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To investigate whether or not duck lens e-crystaliin and duck heart lactate dehydrogenase (LDH) B4are the product of the same gene, we have isolated and sequenced cDNA clones of duck e-crystallin. By using these clones we demonstrate that there is a single-copy Ldh-B gene in duck and in chicken. In the duck lens this gene is overexpressed, and its product is subject to posttranslational modification. Reconstruction of the evolutionary history of the LDH protein family reveals that the mammalian Ldh-C gene most probably originated from an ancestral Ldh-A gene and that the amino acid replacement rate in LDH-C is approximately 4 times the rate in LDH-A. Molecular modeling of LDH-B sequences shows that the increased thermostability of the avian tetramer might be explained by mutations that increase the number of ion pairs. Furthermore, the replacement of bulky side chains by glycines on the corners of the duck protein suggests an adaptation to facilitate close packing in the lens.

The common characteristic of the alpha-crystallin/small heat-shock protein family is the presence of a conserved homologous sequence of 90-100 residues. Apart from the vertebrate lens proteins--alpha A- and alpha B-crystallin--and the ubiquitous group of 15-30-kDa heat-shock proteins, this family also includes two mycobacterial surface antigens and a major egg antigen of Schistosoma mansoni. Multiple small heat-shock proteins are especially present in higher plants, where they can be distinguished in at least two classes of cytoplasmic proteins and a chloroplast-located class. The alpha-crystallins have recently been found in many tissues outside the lens, and alpha B-crystallin, in particular, behaves in many respects like a small heat-shock protein. The homologous sequences constitute the C-terminal halves of the proteins and probably represent a structural domain with a more variable C-terminal extension. These domains must be responsible for the common structural and functional properties of this protein family. Analysis of the phylogenetic tree and comparison of the biological properties of the various proteins in this family suggest the following scenario for its evolution: The primordial role of the small heat-shock protein family must have been to cope with the destabilizing effects of stressful conditions on cellular integrity. The alpha-crystallin-like domain appears to be very stable, which makes it suitable both as a surface antigen in parasitic organisms and as a long-living lens protein in vertebrates. It has recently been demonstrated that, like the other heat-shock proteins, the alpha-crystallins and small heat-shock proteins function as molecular chaperones, preventing undesired protein-protein interactions and assisting in refolding of denatured proteins. Many of the small heat-shock proteins are differentially expressed during normal development, and there is good evidence that they are involved in cytomorphological reorganizations and in degenerative diseases. In conjunction with the stabilizing, thermoprotective role of alpha-crystallins and small heat-shock proteins, they may also be involved in signal transduction. The reversible phosphorylation of these proteins appears to be important in this respect.

The major polypeptide chain of bovine 0-crystallin, BBp, was fragmented by means of cyanogen bromide treatment and by enzymatic digestions. Manual and automated Edman degradation of the resulting peptides provided the complete amino acid sequence of the 0Bp chain. The N-terminal alanine residue was shown to be N-aacetylated by mass spectrometry. The chain has a length of 204 residues and a calculated molecular weight of 23210. There is a considerable degree of homology between the N-terminal and C-terminal halves of the chain, presumably reflecting a tandem duplication of a shorter ancestral gene. The sequence of 0Bp is sufficiently related to that of y-crystallin I1 to place these proteins in the same superfamily. No sequence relationship was found with the a-crystallin chains.The water-soluble eye lens proteins, the crystallins, have proven to be suitable tools for the study of protein biosynthesis, differentiation, aging and molecular evolution [I]. For these purposes a knowledge of the primary stuctures of the crystallins is required, and these have indeed already been established for the a-crystallin A and B chains of several species [2 -41 and for the bovine y-crystallin fraction I1 [5]. We now have dctermined for the calf the complete amino acid sequence of the major polypeptide from the rcmaining class of mammalian crystallins, the 0-crystallin Bp chain. A preliminary report of some of the conclusions of this study has been published [6]. MATERIALS A N D METHODSIsolation and Fragmentation of [IBp /%Crystallin was isolated from calf lenses, and the major polypeptide /IBp purified as previously described [7, 81. The purity of 0Bp was checked by sodium dodecylsulfate/ polyacrylamide electrophoresis and alkaline urea gel electrophoresis [8].S-fi-Aminoethylation [9], carboxymethylation [lo] and cleavage with cyanogen bromide [I 11 (in 70 formic acid and using equal weights of protein and CNBr) were carried out according to standard procedures. Citraconylation of the &-amino groups of lysine [12] was carried out on cyanogen bromide fragments of carboxymethylated PBp.Digestions with trypsin (Worthington TRTPCK), chymotrypsin (Calbiochem A grade) and elastase (Whatman) were carried out in 0.1 M ammonium bicarbonate, brought to pH 8.9 with 1 M ammonia, at 37 'C for 90 min. Substrate concentrations were 10 mg/ml for protein or CNBr fragments, and 1 pmol/ml for peptides. The enzyme:substrate ratio was 2: 100 (w/w), or 0.5 mg/pmol in the case of peptides. Digestion with pepsin (Sigma) was carried out in 0.01 M HCI at a peptide concentration of 1 pmol/ml, using 0.5 mg of enzyme/pmol, during 5 h at 37 'C. Digestion with Stap1zylococcu.s uureus protcase (Miles V 8) was performed at 37 C for 30 h in 0.5 M ammonium bicarbonate, pH 7.8, at a protein concentration of 7 mg/ml and an enzyme : substrate ratio of 2 : 100 (w/w). Digests were either lyophilized (in case of trypsin after lowering the pH to 6.5 with 0.5 M HCl), or directly applied onto a gel filtration column for peptide separation. Purification of PeptidesCNBr fr...

beta-Crystallins are structural lens proteins with a conserved two-domain structure and variable N- and C-terminal extensions. These extensions are assumed to be involved in quaternary interactions within the beta-crystallin oligomers or with other lens proteins. Therefore, the production of beta A3- and beta A1-crystallin from the single beta A3/A1 mRNA by dual translation initiation is of interest. These crystallins are identical, except that beta A1 has a much shorter N-terminal extension that beta A3. This rare mechanism has been conserved for over 250 million years during the evolution of the beta A3/A1 gene, suggesting that the generation of different N-terminal extensions confers a selective advantage. We therefore compared the stability and association behaviour of recombinant beta A3- and beta A1-crystallin. Both proteins are equally stable in urea- and pH-induced denaturation experiments. Gel filtration and analytical ultracentrifugation established that beta A3 and beta A1 both form homodimers. In the water-soluble proteins of bovine lens, beta A3 and beta A1 are present in the same molecular weight fractions, indicating that they oligomerize equally with other beta-crystallins. 1H-NMR spectroscopy showed that residues Met1 to Asn22 of the N-terminal extension of beta A3 have great flexibility and are solvent exposed, excluding them from protein interactions in the homodimer. These results indicate that the different N-terminal extensions of beta A3 and beta A1 do not affect their homo- or heteromeric interactions.

The amino acid sequence of the N-terminal region of the two basic bovine &crystallin B1 chains has been analyzed. The results reveal that @Bib is derived in vivo from the primary gene product @la by removal of a short N-terminal sequence. It appears that them1 chains have the same domain structure as observed in other /3-and y-crystallin chains. They have, however, a very long N-terminal extension in comparison with other &chains. This extension is mainly composed of a remarkable Pro-and Ala-rich sequence, which suggests an interaction of these structural proteins with the cytoskeleton and/or the plasma membranes of the lens cells.

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...

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