A spectrophotometric assay for the determination of the catalytic efficiency of plasminogen activators using a slowly hydrolyzed plasmin substrate.

A simple spectrophotometric assay for the determination of the catalytic efficiency and activity of plasminogen activators is presented. The assay system contains activator, plasminogen, and the chromogenic substrate N-benzoyl-L-arginine-p-nitroanilide (BAPA). Plasmin production is monitored continuously by the hydrolysis of BAPA under non-steady-state, first-order conditions with respect to plasminogen. Apparent catalytic efficiency constants are calculated from the values obtained for the apparent first-order rate constant of activation. The results obtained with the present method were compared with the catalytic efficiency determined through the measurement of kcat and Km, using a different system, under steady-state conditions. Tissue plasminogen activator in the absence and presence of fibrinogen and high-molecular-weight urokinase were used as model activators. Potential applications are discussed.

In vitro stimulation of tissue-type plasminogen activator by Alzheimer amyloid beta-peptide analogues.

We have studied the effects of amyloid beta-peptide analogues on the activity of tissue-type plasminogen activator (t-PA) in vitro. We have found that these peptides have a marked stimulatory effect upon plasminogen activation by t-PA, comparable to that of known stimulators of t-PA. This stimulatory activity appears to increase when beta-peptides form aggregated fibrillar structures similar to those found in amyloid deposits. This finding is significant in that it may provide insights into the pathogenesis of hereditary cerebral haemorrhage with amyloidosis-Dutch type (HCHWA-D) and cerebral amyloid angiopathy-related cerebral haemorrhage. It may also provide an explanation for the deaths resulting from intracerebral haemorrhage that have occurred in patients undergoing t-PA treatment for acute myocardial infarction.

Developmentally regulated alternative splicing of a nematode type IV collagen gene.

A comparison of the genomic DNA sequence that encodes the Ascaris suum alpha 2(IV) collagen chain with the corresponding cDNA sequence led to the identification of a putative exon that was not expressed in the cDNA. The identification of this putative exon raised the possibility that transcripts of the alpha 2(IV) gene may undergo alternative splicing. We have used a reverse transcriptase-polymerase chain reaction assay to establish that such alternative splicing does indeed occur. Our results show that the A. suum alpha 2(IV) collagen gene produces at least two similar, but not identical, transcripts via the selection of two alternative exons. Furthermore, this alternative splicing appears to be developmentally regulated, suggesting that alternative splicing may be used in order to modify the properties of type IV collagen during nematode development.

The complete primary structure of a nematode alpha 2(IV) collagen and the partial structural organization of its gene.

We have isolated and characterized cDNA and genomic DNA clones which encode an alpha 2(IV) collagen chain from the parasitic nematode Ascaris suum. In addition we have determined, by nucleic acid sequence analysis, the structural organization of approximately two-thirds of the gene. This analysis has shown that the gene contains at least 15 introns, and those that have been characterized range in size from 141 to 854 base pairs. The derived protein sequence contains 1763 amino acids and includes a putative 26-amino acid signal sequence. The collagenous triple-helical region contains 17 interruptions, many of which occur in the same positions as those in the human alpha 1(IV) and alpha 2(IV) chains. Comparison of the genomic DNA sequence with the cDNA sequence has revealed the presence of a sequence within the gene which appears to be an intact and normal exon that is not represented in our cDNA sequence. The presence of this putative exon raises the possibility that the A. suum alpha 2(IV) collagen gene may undergo alternative splicing.

Nematode collagen genes.

The collagen genes of nematodes encode proteins that have a diverse range of functions. Among their most abundant products are the cuticular collagens, which include about 80% of the proteins present in the nematode cuticle. The structures of these collagens have been found to be strikingly similar in the free-living and parasitic nematode species studied so far, and the genes that encode them appear to constitute a large multigene family whose expression is subject to developmental regulation. Collagen genes that may have a role in cell-cell interactions and collagen genes that correspond to the vertebrate type IV collagen genes have also been identified and studied in nematodes.

Structure and expression of Ascaris suum collagen genes: a comparison with Caenorhabditis elegans.

We are interested in the structure and organization of collagen and collagen genes in the parasitic nematode Ascaris suum and in the control of collagen gene expression in Ascaris. In this nematode, as in all others studied, collagens constitute the major component of the extracellular cuticle and ultimately we would like to correlate the expression pattern of Ascaris cuticular collagen genes with the structure of the nematode cuticle. We would also like to see if there is any correlation between differential collagen gene expression and the biology and life-cycle of the parasite. In addition, we are also interested in comparing our data with the more extensive data available on collagen genes in the free living nematode Caenorhabditis elegans.

Comparison of collagen gene sequences in Ascaris suum and Caenorhabditis elegans.

The nucleotide sequences of a 3060-bp fragment containing almost the entire coding sequence of one Ascaris suum collagen gene, and a 3019-bp pair fragment containing the 3' end of another A. suum collagen gene have been determined. The polypeptides encoded by these genes show a striking similarity to two Caenorhabditis elegans cuticular collagens, both in the position of the triple-helical regions and in the position of cysteine residues. The results of Northern blot hybridisation experiments together with dot blot analysis of RNA isolated from different adult worm tissues suggest that one of these genes is expressed in the adult nematode and that it encodes a protein of approximately 30 kDa.

