Elution volume (ml) Fig. 1. Isoelectric focusing of DNAases 4 and 5 0 , DNAase activity with denatured DNA; 0 , DNAase activity with native DNA; 1 unit of enzyme activity is that which hydrolyses 1nmol of DNA in 30min at 37°C. ---- PH. 9

fraction with pI3.5 was active only against native DNA and was very unstable. Thus at least five DNAase species are present in Chlamydomonasreinhardii. It is not clear whether any of these activities could be equated with the endonuclease activity described by Small & Sparks (1972). In addition, DNAase activity has been found intimately associated with DNA polymerase a of this organism (Ross & Harris, 1976), and this is likely to be distinct from the above activities. In common with most other organisms therefore, Chlamydomonasreinhardii possesses multiple species of DNAase. We thank the Science Research Council for financial support and the award of a studentship to G. C.L. T. Kates, J. R. & Jones, R. F. (1964) J. Cell. Comp. Physiol. 63, 157-164 Ross, C. A. & Harris, W. J. (1976) Biochem. Soc. Trans. 4, 806-807 Schonherr, 0. Th. & Keir, H. M. (1972) Biochern. J. 129,285-290 Small, G. D. & Sparks, R. B. (1972) Arch. Biochern. Biophys. 153, 171-179

Properties of Pol y(Adenosine Diphosphate Ribose) Polymerase from Baby-Hamster Kidney Cells (BHK-21/C13) HENRY M. FURNEAUX and COLIN K. PEARSON Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen AB9 IAS, Scotland, U.K. All nuclei of eukaryotic organisms so far studied contain an enzymic activity which is capable of covalently binding the ADP-ribose moiety of NAD+ to nuclear proteins, with the concomitant elimination of nicotinamide (Sugimura, 1973; Hilz & Stone, 1976). This modification reaction may utilize only one ADP-ribose unit, or other ADP-ribose units may be linked to the first one (by a 2'+1' glycosidic bond) yielding poly(ADPribose). The biological function of this NAD+-dependent reaction has not been

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elucidated. Since. however, theenzyme modifies various classes of nuclear protein in vitro and displays heterogeneity, both in site of attachment and number of ADP-ribose units bound, it seems likely that this modification wll not have a unique role in nuclear function. In general there Seems ito be a correlation between the activity of poly(ADP-ribose) polymerase and cellular growth rate. Comparatively few studies (Lehmann et al., 1974; Leiber et al., 1973; Stone: & Shall, 1975) have been conducted with systems in which growth rate can be manipulated. Baby-hamster kidney cells (BHK-Zl/C13) grown in vitro offer several advantages in this respect. This cell line has a high proliferation rate, which can be altered by changing the concentration of serum in the growth medium. Transformed derivatives (of BHK cells (PyY) are also readily available. Purified poly(ADP-ribose) polymerase requires both DNA and acceptor proteins for activity (Yoshihara, 1972). Most investigators have therefore studied this enzyme in isolated nuclei, utilizing the endogeneous DNA and acceptor proteins. In the present study we have studied a chromatin preparation in which the specific activity of the enzyme is increased, yet still retains the full complement of modified proteins. Poly(ADP-ribose) polymerase activity was measured by the incorporation of radioactivity from NAD+ (labelled in the adenine moiety) into acid-insoluble material. This technique of detecting AIDP-ribosylation may be criticized on the grounds that acidlabile linkages (should they exist) might remain undetected. If however, the reaction was stopped under neutral conditions [ S ~ M - E D T Aand 1 % (w/v) sodium dodecyl sulphate] and the denatured labelled nucleoprotein isolated by Millipore filtration, no significant difference was observed. We have demonstrated that the acid-insoluble material is poly(ADP-ribose) by virtue of its unique product after degradation with snake-venom phosphodiesterase, namely 2'-(5"-phosphoribosyl)-5'-AMP. As expected (Hilz & Stone, 1976), most of the enzymic activity was found in the nuclear fraction of the cell homogenate. Some activity, however, was detected in the postmitochondria1 fraction, when exogenous DNA and acceptor nuclear proteins were added to the assay medium. It is uncertain whether this cytoplasmic activityis a result of nuclear leakage during isolation, or, as has been sugested (Roberts et al., 1975), a different enzyme. The chromatin-bound enzyme from BHK-21 cells exhibits many of the properties found in other cell lines and tissues. It requires a bivalent cation (MgZ+at 5 m ) , the optimum pH is 8 and the apparent temperature optimum is 25°C. Both B-mercaptoethanol and dithiothreitol stimulate the enzymic activity; on the other hand it is almost completely inhibited by N-ethylmaleimide (91 % inhibition at 0.5m). This suggests that the enzyme requires thiol groups for activity. As reported by other investigators (Hiltz & Stone, 1976), poly(ADP-ribose) polymerase is unstable; our preparation loses half its activity in 15min when incubated at 37°C. We have found, however, that this temperature-dependent loss of activity can be prevented by incubation in the presence of MgZ+. The enzyme is strongly inhibited by nicotinamide (K,7 7 ~ and ) thymidine. It is interesting that this enzyme is inhibited (95% inhibition at 0.5m) by 2-amino-1,3,4thiadiazole, an anti-tumour drug and teratogenic agent. A definite link between NAD+ metabolism and the effects of this compound has been established (Oettgen et al, 1960; Beaudoin, 1976), and it seems likely that its mode of action may involve theinhibition of poly(ADP-ribose) polymerase. We are grateful to the Medical Research Council for support and acknowledge the skilled technical assistance of Mrs. Alison Blair. Beaudoin, A. R. (1976) Terntology 13, 95-100 Hilz, H. & Stone, P. (1976) Rev. Physiol. Biochem. Pharmacol. 76,l-58 Lehmann, A. R., Kirk-Bell, S., Shall, S. &Whish, W. J. D. (1974) Exp. Cell Res. 83,63-72 Leiber, U., Kittler, M., Hilz, H. (1976) Hoppe-Seyler's Z . Physiol. Chem. 354, 1347-1350 Oettgen, H. F., Reppert, J. A, Coley, V. & Burchenal, J. H. (1930) Cancer Res. 20,1597-1601 Roberts, J. H., Stark, P., Giri, C. P. & Smulson, M. (1975) Arch. Biochem. Biophys. 171, 305-3 15




