Proteolysis of Human Ceruloplasmin

Abstract

Highly purified human ceruloplasmin was isolated from fresh donor blood in the presence of inhibitors of proteolysis and from stored retroplacental blood serum both with and without inhibitors of proteolysis. According to the data of electrophoresis. ultracentrifuge sedimentation velocity and sedimentation equilibrium, all the ceruloplasmin samples were homogeneous, their molecular weight being 130 000. The dissociation of the samples treated by dodecylsulphate, guanidine · HCl, and urea was studied by means of quantitative analytica and preparative electrophoresis, and sedimentation equilibrium. The dissociation patterns depended on whether inhibitors were used in the isolation procedure. Polypeptides with molecular weights of 130 000, 112 000 and 16 000 (minor component) were obtained, if phenylmethylsulfonyl fluoride and/or 6‐aminohexanoic acid were used; if these compounds were not used, the dissociation yielded additional polypeptides of molecular weights of 100 000 (minor component), 64 000 and 48 000. Under proteolysis‐favouring conditions the relative amount of these polypeptides increased. Prolonged storage of samples under sterile conditions without inhibitors of proteolysis resulted in a decrease of the relative amount of polypeptides with molecular weights of 130 000, 112 000, 100 000, 64 000 and 48 000, contrary to that of the 16 000‐Mr polypeptide, which increased. At the same time new polypeptides appeared with molecular weights of 42 000 and 21 500–23 000. Spontaneous specific fragmentation of the ceruloplasmin molecule is due to trace amounts of proteases, which seem to originate from blood plasma. Limited tryptic hydrolysis of the ceruloplasmin globule resulted in the appearance of polypeptides with the same molecular weights which were observed in spontaneous fragmentation. A conclusion is drawn that the ceruloplasmin molecule in vivo is a single polypeptide chain with at least five bonds which in vitro are the points of specific proteolytic fragmentation, yielding six principal fragments.

Internal triplication in the structure of human ceruloplasmin.

“…1). This model is based on the results of amino acid sequence analysis ofthe 67-kDal fragment reported here, the sequences ofthe 50-kDal and 19-kDal fragments determined earlier (2)(3)(4)(5)(6), and the sites of limited proteolytic cleavage (4)(5)(6)(7)(8). The model predicts the presence of a series of two (A, B) or three (A1, A2, B) homologous domains, each type being repeated three times in the structure.…”

“…Although subject to spontaneous proteolysis, it consists of a single polypeptide chain (MAr 135,000) that exhibits internal duplication in amino acid sequence (2). Three types ofobservations suggest that the ceruloplasmin molecule may be divided into a series of homologous domains: (i) the internal homology in primary structure (2); (ii) the existence of sites of limited proteolytic cleavage resulting in a series of fragments, the principal ones having approximate molecular weights of 67,000, 50,000, and 19,000 (3)(4)(5)(6)(7)(8); and (iii) the presence of six copper atoms of three different kinds, as distinguished by spectroscopic and other physical properties (9,10).…”

Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites

“…Samples of hCP were obtained as described previously (Moshkov et al, 1979). However, prior to crystallization a final purification stage was undertaken using size-exclusion chromatography.…”

Coagulation factors V and VIII and ceruloplasmin constitute a family of structurally related proteins.

“…All three proteins are relatively sensitive to proteolysis. Factor V and factor VIII undergo cleavage by serine proteases to form their active forms factor Va and factor VIIIa, whereas early purifications of HCp often resulted in preparations having three predominant polypeptides of mass 67-, 50-, and 19-kDa fragments (18,19). The biological role, if any, as well as the enzyme responsible for this fragmentation in ceruloplasmin are unknown.…”