No abstract

Irradiation by UV-A of E. coli DNA polymerase I in the presence of 8-methoxypsoralen leads to a similar inactivation of the 5' -+ 3' polymerase and 3' 4 5' exonuclease activities of the enzyme. The kinetics of inactivation depend on the psoralen concentration and on the duration of irradiation. The 5' + 3' exonuclease activity is first slightly stimulated at short irradiation time in the presence of 8-MOP; further irradiation leads to inhibition of this activity. The mechanism of the reactions involves oxygen as shown by the absence of any effect of UV irradiation when oxygen is removed. The results obtained in D20, in oxygen-saturated buffer or in the presence of singlet oxygen ('0,) quenchers show that '0, is one of the reactive intermediate species. Irradiation of homopolymers primed with oligomers in the presence of 8-MOP inhibits their template and initiating capacity in the same dose range as that required to inactivate DNA polymerase I. Mono-adducts of 8-MOP with nucleic acid bases can act as inhibitors of both replication and initiation.

The present study describes the acetylation by an enzyme present in calf lens of a synthetic tridecapeptide [analogous to a-melanotropin (a-melanocyte stimulating hormone) but lacking the naturally occurring NH2-terminal acetyl group: des-Nal-Ac-a-melanotropin]. The reaction is specific for the a-amino group of the NH2-terminal amino acid. The minimum length required for the substrate to become acetylated appears to be a sequence of five to eight amino acid residues. Modification of the internal lysine decreases the incorporation of acetate, irrespective of the size of the blocking group. The number of NH2-terminally acetylated proteins described in the literature is considerable, but virtually nothing is known concerning the underlying mechanism and possible functional implications. NH2-terminal acetylation of Escherichia coli ribosomal protein L12 (1, 2) and of ribosome-associated proteins from rat liver (3, 4) is under enzymic control. A conspicuous feature of a-NH2-acetylated proteins is that, with rare exceptions, only NH2-terminal alanyl, glycyl, methionyl, seryl, and threonyl residues are involved (5, 6). The lens cell-free system explored previously in our laboratory for the solution of a variety of other problems in protein biosynthesis (7,8) appeared to be a useful system for the study of NH-terminal acetylation. This system must have a powerful acetylating capacity in view of the fact that approximately 75% of the water-soluble lens proteins are NH2-terminally acetylated. The experimental approach of NH2-terminal acetylation was until now seriously hampered by the lack of a suitable substrate. Obviously, all potential substrates isolated from natural sources, with exception of the ribosomal protein L12 (9), are already acetylated in NH2-terminal position. We were able to circumvent this difficulty by using a synthetic tridecapeptide, with the same sequence as a-melanotropin (a-melanocyte stimulating hormone, a-MSH) but lacking the a-NH2-acetyl group normally present in the native hormone. This substrate becomes readily acetylated by the lens cell-free system using acetyl-CoA as acetate donor. The acetylation is an enzymatic process and is specific for the NH2-terminal amino group of the substrate.MATERIALS AND METHODS Substrates. Peptides with sequences related to the sequence of melanotropin (melanocyte stimulating hormone, a-MSH) were synthesized as described (10). The main substrate used was des-Nal-acetyl-a-MSH: Preparation of Lens Extract. The epithelial cell layer was removed from fresh calf lenses, and 12 mm of the outer cortex at the equator was punched off with the aid of a glass trephine. Homogenization was carried out in two volumes of buffer (50 mM Tris-HCl, pH 7.4, 300 mM sucrose, 50 mM KC1, 3 mM magnesium acetate) by applying 10 strokes with a Teflon homogenizer. The homogenate was centrifuged at 15,000 X g for 10 min at 2°. The supernatant fraction (S15) was used for subsequent experiments. In some cases an aqueous lens extract was lyophilized and used as a source of the acety...