Serum ferritin isolated from the horse was structurally compared with horse spleen ferritin and was found to differ markedly in molecular weight, iron content, carbohydrate, subunit size and amino acid sequence. The results are summarized and initial results obtained with candidate clones of pieces of two serum ferritin subunits are described.
Numerous aminoacyl-tRNA synthetase sequences have been aligned by computer and phylogenetic trees constructed from them for the two classes of these enzymes. Branching orders based on a consensus of these trees have been proposed for the two groups. Although the order of appearance can be rationalized to fit many different scenarios having to do with the genetic code, the invention of a system for translating nucleic acid sequences into polypeptide chains must have predated the existence of these proteins. In the past, a variety of schemes has been proposed for matching amino acids and tRNAs. Most of these have invoked direct recognition of one by the other, whether or not the anticodon was involved. Often ignored is the possibility of a nonprotein (presumably RNA) matchmaker for bringing the two into conjunction. If such had been the case, then the contemporary aminoacyl-tRNA synthetases could have entered the system gradually, each specific type replacing its matchmaking RNA counterpart in turn. A simple displacement scheme of this sort accommodates the existence of two different families of these enzymes, the second being introduced well before the first had undergone sufficient genetic duplications to specify the full gamut of amino acids. Such a scheme is also consistent with similar amino acids often, but not always, being the substrates of enzymes with the most similar amino acid sequences.
Sequence segments of about 140 amino acids in length, each containing a selected consensus region, were used in alignments of the amiyl-tRNA synthetases with the aim of discerning their evolutionary relationships. In all cams tested, enzymes specific for the same amino acid from a variety of organisms grouped together, reinforcing the supoton that the aminoacyl-tRNA synthetases are very ancient enzymes that evolved to indude the full complement of 20 amino acids long before the divergence leading to prokaryotes and eukaryotes. The enzymes are divided into two mutually exclusive groups that appear to have evolved from independent roots. Group I, for which two sequence segments were analyzed, contains the enzymes specific for amic acid, glutine, tryptophan, tyrosine, valine, leucine, methionin, and argmine. Group II enzymes include those activating threonine, proline, serine, lysine, aspartic acid, hisidine, alanine, glycine, and phenylaanine. Both groups contain a spectum ofamino acid types, s ing the pobity that each could have once supported an independent system for protein synthesis. Within each group, enzymes specific for c ly similar amino aids tend to duster together, indicating that a major theme of synthetase evolution involved the a ion of binding sites to accommodate related amino acids with subsequent specialization to a Asle amino acid. In a few cases, however, synthetases activating diimiar amino acids are grouped together.Aminoacyl-tRNA synthetases catalyze the esterification or "charging" of a single amino acid to its cognate tRNA; thus, 20 such enzymes, one enzyme specific for each amino acid found in proteins, constitute a minimum set for protein biosynthesis. The evolution and structural relatedness of these enzymes has been a subject of intense interest for many years (for review, see refs. 1-3). Because they appear to participate universally in protein synthesis, the origins of these "activating" enzymes must be very ancient (4) and studies of their divergence may shed sigificant light onto the development of the genetic code and its expression. In addition, the structural basis for nucleic acidprotein recognition and the manner in which enzymes have come to activate only a single amino acid and cognate tRNA while excluding a large number of structurally similar molecules is a subject of great interest. As the sequences of more and more of the aminoacyl-tRNA synthetases became available, we undertook a study of the interrelationships within the more than 50 reported sequences with the aims of tracing their evolution and of identifying more conserved sequence segments that are presumably essential to their biological function.Because these enzymes catalyze the same overall reaction and utilize a common strategy for chemical activation of their amino acids, aminoacyl-AMP being formed at the expense of ATP, it has long been supposed that all these enzymes had a common ancestral root, even though large differences in polypeptide chain length (303-1104 amino acids) and quaternary structure...
