1-Methylguanine and 7-methylguanine increase cell agglutinability

Summary 1-Methylguanine and 7-methylguanine, both metabolic products of tRNA degradation, are known to induce transformation of Chinese hamster fibroblasts in culture. The effects of these compounds on the cell membrane have been studied by the method of Concanavalin A-mediated hemadsorption. 1-Methylguanine or 7-methylguanine induced a 50% increase of Con A-mediated hemadsorption within 20 hours of exposure of the cells to the agent at a concentration of 10^-5 M. This alteration was reversed within 13 days when the cells were grown in the control medium. Prolonged treatment with 1-methylguanine or 7-methylguanine resulted in changes which were only slowly reversed during growth of the cells in the control medium. The effect of the methylated purines on the cell membrane could be completely inhibited by simultaneous addition of dibutyryl-cAMP at a concentration of 10^-5 M. The possible mechanism of cell membrane alteration by methylated purines and its relevance to transformation in vitro are discussed.     

Modification of the gene 5 DNA unwinding protein with ruthenium

A chemical modification of the gene 5 DNA binding protein (G5BP) from bacteriophage fd was investigated using X-ray diffraction and difference Fourier analysis. The crystalline protein was reacted with pentaammineruthenium (III) trichloride, Ru(NH₃)₅Cl₃, a reagent believed specific for histidine residues and useful in NMR and chemical modification studies of proteins. The major ruthenium site was found by difference Fourier analysis to be 4 Å from histidine 64, the only histidine residue in the molecule. A second bipartite site, believed to be a ruthenium-anion pair, appeared to be salt-bridged to glutamic acid 40 and arginine 16. Indications were present in the difference Fourier results to suggest that the loop containing tyrosine 41 had undergone a slight conformational rearrangement to accommodate this interaction.     

The regulation of ferredoxin-dependent nitrogenase activity in Rhodospirillum rubrum and Rhodopseudomonas capsulata

Abstract The regulatory properties of Rhodospirillum rubrum nitrogenase reduced by either the endogenous electron donor (ferredoxin) or an artificial donor (dithionite) were examined. The nitrogenase obtained from glutamate-grown cells required activating enzyme for maximum activity with either reductant. The activating enzyme requirement of ferredoxin-dependent nitrogenase activity implies a physiological significance of the activating enzyme in R. rubrum. Rhodopseudomonas capsulata nitrogenase also required activating enzyme when dithionite was the reductant, but there appeared to be no activating enzyme requirement with ferredoxin as the reductant. Because the catalytic activity of the enzyme was very low under these conditions, the physiological significance of activating enzyme in this organism remains in question.     

Effects of chemical modification of carboxyl groups on the voltage-clamped nerve fiber of the frog

Summary Voltage-clamped single nerve fibers of the frogRana esculenta were treated with the carboxyl group activating reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of different primary amines and without added amine. Carboxyl groups form stable amide bonds with primary amines in the presence of EEDQ. EEDQ treatment reduced the sodium current considerably and irreversibly, regardless of the presence of a primary amine in the Ringer's solution. The potassium current was also reduced. After modification the reduced sodium currents inactivated slowly and incompletely. The descending branch of the sodium current-voltage relation,I _Na(E), was shifted along the voltage axis in the depolarizing direction. The size of the shift was strongly dependent on the amine present during modification with EEDQ. The voltage-dependence of sodium inactivation,h _x (E), was shifted to more positive values of membrane potential by EEDQ in the presence of ethylenediamine (11 mV) and glucosamine (3 mV). In contrast, a small shift to more negative potentials occurred in the presence of taurine (–3 mV) or without the addition of an amine (–2 mV). A tenfold increase of the calcium concentration still shifted theI _Na(E) andh _x (E) curves of the chemically modified fibers. However, these shifts were smaller than those observed on untreated fibers. The currents remaining after the modification were completely blocked by tetrodotoxin; no change of the reversal potential occurred.     

An enzyme-probe study of motile domains in the subfragment-2 region of myosin

The temperature-dependence of local melting within the subfragment-2 region of rabbit skeletal muscle myosin has been investigated using an enzyme-probe technique. Rate constants of fragmentation of two long subfragment-2 particles (61,000 Mr and 53,000 Mr per polypeptide chain) and a short subfragment-2 particle (34,000 Mr per polypeptide chain) by three different enzymes (alpha-chymotrypsin, trypsin and papain) have been determined over the temperature range 5 to 40 degrees C. We followed the time-course of digestion at specific sites at high (I = 0.50, pH 7.3) and low (physiological, I = 0.15, pH 7.3) ionic strengths by electrophoresis of the digestion products on sodium dodecyl sulfate-containing gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzymes by comparison with model substrates. Normalized rate constant versus temperature profiles for the three enzyme-probes are similar in showing that local melting in long subfragment-2 (61,000 Mr) occurs in two distinct stages as was observed earlier for the intact myosin rod. Over the temperature range 5 to 25 degrees C a restricted region at Mr = 53,000 to 50,000 from the N terminus of the rod (the light meromyosin/heavy meromyosin junction) shows the highest susceptibility to proteolytic cleavage. At temperatures above 25 degrees C local melting was detected by all three enzymes at several specific sites within the hinge domain (Mr = 53,000 to 34,000). Activation energies for cleavage at the susceptible sites were similar for the three enzyme probes. They suggest that this region of the myosin rod has significantly lower thermal stability than the flanking light meromyosin and short subfragment-2 segments. These results, together with other physico-chemical studies, point to the hinge domain of the myosin cross-bridge as an important functional element in the mechanism of force generation in muscle.     

Dolichol and Dolichyl Phosphate Levels in Brain Tissue from English Setters with Ceroid Lipofuscinosis

Human ceroid lipofuscinosis (CL) is an inherited disease marked by cerebromacular degeneration and early death. We have utilized the canine model to investigate the possible role of dolichol and dolichyl phosphate in the developmental pathology of CL. We found that while brain levels of dolichol increase with age in both affected and unaffected dogs, the amount in the diseased animal was similar to that in controls. Brain levels of dolichyl phosphate ranged from 20 to 35 micrograms/g in control dogs at all ages examined, but increased substantially during development in the affected dogs, a value of 113 +/- 24 micrograms/g (mean +/- SD) being obtained in the end-stage animals. In addition to the results obtained in the canine model, dolichyl phosphate levels in human brain tissues from a 5-year-old with late infantile CL and from a 19-year-old with juvenile CL were found to be 153 and 382 micrograms/g, respectively, compared with a control that assayed 26 micrograms/g. The preliminary findings with human tissues provide further evidence for an association of elevated brain dolichyl phosphate levels with CL. Whether the increase is primary or secondary remains to be determined.     

Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.     

Selenium modifies carcinogen metabolism by inhibiting enzyme induction

Since selenium has been found to exert a protective action against carcinogenesis in various systems, the mechanism where-by sodium selenite inhibits DNA binding of the carcinogen, 7,12-dimethylbenz[a]anthracene, was investigated. It was found that selenite preferentially reduced DNA binding occurring through ananti-dihydrodiol epoxide metabolite of this carcinogen by inhibiting the induction of an enzyme system that generates this specific reactive metabolite.