alkaline phosphatase and by resistance to snake venom phosphodiesterase. cAdo-(2')P(5')PP-Rib dose-dependently released Ca2+ from an intracellular, non-endoplasmic reticular Ca2+ pool of permeabilized Jurkat and HPB. ALL T-lymphocytes. In contrast, the closely related compounds 1-(5-phospho-beta-D-ribosyl)3'phosphoadenosine 5'-phosphate cyclic anhydride and 1-(5-phospho-beta-D-ribosyl)cyclic 2',3'-phosphoadenosine 5'-phosphate cyclic anhydride did not induce Ca2+-release from permeabilized T cells. The Ca2+ pool sensitive to cAdo(2')P(5')PP-Rib partially overlapped with the Ca2+ pool sensitive to cADP-Rib recently described in T cells [Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L. & Mayr, G. W. (1995) Characterization of cyclic adenosine diphosphate-ribose-induced Ca2+-release in T-lymphocyte cell lines, J. Immunol. 155, 3353-3359]. Control experiments suggest that the results were neither due to Ca2+ contaminations in the cADP-Rib preparation nor to catabolism of cAdo(2')P(5')PP-Rib to cADP-Rib.

ADP-ribose. A historical overview.

ADP-ribosylation of proteins was first detected as a modification of nuclear proteins by polymeric ADP-ribose residues derived from NAD+. Subsequently, the field developed into three destinct sections: (i) Possible function(s) of poly-ADP-ribosylation with increasing evidence for participation in DNA excision repair and perhaps in DNA recombination; (ii) Mono-ADP-ribosylation as a mechanism to inactivate (by some toxins) or to regulate enzymes/proteins (e.g. in bacterial nitrogen fixation, in protein traffic through membranes, in intercellular communication); (iii) Intramolecular ADP-ribosylation converting NAD+ to cyclic ADP-ribose, a possible Ca(2+)-mobilizing agonist. Thus, NAD+ first known as a cofactor of oxidoreductases has experienced an impressive metamorphosis from a housekeeping coenzyme to a multifunctional group transfering metabolite involved in an increasing number of intracellular and intercellular regulatory loops.

The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex.

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.

[Molecular forms of prostate-specific antigen and their clinical significance]. / Molekulare Formen des PSA und ihre klinische Signifikanz.

PSA is a proteolytic enzyme produced in the prostatic epithelium and secreted into the seminal fluid. PSA can also be a constituent of the serum even under apparently normal conditions. In many cases of prostate cancer (PCa), increased serum concentrations are found. Elevated serum levels, however, are also observed in benign prostatic hypertrophy (BPH), thus limiting the specificity of serum PSA as a marker for prostate cancer. Recently, subfractions of PSA ("free PSA"; PSA-antichymotrypsin complex) were analyzed in order to gain a higher specificity of PSA as a tumor marker. The present paper describes biochemical features and clinical significance of the various PSA subfractions, pointing out an improved discrimination of PCa and BPH by evaluating the ratio "free PSA": total PSA.

Isolation of the myc transcription factor nucleoside diphosphate kinase and the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase by cAMP affinity chromatography.

Cyclic AMP affinity chromatography applied to various mammalian tissue extracts yielded two proteins in addition to the regulatory subunits of protein kinase. This paper characterizes these proteins and provides a simple procedure for their preparation. The polypeptides (36 kDa and a 19 kDa/21 kDa doublet) were isolated from the cAMP matrix by sequential elution with cAMP solutions of increasing concentrations. Microsequencing was accomplished following chemical or enzymic degradation of isolated polypeptides. Partial amino acid sequences of the 36 kDa protein and analyses of its enzymic activity indicated identity with glyceraldehyde-3-phosphate dehydrogenase whilst the lower MW protein proved to be identical with mammalian nucleoside diphosphate kinase subunits. In both cases, binding to cAMP appeared to occur at the nucleotide (NAD and ATP, respectively) sites. In conclusion, we present a one step-procedure, applicable to tissue and cell extracts, which allows the simultaneous isolation of both glyceraldehyde-3-phosphate dehydrogenase and nucleoside diphosphate kinase. This procedure may help to elucidate the multiple functions of these two important enzymes.

Purification of a novel G-protein alpha 0-subtype from mammalian brain.

