Model studies of enzymatic NH2-terminal acetylation of porteins with des-Nalpha1-acetyl-alpha-melanotropin as a substrate.

Abstract

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...

The Vertebrate Eye Lens

“…1 at the NH2-terminus is Acetyl coenzyme A serves as donor of 1. We found that the ace-the acetyl group (57). Our experiments ery of heterologous pro-showed that a hexapeptide can serve as ig systems acetylates the substrate, whereas tetrapeptides are not luct of 14S lens mRNA.…”

Targeted mass spectrometric analysis of N‐terminally truncated isoforms generated via alternative translation initiation

“…N α ‐acetyltransferase anchors to the ribosome and interacts with nascent polypeptides while they are within 20–50 amino acids in length [17,18]. N‐terminal acetylation is almost exclusively found to occur cotranslationally, and N a ‐acetylation following translation has been reported only in the particular case of short peptides such as α ‐melanocyte‐stimulating hormone (13 residues) [19], leading us to postulate that acetylation of p44 dok most likely occurs cotranslationally. The N α ‐acetylation of N‐terminally truncated proteins could be common feature to isoforms generated via alternative translation initiation – and possibly alternative splicing, as well.…”

Synthesis of Human Ultraviolet Filter Compounds: O-β-d-Glucopyranosides of 3-Hydroxykynurenine and 2-Amino-3-hydroxy-γ-oxobenzenebutanoic Acid

“…2,3 A number of as yet unidentified UV filters are contained in the human lens. As enzymes capable of acetylating amino groups of lens proteins (such as Rand β-crystallins) and small peptides are known to be active in the lens, 4 the N-acetyl derivative of the most abundant UV filter 1 may also be present. An acetylated derivative, N-acetyl-3-hydroxykynurenine, is the major low molecular weight pigment found in the lens of the gray squirrel.…”