Bovine thymus

Thymine was isolated from hydrolyzates of bovine thymus or spleen in 1893, several years before uracil, but it was not made synthetically until 1901. Unlike uracil, it comes not from ribonucleic but from deoxyribonucleic acids via thymidine (3-D-2 -deoxyribofuranosidothymine). 

Cytosine and thymine were first isolated by hydrolysis of calf thymus tissue by Albrecht Kossel (1853-1927) and A. Neumann during 1893-1894. Thymine s structure was published in 1900 and confirmed over the next several years when it was synthesized by several investigators. In 1903, cytosine was synthesized by Henry Lord Wheeler (1867-1914) and Treat B. Johnson, confirming its structure. Uracil was first isolated in 1900 from yeast nucleic acid found in bovine thymus and herring sperm. The methylation of uracil produces thymine thymine is also called 5-methyluracil because methylation takes place at the fifth carbon in uracil to produce thymine. 

All of the core histones share a conserved 65-residue histone fold.27 28 The arginine-rich histones have a strongly conserved amino acid sequence, histone H4 from pea seedlings differing from that of the bovine thymus by only two amino acids. On the other hand, the lysine-rich HI is almost species-specific in its sequence. Differentiated tissues contain at least seven variant forms of histone HI including proteins designated HI0, Hit, and H5 29-31... 

Thymosin otj 56, isolated by A. L. Goldstein et al. 134) from bovine thymus glands, is important for the development of thymus-dependent T-lymphocytes. 

The amino acid sequences of the cyclophilins remained highly conserved during evolution. This holds in particular for the proteins from eukaryotes. The cyclophilins from bovine thymus and from porcine kidney are identical in sequence (Takahashi et al., 1989), and the human and the bovine cyclophilins share 98% identical amino acids (Haendler et al., 1987). The homology between the mammalian cyclophilins and the cytosolic PPl from E. coli is about 25% (Hayano et al., 1991). The PPIs from porcine kidney and E. coli cytoplasm were used in most of the work on the function of prolyl isomerases as catalysts of protein folding that will be discussed herein. 

Emodin (l,6,8-trihydroxy-3-methylanthraquinone), the active principle of Polygonum cuspidatum (Polygonaceae), was reported to be an inhibitor of the p56 -PTK activity from bovine thymus, with an IC50 of 18.5 pM. When the hydroxyl functions at C-6 or C-8 were blocked by methylation or glycosylation, respectively, the effect disappeared. The inhibition was competitive with respect to ATP and non competitive with respect to the substrate [64]. In a bioassay-guided separation of the anthraquinones found in rhizomes of another Polygonaceae species, rhubarb Rheum... 

Some simple stilbenes isolated from the roots of Polygonum cuspidatum (Polygonaceae) showed moderate inhibitory activity of the bovine thymus P56 " tyrosine kinase activity, when angiotensin 1 was used as a substrate. The most potent were resveratrol (26) and its cis form obtained by photochemical isomerisation. Both compounds showed similar potency against rat brain PKC, whereas resveratrol monoglucosides were less active [81]. 

Beullens M, Van Eynde A, Stalmans W et al (1992) The isolation of novel inhibitory polypeptides of protein phosphatase 1 from bovine thymus nuclei. J Biol Chem 267 16538-16544... 

Uracil was first isolated from herring sperm in 1900 and it originates from ribonucleic acids hydrolytic breakdown gives initially the nucleotide which is further cleaved to uridine (1- -D-ribofuranosidouracil) and finally to uracil. Thymine was originally isolated in 1893 from hydrol-yzates of bovine thymus or spleen. It is one of the four bases in deoxyribonucleic acids which on hydrolysis give thymine via thymidine [3-(2 -deoxy-D-ribofuranosido)-thymine]. 

Amino add Ehrlich asdtes cells (93) (mole/100 mole) Bovine thymus (158) (mole/100 mole) Calf thymus UOO) (mole/100 mole) Pig thymus (92) (mole/100 mole)... 

Histones have been reported to be required for stimulation of synthetase activity in the presence of DNA (43, 60, 93,100,105,117,125, 148,158,159,220,221,233). As noted, however, near maximal rates of poly(ADP-ribose) formation could be obtained utilizing poly(dA) poly(dT), or "active DNA in the absence of added histones (83). Stimulation of poly ADP-ribosylation was observed when histones were added to intact (100%) or partially denatured calf thymus DNA (400%). Yoshihara suggested (83) that added histone binds to denatured DNA and masks its inhibitory action. Histones did not activate by serving as ADP-ribose acceptor in the enzyme reaction catalyzed by the purified bovine thymus poly(ADP-ribose) synthetase. It was known, however. 