Three distinct G-protein alpha o-subtypes, i.e. alpha o1, alpha o2 and a newly observed 'alpha o3', are present in membranes of mammalian brain, each appearing as two isoforms on SDS/PAGE. Only alpha o1 and alpha o2 appear to be substrates for pertussis toxin (PTX) when membranes or partially purified proteins are examined. In order to elucidate the apparent PTX-resistance of the third alpha o-subtype, we purified alpha o3 from porcine and bovine brain membranes. During the purification procedures, alpha o3 occurred in its dissociated monomeric form and, together with beta gamma-complexes, as a heterotrimer. In a first attempt, we used purified G-protein alpha i/alpha o-mixtures to obtain a final separation of alpha o3. By using f.p.l.c. anion-exchange chromatography on a Mono Q column, complete separation of alpha i1 and alpha o2 was achieved. Partial resolution of alpha o1, alpha i2 and alpha o3 was observed; alpha o3 was eluted between alpha o1 and alpha i2. If alpha o-subunits free from alpha i contaminants were loaded on to the Mono Q column, all three alpha o-subtypes were resolved. The identity of the third subtype as an alpha o-subtype was confirmed by sequence analysis of tryptic fragments. All three alpha o-subtypes bound GTP[S]. Purified alpha o3 was ADP-ribosylated when subjected to PTX treatment in the presence of beta gamma-subunits, and on SDS/PAGE the mobility of alpha o3 was similar to that of ADP-ribosylated alpha o1. On the basis of results obtained with subtype-specific antibodies, the third alpha o-subtype is immunologically more related to alpha o1 than to alpha o2. Purified alpha o3 failed to reconstitute carbachol-mediated inhibition of Ca2+ current in PTX-pretreated SH-SY5Y-cells, whereas alpha o1 and alpha o2 did successfully restore this effect. We conclude that the novel alpha o3 forms differs from alpha o1 and alpha o2 in its primary structure and may be involved in signal-transduction pathways other than those described for alpha o1 and alpha o2.

Posttranslational modification of nitrogenase. Differences between the purple bacterium Rhodospirillum rubrum and the cyanobacterium Anabaena variabilis.

In the photosynthetic bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulatus post-translational regulation of nitrogenase is due to ADP-ribosylation of the Fe-protein, the dinitrogenase reductase [Burris, R. H. (1991) J. Biol. Chem. 266, 9339-9342]. This mechanism has been assumed to be responsible for nitrogenase modification in a variety of organisms. In the present study, we examined whether ADP-ribosylation holds true for the filamentous cyanobacterium Anabaena variabilis. Genes coding for the nitrogenase-modifying enzymes dinitrogenase reductase-activating glycohydrolase (DRAG) and dinitrogenase reductase ADP-ribosyl transferase (DRAT) from R. rubrum have been subcloned and overexpressed in Escherichia coli. After isolation under anaerobic conditions, both proteins were functional as determined by in-vitro assays using nitrogenase from R. rubrum as substrate. In contrast to the R. rubrum enzyme, nitrogenase from A. variabilis was not affected by DRAG or DRAT. Neither could inactive nitrogenase be restored by DRAG, nor nitrogenase activity suppressed by DRAT. Using specific antibodies against arginine-bound ADP-ribose [Meyer, T. & Hilz, H. (1986) Eur. J. Biochem. 155, 157-165], immunoblotting of the inactive, modified form of the Fe-protein from R. rubrum but not that from A. variabilis showed a strong cross reaction. Furthermore, differently to R. rubrum no ADP-ribosylated proteins could be detected at all, indicating the absence of this posttranslational modification in A. variabilis.

Epigenetic activation of Gi-2 protein, the product of a putative protooncogene, mediates tumor promotion in vitro.

Promotion of 'initiated' JB6 epidermal cells to the tumor phenotype can be effected by 12-O-tetradecanoylphorbol-13-acetate treatment, by stimulation of epidermal growth factor (EGF) receptor activity with EGF or transforming growth factor alpha and by exposure to the isoquinoline derivative H7. When these cells were incubated with pertussis toxin (PTX), induction of anchorage-independent growth by all four promoting substances was suppressed. The inhibition is specific since cell proliferation is not affected, suggesting that activation of a Gi protein is essential for promotion of the epidermal cells. This interpretation is strongly supported by the observation that the wasp poison mastoparan, which is known to mimic receptor-mediated activation of certain Gi proteins, also promoted anchorage independence. Immunological data and partial amino acid sequence analysis of ADP-ribosyl alpha i isolated from PTX-treated JB6 cells indicate that a Gi-2 protein is a mediator to tumor promotion in this system. The inhibitory action of 4-bromophenacyl bromide may point to a coupling of the Gi protein to phospholipase A2. From our data we infer that promoters induce the tumor phenotype in 'initiated' JB6 epidermal cells by activating epigenetically the same Gi protein that in a number of adrenal and ovarian tumors appears to be persistently activated by mutational events.

