Nine samples of human ceruloplasmin [iron (II).oxygen oxidoreductase; EC 1.16.3.1] prepared by different procedures have been examined for heterogeneity; gel electrophoresis showed that seven contained a number of components with molecular weights ranging from 20,000 to 130,000, and two contained largely a single component of molecular weight 130,000. Digestion of a single-component preparation with plasmin produced fragments with molecular weights similar to those found in the multicomponent preparations. Amino-terminal analysis, peptide mapping, and amino acid analysis showed that plasmin digestion generated a fragment of 20,000 molecular weight, which corresponded to a component present in a multicomponent ceruloplasmin preparation. The 20,000 molecular weight fragment appears to correspond to the so-called a-subunit or L-chain of human ceruloplasmin. Chemical evidence is thus provided that ceruloplasmin is a single-chain protein and that the so-called subunits are fragments.The 20,000 molecular weight fragment contains a single cysteine; amino acid sequence studies have shown that the sequence in the vicinity of this residue is similar to that around the single cysteine residue in plant plastocyanins and bacterial azurins, which are small, blue, copper-containing proteins. Despite Ryd6n's evidence (1, 2) for a single-chain structure for human ceruloplasmin (ferroxidase) [iron(II):oxygen oxidoreductase; EC 1.16.3.1], several authors continue to propose subunit structures (3, 4). The dilemma is posed by the following quotation from a recent review on ceruloplasmin (5); on the question of subunits the authors conclude "one, two, four, eight-only the future will tell." Furthermore, although many spectroscopic and magnetic investigations have been made of ceruloplasmin copper, little is known about its copper-binding sites or their possible structural similarity to sites in other copper-containing oxidases or electron transport proteins. In this paper we give chemical evidence for a single-chain structure for ceruloplasmin and report an amino acid sequence that may be involved in copper binding.We have found that ceruloplasmin prepared by different procedures contains varying amounts of discrete subunit-like fragments. Limited plasmin digestion of essentially undegraded ceruloplasmin has shown that cleavage by this enzyme, or by one with a similar specificity, is responsible for the variety of molecular forms reported for ceruloplasmin. We have also shown that plasmin produces a fragment of 20,000 molecular weight (Mr) that appears to correspond to the so-called a-chain (4) or L-chain (3). We have almost completely determined the amino acid sequence of the corresponding 20,000 Mr fragmentThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5377 isolated from partially degraded ceruloplasmin. A single cysteine residue is located in the COO...

The (6) and probably corresponds to the a subunit proposed by Simons and Bearn (4) and the light chain of Freeman and Daniel (5).The relation of Cp F5 to the structure of the intact ceruloplasmin chain has to be deduced from indirect observations because of the small amount of single-chain ceruloplasmin available to us and the strong interaction of the fragments. From the kinetics of the proteolytic cleavage of intact ceruloplasmin and the chemical properties of the fragments, we have suggested that Cp F5 is from the COOH terminus of the single chain (8). We have obtained a unique amino acid sequence and conclude that Cp F5 is probably an intact domain; it appears to be attached to the COOH-terminal end of the ceruloplasmin chain by a labile interdomain peptide bond. Prediction of the secondary structure by the empirical method of Chou and Fasman (9, 10) leads to a model in which approximately 30% of the residues occur in f3 sheets, 25% in a helices, and 45% in j turns and other structure.Because the ceruloplasmin preparation studied is derived from a pool of plasma from more than 10,000 donors from the ethnically mixed American population, the finding of a unique amino acid sequence indicates that the frequency of genetic polymorphism is too low to interfere with sequence determination of this fragment.In a previous report (8)

No abstract

No abstract