Human fibrinogen and the plasmin-generated fibrinogen fragment D were photoaffinity labeled specifically with the peptide [14C]Gly-Pro-Arg-N(4-azido-2-nitrophenyl)Lys amide. In the case of fibrinogen, >85% of the incorporated radioactivity was found in the y chain. Similarly, when fragment D (Mr, 90,000) was labeled with the same derivatized peptide, virtually all the radioactivity was found in the y-chain portion. The labeled fragment D was treated with CNBr and an initial purification was achieved by two gel.filtration steps. The labeled material was purified further by HPLC and was also compared with CNBr digests of unlabeled material. Amino acid analysis and gas-phase sequencing showed the labeled fragment to be y-chain residues 337-379.In vertebrates, the conversion of fibrinogen into fibrin is initiated by the thrombin-catalyzed removal of the fibrinopeptides A, the immediate consequence of which is the exposure of a pair of new amino termini beginning with the sequence Gly-Pro-Arg (for a review, see ref. 1). Synthetic peptides beginning with that sequence bind strongly to fibrinogen and are able to block fibrin formation (2). The binding has been localized to the terminal domains corresponding to the large plasmin-generated fragment designated D1 (3). In contrast, synthetic peptides beginning with the sequence Gly-His-Arg, which corresponds to the amino terminus of chains after release of the fibrinopeptides B, do not block fibrin formation, even though they do bind to fibrinogen. The Gly-His-Arg peptides bind to both fragments D1 and D3, the latter being a further degradation product of the former. Peptides with the sequence Gly-Pro-Arg do not bind to fragment D3, however, which lacks the carboxylterminal 109 residues of the y chain, leading to the supposition that the complementary binding site for the Gly-Pro-Arg "knob" is situated in the carboxyl-terminal third of the y-chain portion of fragment D (4). Previous efforts to localize further the complementary binding sites for the two sets of peptides have been a matter of dispute. Thus, Olexa and Budzynski (5) reported that the carboxylterminal 38 residues ofthe y chain (i.e., 'y374411) embodied the principal binding site; a similar claim about residues y374-396 was made by Horwitz et aL (6). Subsequently, Southan et al. (7) and Varadi and Scheraga (8) reported experiments indicating that these results were likely artifactual. We now report the results of a photoaffinity labeling study with the peptide Gly-Pro-Arg-N(4-azido-2-nitrophenyl)Lys amide that implicates residues 337-379 from the y chain. MATERIALS AND METHODSHuman fibrinogen was prepared from blood bank plasma as described (9). Fragment D was prepared by digesting the fibrinogen with plasmin in the presence of calcium ions (10), thereby limiting the digest to the large molecular weight D usually referred to as fragment D1. Digestions were stopped by the addition of the plasmin inhibitor Trasylol (aprotinin from bovine lung), and fragments were purified either by ion-exchange chromato...
Reconstitution of aspartate transcarbamoylase (EC 2.1.3.2) from dilute solutions of the isolated regulatory and catalytic subunits, with the latter in large excess, led to the formation of appreciable amounts of a second, stable component in addition to the reconstituted enzyme. The purified component, designated r4c6, was found to have a molecular weight about 3 X 104. less than that of the native enzyme, and it combined with isolated regulhtory subunit to form aspartate transcarbamoylase.It also combined with one succinylated regulatory subunit to form a hybrid species that was identified electrophoretically. These findings indicate that r4cs differs from the native enzyme in that only two (rather than three) regulatory subunits participate in "crosslinking" the two catalytic trimers. The "incomplete" enzyme, r4c6, exhibits the characteristic sigmoidal saturation behavior and CTP inhibition of aspartate transcarbamoylase; however these allosteric effects are reduced in extent by about one-third in comparison to the native enzyme and free catalytic subunits. The complex, which may be an intermediate in the assembly and dissociation of the native enzyme, is useful in assessing the role of the various bonding domains responsible for the stability and regulatory properties of the native enzyme.Following the discovery that the regulatory enzyme, aspartate transcarbamoylase (EC 2.1.3.2; carbamoylphosphate: 'A aspartate carbamoyltransferase) from Escherichia coli, is composed of discrete subunits for catalysis and regulation, each containing a unique polypeptide chain (1), there have been many studies aimed at determining the structure and mechanism of action of the enzyme (2, 3). It is now known that it is composed of six catalytic (c) and six regulatory (r) polypeptide chains (4-7) arranged in a molecule having 2-fold and 3-fold axes of symmetry (8-11). The catalytic chains are organized as trimers (C) both in the intact enzyme and after its dissociation with certain mercurials (5). Although the C subunits show little tendency to associate to form discrete species, they combine readily with free regulatory subunits (R), yielding reconstituted molecules with the unusual physical and kinetic properties of the native enzyme (1, 12). The R subunits, as a result, have been viewed as "crosslinks" that bind the two C subunits in the intact enzyme molecules. Various experiments have shown that the r chains are organized as dimers both in the native enzyme and in the R subunit released by treating the enzyme with mercurials (7). In addition, considerations of the weight composition of the enzyme in terms of c and r chains (1, 6, 7) plus hybridization experiments with native C and mixtures of native and succinylated R (13) have led to the conclusion that there are three R dimers in each enzyme molecule. Recent evidence from electron microscopy (14) and x-ray diffraction studies (10, 11) has provided support for a model of the enzyme as a complex containing two catalytic trimers bonded through three regulatory dimers;...
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