Suppression of c-fos precursor RNA splicing by the protein kinase C inhibitor H7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine].

In JB6 epidermal cells, induction of fos proto-oncogene expression by phorbol 12-myristate 13-acetate can be inhibited by the protein kinase C (PKC) inhibitor H7 [1-5(isoquinolinesulphonyl)-2-methylpiperazine]. The compound causes also a dose-dependent suppression of fos precursor RNA splicing which, however, appears to react somewhat less sensitively to H7 than does PKC activity. This indicates that H7-induced accumulation of fos precursor RNA is not due to inhibition of PKC. Support for this interpretation comes from the finding that other inhibitors of PKC, such as N-(2-guanidinoethyl)-5-isoquinolinesulphonamide dihydrochloride, sphingosine, staurosporine or N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide, do not suppress splicing when applied at PKC-inhibiting concentrations.

3-Aminobenzamide inhibits cytotoxicity and adhesion of phorbol-ester-stimulated granulocytes to fibroblast monolayer cultures.

Damage of 3T3 fibroblasts as induced by short-term co-cultivation with O2(-)-producing granulocytes, stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was compared with that induced by treatment with enzymically generated O2- and with the alkylating agent dimethyl sulfate. The action of stimulated granulocytes was different in several aspects: (a) DNA fragmented by the products of TPA-stimulated granulocytes showed a biphasic alkaline elution pattern while fragmentation induced by alkylation or by enzymically produced O2- was monophasic. (b) Poly(ADP-ribosyl)ation of nuclear proteins after treatment with TPA-stimulated granulocytes exhibited a lag phase and was, in most experiments, less pronounced than after equitoxic dimethyl sulfate treatment. (c) 3-Aminobenzamide, the most widely used inhibitor of ADP-ribosylation, partially protected target cells from the cytotoxic effects of TPA-stimulated granulocytes, while it enhanced alkylation-induced and O2(-)-induced cytotoxicity. Protection by 3-aminobenzamide in the granulocyte system was apparently not mediated by an inhibition of nuclear poly(ADP-ribosyl)ation. Other inhibitors, like benzamide and nicotinamide, augmented cytotoxicity of TPA-stimulated granulocytes. The unique effect of 3-aminobenzamide in this system appeared to relate to TPA-induced adhesion of the neutrophils to surfaces. In the presence of 1 mM 3-aminobenzamide, but not of benzamide, the adhesion of stimulated granulocytes to 3T3 monolayer cultures was markedly reduced or even abolished. This effect was also seen in granulocyte preparations depleted of monocytes. Since 3-aminobenzamide at the doses applied does not inhibit TPA-induced superoxide production in isolated granulocytes, its specific anticytotoxic effect appears to result from a 'dilution' of granulocyte-derived damaging agents into the medium. Our data suggest that prevention of granulocyte adhesion is likely to reduce tissue damage and carcinogenesis in areas of chronic inflammation.

Nicotinamide and nicotinamide analogues as antitumor promoters in mouse skin.

Phorbol ester-induced promotion of initiated NMRI mouse skin keratinocytes to papillomas could be largely prevented when nicotinamide-like inhibitors of poly(ADP-ribose)polymerase (nicotinamide, benzamide, 3-aminobenzamide) were applied simultaneously with 12-O-tetradecanoylphorbol-13-acetate (TPA). A similar suppression of tumor promotion by nicotinamide analogues was demonstrated in clone 41 JB6 epidermal cells which are promotable by TPA to anchorage-independent growth. The antipromotion effect of nicotinamide analogues, however, does not appear to come about by an inhibition of poly(ADP-ribose)polymerase. Acid analogues of nicotinamide, such as benzoic acid or 3-aminobenzoic acid which do not inhibit the polymerase, showed antipromotion activity similar to that of their corresponding amides. It could also be ruled out that these antipromoters mediate their effect on keratinocytes by a cytostatic action, by scavenging the promoter TPA in a chemical reaction, or by inhibiting protein kinase C. In initiated mouse skin, nicotinamide analogues strongly suppressed TPA-induced accumulation of inflammatory cells and vascular permeability, while epidermal hyperplasia was not significantly affected.

Tumor promotion and depletion of protein kinase C in epidermal JB6 cells.