In work with the bovine thymus synthetase, Tanaka et al. 212) demonstrated that the enzyme was completely dependent on histone when Mg + was omitted from the assay histone HI was ADP-ribosylated under these reaction conditions. Maximum stimulation and ADP-ribosylation occurred when the ratio of DNA to histone HI was 1 to 10 on a weight basis stimulation was lost when the amount of DNA was increased to 50% of histone HI. All other histone fractions were efiective in stimulating the reaction but none was as active as histone HI. Kawaichi et al. 109) and Ueda and co-workers 222) also observed synthesis of poly(ADP-ribosyl) histone using an apparently homogeneous preparation of rat liver poly( ADP-ribose) synthetase. As opposed to the requirement for a large excess of histone over DNA used by Tanaka et al. 212) to demonstrate modification of Hi, a ratio of DNA to histone of 1 1 (on a weight basis) appeared to be optimal. The amount of ADP-ribose incorporated into histone Hi increased linearly as the DNA to histone Hi ratio was fixed at unity and their concentrations increased from 25 to 150 jug/ml. The ADP-ribose incorporated into histone HI represented, however, only about 50% of the total poly ADP-ribosylation the remainder was polymer associated with the synthetase itself. In these studies 212), Mg + was present at a concentration of 10 mAf. All histone subfractions were tested as acceptor proteins Hi was best, followed by H2B H2A, H3, and H4 were poor acceptors. This order of effectiveness is nearly identical to that found in experiments with intact nuclei (2,28,30, 64, 76,102,103,149,162,164,178-180, 200, 215, 229). 

Yoshihara K, Hashida T, Tanaka Y, Ogushi H, Yoshihara H, KamiyaT (1978) Bovine thymus poly(adenosine diphosphate ribose) polymerase. J Biol Chem 253 6459-6466... 

Bovine thymus poly(ADP-ribose) polymerase was purified to near homogeneity (95%) as described previously [11]. The enzyme reaction was carried out principally as described in a previous report [5], except that the buffer concentration of the reaction mixture was decreased to 5 roM. The reaction mixture contained 5 m/lf Tris-HCl buffer, pH 8.0, 1 vaM dithiothreitol, 10 roM MgQ, 10 Mg of calf thymus DNA, 2 mM NAD", 5 Mg of purified poly(ADP-ribose) polymerase, and an appropriate amount (1 to 15 Mg protein) of various nuclear enzymes in a total volume of 0.2 ml. The mixture was incubated at 25°C for 40 min and the reaction was terminated by chilling the sample on ice or by the addition of a final concentration of 50 mM nicotinamide. 

Fig. 3A-C. Inhibition of DNA ligase II, DNA polymerase jS, and terminal deoxynucleotidyl transferase by poly(ADP-ribos)ylation. Highly purified DNA ligase II (5 Mg, A), DNA polymerase ]3 (5 Mg, B), and terminal deoxynucleotidyl transferase (0.65 Mg, Q from bovine thymus were incubated in a poly(ADP-ribos)ylating reaction mixture as described in Sect. 2 except that the concentration of NAD was changed as indicated. After incubation, respective enzyme activity was assayed. The activity of a control sample incubated without NAD in each experiment was set at 100%...

We also tried to inhibit bovine thymus DNA ligase I [12] using the ADP-ribosylat-ing system. In the preliminary trial, however, the enzyme activity was not affected by poly(ADP-ribos)ylation under the condition employed. Thus, DNA ligase I may not be the acceptor of the poly(ADP-ribose) polymerase reaction, although the final conclusion in this point should be reserved until further examination of this enzyme is completed. 

Yagura et al. [20] have reported that various eukaryotic cells contain at least two forms of DNA polymerase a. One possessed an ability to synthesize poly(dA) with poly(dT) as template and without primer in the presence of ATP and dATP [21]. They also showed that the unique property of this enzyme was due to the association of DNA primase activity with this enzyme [22]. As shown in Fig. 5, when bovine thymus DNA polymerase a fraction obtained from phosphocellulose column chromatography was further purified by DEAE-Sephadex A-50 column chromatography, the enzyme activity was separated into two major sharp peaks. Since one of the peaks was associated with the primase activity, we call this fraction DNA polymerase a with primase (DNA pol a-primase) and the other (fraction II in Fig. 5) DNA polymerase a in this study. When these two enzyme fractions were incubated in a reconstituted poly(ADP-ribos)ylating system, both DNA pol a-primase and DNA polymerase a were strongly inhibited (77 and 50% inhibition, respectively, Table 1). Thus, in order to clarify the mechanism of the inhibition, further study was carried out using mainly the DNA pol a-primase fraction. All the components of poly(ADP-ribos)ylation... 

Fig. 5. DEAE-Sephadex A-50 column chromatography of DNA polymerase a. A crude preparation of DNA polymerase a fraction obtained from phosphocellulose column chromatography of bovine thymus extracts was further fractionated by DEAE Sephadex A-50 column chromatography. Details of the purification procedure will be described elsewhere [33]. Dotted and broken lines indicate absorbance at 290 nm and the concentration of NaHSOj in the elution buffer, respectively. Open and closed circles indicate DNA pol a-primase and DNA polymerase ot activities, respectively...

The effect of poly(ADP-ribos)ylation on DNA polymerase jS was also examined with partiaDy purified enzyme from bovine thymus. In spite of our expectation that the enzyme may not be inhibited by poly(ADP-ribos)ylation, since the enzyme is considered to be main DNA polymerizing enzyme working in DNA repair [24] and poly(ADP-ribos)ylation in vivo stimulates the process [9], DNA polymerase jS was also... 

Teraoka H, Tsukada K (1982) Eukaryotic DNA ligase purification and properties of the enzyme from bovine thymus and immunochemical studies of the enzyme from animal tissues. J Biol Chem 257 4758-4763... 

---