Takeyoshi Minaga

Analysis of larger than tetrameric poly(adenosine diphosphoribose) by a radioimmunoassay in nuclei separated in organic solvents

Anitibodies were prepared against poly(adenosine diphosphoribose) of an average chain length of 40 adenosine diphosphoribose units by repeated injection of the polymer mixed with methylated albumin and adjuvants into rabbits. The antibody was present mainly in the 7 S fraction of the immunoglobulins. A membrane binding assay was developed, and its specificity determined for the detection of (adenosine diphosphoribose)ngreater than4 in organs. The method is suitable for the study of the variation of the polymer content of nuclei. The size recognition of the anti-poly(adenosine diphosphoribose) globulin fraction was the same for polymers composed of 4--40 adenosine diphosphoribose units, but smaller oligomers were not detectible. A quantitative extraction technique was developed and applied for radioimmunoassay of nuclear (adenosine diphosphoribose)n greater than 4. Organs were freeze-clamped, freeze dried, broken into subcellular fragments in a colloid mill, and the nuclear fraction was subsequently separated in organic solvents in order to preserve the polymer. Nicotinamide and nicotinic acid, when administered in vivo, augmented the (adenosine diphosphoribose)n greater than 4 content of rat liver and heart. Tissues of infant pigeons contained larger quantites of (adenosine diphosphoribose)ngreater than4 than tissues of adult rats.

Probable Helical Conformation of Poly(ADP-Ribose)

It is well established that helical conformations of nucleic acids are recognizable from a proportionality between the UV absorbance and CD spectrum [1]. For a helix the component of the absorption band polarized perpendicular to the helix axis gives rise to a unique CD curve. The X max at 258 nm and the crossover point through the 0 line of the CD spectrum at the same wave-length fulfill the theoretical prediction of the helical conformation of poly(ADP-ribose) [2, 3]. Based on the following results, helical conformation of poly(ADP-ribose) was postulated.

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.

[30] Method of induction of cardiac l-ornithine decarboxylase by nicotinamide

Publisher Summary This chapter discusses the method of induction of cardiac L-ornithine decarboxylase by nicotinamide. In this method, male Sprague Dawley rats are fasted 20 h before experiments. Intraperitoneal injections of substances to induce cardiac L-ornithine decarboxylase activity are carried out, by one of the following schedules: nicotinamide or nicotinic acid 5′-methylnicotinamide and thymidine are injected twice at hourly intervals, and 3-isobutylmethylxanthine is administered, by a single injection. Rats are sacrificed, by decapitation, and the hearts are rapidly minced, with scissors, before homogenization at 0°. Homogenates are prepared at 0–4° with a Potter-Elvehjem homogenizer. The time course of induction of cardiac ornithine decarboxylase is described in this chapter. The top curve describes the effect of nicotinamide and the lower curve describes the effect of nicotinic acid. The dose–response relationship between induction and the dose of nicotinamide is explained in this chapter. The rate of decay of cardiac nicotinamide follows first-order kinetics. Other inhibitors of the polyadenosine diphosphoribose system or of NAD+ glycohydrolase have an enzyme-inducing effect similar to nicotinamide, except different agents act, on liver and heart preferentially, in accordance with the selective uptake of these drugs.

[26] Extraction and quantitative determination of larger than tetrameric endogenous polyadenosine diphosphoribose from animal tissues

The validity of the extraction technique was tested by recovery of added 14C-labeled polymer. Recovery was always close to 90%. It is apparent that the predominant chromatin fraction, containing more than 99% of (ADP-R)n>4 is the nonhistone fraction.