Promotion of JB6 epidermal cells to anchorage-independent growth requires exposure to TPA for greater than 4 days. Over a similar time span, a practically complete loss of enzymic and immunoreactive proteinkinase C (PKC) equivalents was observed at greater than 10 nM TPA. Promotion did not appear to require (transient) activation of PKC since PKC inhibitors H7 and HA1004 did not prevent but enhanced colony formation in soft agar at concentrations greater than IC50-values. The efficacy of the inhibitors in vivo was shown by their ability to suppress PKC-induced transcription of c-fos gen. PKC inhibitors that interfered with cell proliferation at lower concentrations than those required for PKC inhibition (sphingosine, staurosporin, sangivamycin, trifluoperazine) did not stimulate anchorage-independent growth. As H7 as well as HA1004 were able to promote JB6 cells in the complete absence of TPA, and induced neither depletion nor processing of PKC we postulate that depletion/inactivation rather than activation of PKC correlates with the promotion of epidermal JB6 cells to anchorage-independent growth.

Differentiation of 3T3-L1 pre-adipocytes induced by inhibitors of poly(ADP-ribose) polymerase and by related noninhibitory acids.

To analyze a possible involvement of ADP-ribosylation reactions in 3T3-L1 pre-adipocyte differentiation. ADP-ribosyltransferase activities is permeabilized cells as well as endogenous amounts of protein-bound mono- and poly(ADP-ribose) residues were determined. Also, in vivo labeling with [3H]adenosine of ADP-ribose residues linked to high-mobility-group (HMG) proteins was performed. As an additional probe, the effects of ADP-ribosylation inhibitors and non-inhibitory analogs were studied. Basal and total poly(ADP-ribose) polymerase activities markedly increased prior to the appearance of the differentiation marker glycerol-3-phosphate dehydrogenase. Despite these apparent changes in activity, however, neither protein-bound poly(ADP-ribose) residue nor mono(ADP-ribosyl) groups in histones, nor the NAD content, changed significantly under these conditions. Furthermore, although HMG protein-associated [3H]ADP-ribose was reduced in differentiating [3H]adenosine-labeled cells, the data suggest altered precursor pool labeling rather than a specific decrease in ADP-ribosylated HMG proteins. Non-participation of ADP-ribosylation reactions in 3T3-L1 differentiation is further supported by experiments with inhibitors and non-inhibitory analogs. Benzamide at 0.3-3 mM per se without effect on differentiation, was able to induce specific gene expression when combined with insulin (10(-12)-10(-7) M). Similar effects were seen with benzoate as well as with nicotinamide, 3-aminobenzamide and their corresponding acids. The data indicate that benzamide and analogs have profound effects on chromatin functions that are not mediated by ADP-ribosylation reactions.

ADP-ribosyl proteins formed by pertussis toxin are specifically cleaved by mercury ions.

Various types of ADP-ribosyl protein conjugates were synthesized and their chemical stability was compared with that of cysteine-linked ADP-ribosyl groups as formed by incubation of transducin or Gi/Go proteins with NAD and pertussis toxin. Treatment with 0.1 mM HgCl2 specifically cleaved the cysteine-linked conjugates. This may provide a tool for the quantitation of modified Gi/Go proteins as well as of other acceptors modified by ADP-ribose at cysteine residues in the presence of other ADP-ribosyl proteins.

Immunoquantitation and size determination of intrinsic poly(ADP-ribose) polymerase from acid precipitates. An analysis of the in vivo status in mammalian species and in lower eukaryotes.

Antibodies against pig thymus poly(ADP-ribose) polymerase were obtained with enzyme-hemocyanin conjugates and used for immunoquantitation. The quick-blot procedure used allowed the determination of amounts as low as 1 ng of enzyme from whole cell trichloracetic acid precipitates. When applied to analysis of various human, rodent, and bovine cell types, surprisingly similar amounts of polymerase were found (1-5 ng of pig thymus polymerase equivalents/micrograms of DNA, 2 X 10(5) polymerase molecules/HeLa cell). Also, no significant difference was seen between normal and transformed cells. Polymerase tended to decline in several fibroblast cultures upon reaching confluency, which was not reflected by total polymerase activity. Divergence between total activity and immunogenic equivalents was also seen in alkylated cells and in rat liver treated with phenobarbital. Trichloroacetic acid-insoluble fractions dissolved in sodium dodecyl sulfate buffer could also be used to analyze, by Western blotting, the size distribution of poly(ADP-ribose) polymerase in vivo. Application to various cell types revealed that all mouse and rat cells tested had two immunogenic bands (116 and 98 kDa) of similar intensity. A highly conserved structure of poly(ADP-ribose) polymerase may be deduced from the existence of immunogenic and renaturable 116-kDa polypeptide bands even in the low eukaryotes Physarum polycephalum and Dictyostelium discoideum.