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At least 991 records · Page 55Linked to original sources
PubMed1999

Sensitized photomodification of mammalian DNA polymerase beta. A new approach for highly selective affinity labeling of polymerases.

To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base-substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site-bound photosensitizer for photoaffinity modification of DNA polymerase beta (pol beta). 5'-[32P]-labeled primer was elongated in situ by pol beta with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365-450 nm), can activate the photoreagent, cross-linking it to pol beta as a result of fluorescence resonance energy transfer. The initial rate of pol beta photomodification was shown to increase by a factor of ten. The selectivity of pol beta photosensitized modification was proved by adding human replication protein A.

Animals↗
PubMed1994

Heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26: heterotropic activation of deoxyadenosine kinase subunit implicated by limited proteolysis and affinity labeling.

The deoxyguanosine (dGuo) kinase/deoxyadenosine (dAdo) kinase complex of Lactobacillus has been purified to homogeneity by using a newly constructed dATP-Sepharose column as a final step (2700-fold purification). A heterodimeric structure for the complex has recently been established [Ikeda et al. (1994) Biochemistry 33, 5328-5334]. On the basis of the kinetic and structural data accumulated so far, a model for the heterotropic activation of the dAdo kinase subunit by dGuo or dGTP is proposed: (1) there is an intrinsic difference in the enzyme conformation of the two subunits, with the dAdo kinase subunit being in a constrained (closed) state and the counterpart dGuo kinase subunit being in a relaxed (open) state, as reflected in their relative Vmax values and in the presence or absence of heterotropic activation, and (2) the conformational change induced by the binding of dGuo or dGTP to the active site of the dGuo kinase subunit causes the activation of the dAdo kinase subunit through subunit--subunit interactions. These proposed mechanisms are strongly supported by the following new findings made in this work: (1) low concentrations of chaotropic agents such as guanidine--HCl were found to increase the Vmax of dAdo kinase up to 2-fold--in the same kinetic fashion, apparently, as the activation by dGuo--while showing no effect on dGuo kinase; (2) the proteolytic inactivation of dAdo kinase by trypsin is significantly slower than that of dGuo kinase, but its rate of inactivation is stimulated by dGTP to the same level as for dGuo kinase; (3) the activating effect of dGuo on dAdo kinase was abolished in the course of differential proteolytic inactivation of the dGuo kinase by trypsin in the presence of dATP; and (4) photoaffinity labeling with [8-14C]-8-azido-Ade produces a new species of kinase heterodimer in which the dAdo kinase subunit is permanently activated as a result of specific labeling of the dGuo kinase active site.

Enzyme Activation↗