Stone, P. & Shall, S. (1975)Exp. Cell Res. 91, 95-100 Sugirnura, T. (1973)Prog. NucIeic Acid Res. Mol. Biol. 13, 127-151 Yoshihara, K.(1972)Biochem. Biophys. Res. Commun. 47, 119-125

Changing-pH Assay for Study of Glycogen Phosphorylase Activity under Conditions Simulating those in vivo JOHARI M. SAAD and JOHN C. KERNOHAN Department of Biochemistry, University of Dundee, Dundee DDI 4HN, Scotland, U.K.

Glycogen phosphorylase (1,4-a-~-glucan-orthophosphatea-glucosyltransferase, EC catalyses the reversible reaction (Glucosyl).+PI

+ (glucosyl). - +glucose 1-phosphate

At equilibrium at pH6.8 the PJglucose I-phosphate molar ratio is 3.6, indicating that glycogen synthesis is slightly favoured (Hesrin, 1949). Under physiological conditions the enzyme promotes glycogen degradation almost entirely (Karpatkin et al., 1964). The classical assay for phosphorylase (Cori et al., 1943), based on the release of PI from glucose 1-phosphate, is of limited use for studying the physiological role of the enzyme and the effects of allosteric modulators, since glucose 1-phosphate is itself an activator. Because of the unfavourable equilibrium, assay of phosphorylase action in the direction of glycogen degradation generally involves removal of the product, glucose 1-phosphate, in a coupled assay. The coupled assay devised by Lowry et al. (1964), which uses glucose 6-phosphate dehydrogenase, is limited to use at very low glycogen concentrations because of light-scattering from glycogen. This coupled assay cannot be used to examine the effect of glucose 6-phosphate, which is known to be a potent inhibitor of phosphorylase b (Morgan & Parmeggiani, 1964). We here present a simple and direct continuous-assay procedure for phosphorylase acting in the physiological direction. We aIso present some preliminary observations on the effectsof varying the glycogen concentration and of ATP and glucose 6-phosphate on phosphorylase activity. The changing-pH assay has the same basis as the titrimetric assay described by Palter & Lukton (1973) and exploits the difference between the pK values for the second ionizations of PI (pK 6.77) and of glucose 1-phosphate (pK 6.14) at 37°C and Z 0.1. Calculation shows that the relationship between the change in pH from its value at the start of the reaction and the amount of PI which has reacted is virtually linear. If no buffer except PI is present originally, then the expected pH change would be 0.63 unit if all the PI was converted into glucose 1-phosphate. If other buffering species such as glucose 6-phosphate are present, corrections for the increased buffering power must be made. The validity of these corrections was tested by adding glycerol 2-phosphate to the reaction mixture. This substance has little if any effect on phosphorylase activity. The experimentalarrangement was as described by Taylor et al. (1975), except that the temperature was 37°C and thereaction volume was decreased to 1.5ml. As in that study, it was necessary to include 0.1wKCl in the reaction mixture to decrease electrode ‘noise’ to an acceptable value. The reaction mixture also contained 2 m - P I , pH6.80, and 0.1m - A M P except where indicated. These conditions approximate to those in resting muscle. The effect of varying the glycogen concentration on the activities of the a and b forms of phosphorylasefrom rabbit muscle, in the absence and presence of AMP, is shown in Fig, 1. AMP activation of phosphorylase a occurs at all glycogen concentrations, but becomes progressively more pronounced as the glycogen concentration decreases. This is in accord with the observation by Lowry et al. (1964) of 80-fold activation of phos-

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C13) [proceedings].

568th MEETING, ABERDEEN I40 743 r 10 5E 0 50 I00 I50 Elution volume (ml) Fig. 1. Isoelectric focusing of DNAases 4 and 5 0 , DNAase activity...
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