Sequences encoding two trypsin inhibitors occur in strikingly similar genomic environments.

The nucleotide sequences of two approx. 4 kilobase pair segments of the bovine genome are presented. One segment contains a coding region for bovine pancreatic trypsin inhibitor (BPTI) and the other segment contains a coding region for a BPTI homologue. The two 4 kilobase pair sequences are strikingly similar over approx. 3.4 kilobase pairs of their sequence, including putative intron sequences, suggesting that they have evolved from a gene duplication event.

Isolation of a genomic clone for bovine pancreatic trypsin inhibitor by using a unique-sequence synthetic DNA probe.

Unique-sequence synthetic DNA probes, based on the known amino acid sequence of bovine pancreatic trypsin inhibitor, were constructed from oligodeoxynucleotides. In genomic Southern blot experiments, these probes were shown to hybridize specifically to discrete restriction fragments. A synthetic probe also was used to isolate a cloned BPTI gene from a bovine genomic library. DNA sequence analysis of this clone indicated that the BPTI coding region was neither preceded by a start codon nor immediately followed by a termination codon. This suggests that the mature form of BPTI may be produced through proteolytic processing from a larger polypeptide precursor.

The primary structure of hen ovotransferrin.

Peptide sequences obtained from hen ovotransferrin are compared with the complete amino acid sequence of the protein deduced from a cDNA sequence (Jeltsch and Chambon, preceding paper). Of the 705 positions of the whole protein 605 can be matched by the peptide sequences. Some possible discrepancies between the two methods are pointed out. The two halves of the chain show marked similarities in their sequences with 37% identical residues. The positions of the 15 disulphide bridges are shown; there are 6 homologous bridges in each half of the molecule and 3 extra bridges which occur only in the C-terminal half. The terminal residues of the half-molecule fragments obtained by limited proteolysis are identified. The two domains are joined by a 9-residue connecting peptide. Sequence variability has been found at 9 positions. The sequence of hen ovotransferrin is compared with the partial available for human transferrin. From this some tentative conclusions about the identities of the metal-binding residues and about the evolution of transferrin are reached.

Primary structure of a histidine-rich proteolytic fragment of human ceruloplasmin. I. Amino acid sequence of the cyanogen bromide peptides.

A histidine-rich fragment, Cp F5, with a molecular weight of 18,650 was isolated from human ceruloplasmin. It consists of 159 amino acids and contains a possible copper-binding site. The sequence of the first 18 NH2-terminal residues of Cp F5 was determined by automated Edman degradation. Cp F5 was cleaved by cyanogen bromide to produce nine fragments of from 2 to 63 residues. The amino acid sequence of all of the cyanogen bromide fragments was investigated using automated and manual Edman degradation, the fragments being digested with trypsin, chymotrypsin, thermolysin, staphylococcal protease, and pepsin as appropriate. The results, in conjunction with the data on the tryptic peptides reported in the accompanying paper (Kingston, I.B., Kingston, B.L., and Putnam, F.L. (1980) J. Biol. Chem. 255, 2886-2896), establish the complete amino acid sequence of Cp F5.

Primary structure of a histidine-rich proteolytic fragment of human ceruloplasmin. II. Amino acid sequence of the tryptic peptides.

Amino acid sequence studies of tryptic peptides isolated from a histidine-rich fragment (Cp F5) of human ceruloplasmin are described. Nineteen tryptic peptides were isolated from unmodified Cp F5 and five tryptic peptides were isolated from citraconylated Cp F5. These peptides, together with the cyanogen bromide fragments reported previously, allowed the assembly of the complete sequence of Cp F5. The fragment has 159 residues and a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains 1 free cysteine that may be part of a copper-binding site. Human ceruloplasmin is a single polypeptide chain with a molecular weight of about 130,000 that is readily cleaved to large fragments by proteolytic enzymes; the relationships of Cp F5 to intact ceruloplasmin and to structural subunits earlier proposed is described. Cp F5 probably is an intact globular domain that is attached to the COOH-terminal end of ceruloplasmin by a labile interdomain peptide bond.

Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin.

The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in alpha helices, about a third in beta-sheet structure, and the remainder in beta turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication.

Chemical evidence that proteolytic cleavage causes the heterogeneity present in human ceruloplasmin preparations.

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 alpha-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.

The amino acid sequence of a carbohydrate-containing fragment of hen ovotransferrin.

1. Hen ovotransferrin was treated with CNBr and fractionated by gel filtration. 2. After further treatment by reduction and carboxymethylation a carbohydrate-containing fragment of molecular weight 11990 was obtained (fragment BCd). 3. The amino acid sequence of this fragment was determined. It consists of a single chain of 94 residues. 4. The structure of a tryptic glycopeptide derived from whole ovotransferrin permitted a further eight residues to be assigned at the N-terminus of fragment BCd. 5. Heterogeneity was found at two positions. 6. Further evidence has been deposited as Supplementary Publication SUP 50045 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1975), 145, 5.

The amino acid sequence around the reactive cysteine residue in human plasma Factor XII.

1. Activated human plasma factor XIII was 65% inhibited by iodo[(14)C]acetate and incorporated 0.6 mol of label into the alpha subunit, which eventually was allowed to form a precipitate. 2. All the label was recovered as S-carboxymethylcysteine in a tetra-peptide of sequence Gly-Gln-Cys-Trp.