The complete amino acid sequence of low molecular mass urokinase from human urine

The sequence of all 253 amino acids of the heavy (B-) chain of human urinary urokinase was determined. The fragmentation strategy employed included cyanogen bromide cleavage of S-carboxymethylated B-chain at Met and/or Trp residues, cleavage of acid-labile Asp-Pro bonds, and the use of the specific endoproteinases Lys-C and Arg-C for generation of overlapping fragments. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. The amino acid sequence obtained substantiates the serine protease character of the B-chain of urokinase: a considerable homology with other serine proteinases, especially with the B-chain of human plasmin, was proved. The pertinent active site amino acids were localized: His-46, Asp-97, and Ser-198. A carbohydrate side chain, containing at least 4 glucosamine and 2 galactosamine residues, was demonstrated to be fixed at asparagine in position 144. The sequence data presented, together with the sequence of the second (A1-) chain of low molecular mass urokinase which was reported by us in an earlier communication, complete the knowledge of the whole primary structure of an active form of human urinary urokinase.     

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.     

A functionally active heavy chain derived from human high molecular weight urokinase

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.     

The complete amino acid sequence of human complement factor H.

The complete amino acid sequence of the human complement system regulatory protein, factor H, has been derived from sequencing three overlapping cDNA clones. The sequence consists of 1213 amino acids arranged in 20 homologous units, each about 60 amino acids long, and an 18-residue leader sequence. The 60-amino-acid-long repetitive units are homologous with those found in a large number of other complement and non-complement proteins. Two basic C-terminal residues, deduced from the cDNA sequence, are absent from factor H isolated from outdated plasma. A tyrosine/histidine polymorphism was observed within the seventh homologous repeat unit of factor H. This is likely to represent a difference between the two major allelic variants of factor H. The nature of the cDNA clones indicates that there is likely to be an alternative splicing mechanism, resulting in the formation of at least two species of factor H mRNA.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase.

The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase, comprising 363 residues, was determined. The sequence was deduced by automated sequencing of CNBr-cleavage, o-iodosobenzoic acid-cleavage, trypsin-digest and staphylococcal-proteinase-digest fragments. Comparison of the sequence with other class I aldolase sequences shows that the mammalian muscle isoenzyme is one of the most highly conserved enzymes known, with only about 2% of the residues changing per 100 million years. Non-mammalian aldolases appear to be evolving at the same rate as other glycolytic enzymes, with about 4% of the residues changing per 100 million years. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Freemont (1988) Biochem. J. 249, 789-793].     

Human adenosine deaminase. cDNA and complete primary amino acid sequence

A previously cloned partial adenosine deaminase cDNA insert (0.8 kilobase) was used to clone additional nucleotide sequences from human HPB ALL cDNA libraries. cDNA encompassing the entire coding, and 3'-untranslated regions as well as nearly all of the 5'-untranslated region was obtained. The complete amino acid sequence of the enzyme deduced from the cDNA sequence and protein sequencing consists of 362 amino acids, excluding the initiator Met, and accounts for Mr = 40,638. Secondary structure predictions assign adenosine deaminase to the alpha/beta class of proteins. Northern blot analysis with a cDNA probe showed adenosine deaminase mRNA to be present in normal to above normal amounts in B-lymphoblasts derived from adenosine deaminase-deficient patients with severe combined immunodeficiency disease. Knowledge of the cDNA and primary amino acid sequence of adenosine deaminase will be pivotal in further defining the genetic abnormality and its functional consequences in adenosine deaminase expression defects.     

Primary structure of yeast cytochrome c peroxidase. II. The complete amino acid sequence

The complete amino acid sequence of 293 residues in the single polypeptide chain of yeast cytochrome c peroxidase has been determined. Sequence analyses were performed on fragments obtained by cleavage with cyanogen bromide and by tryptic digestion of citraconylated protein. These fragments were aligned with sequential and compositional data as well as those obtained with intact protein, tryptic, and chymotryptic peptides (Takio and Yonetani, 1980, Arch. Biochem. Biophys.203, 605–614). Residues critical for catalysis by the enzyme were identified as arginine 48, tryptophan 51, histidine 52, and histidine 174 by fitting the sequence to the electron density map derived by others. Sequence comparison with other proteins show limited homology with horseradish peroxidase and myoglobin.     

The covalent structure of hepatic microsomal epoxide hydrolase. II. The complete amino acid sequence

The complete amino acid sequence of hepatic microsomal epoxide hydrolase has been determined. The protein contains 455 amino acid residues in a single polypeptide chain and has Mr = 52,691. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. This is the first epoxide hydrolase and the third microsomal enzyme for which the complete sequence has been determined.     

The amino acid sequence of the light chain of human high-molecular-mass kininogen

The complete amino acid sequence of the light chain of human high-molecular-mass kininogen has been determined. The peptide chain contains 255 amino acid residues. The half-cystine, which forms the disulfide bridge to the heavy chain, was identified in position 225. Nine carbohydrate attachment sites were found. All carbohydrate side chains are O-glycosidically linked. Alignment of the present sequence with the bovine kininogen light chain sequence shows a high degree of homology, except for an extension of 22 amino acids within the histidine-rich part of the sequence. The histidine-rich region may have arisen by gene multiplication during evolution.     

The complete amino acid sequence of monkey progastricsin

The complete amino acid sequence of progastricsin from the Japanese monkey (Macaca fuscata) was determined. Progastricsin is composed of 374 residues, including the gastricsin moiety of 331 residues and the activation segment of 43 residues. Upon activation under acidic conditions, progastricsin was converted to gastricsin via the intermediate protein species. NH2-terminal sequence determination of these protein species enabled us to deduce the NH2-terminal 78-residue sequence of progastricsin, including the 43-residue activation segment. The complete sequence of the gastricsin moiety was determined using peptide fragments obtained by several chemical and enzymatic cleavages. The molecular weight of progastricsin was determined to be 40,785. As compared with pepsinogen A of the same monkey species, deletion of 4 residues and insertion of 5 residues were observed. Although monkey progastricsin and pepsinogen A have highly homologous sequences around the two active site aspartyl residues, the homology between these proteins is rather small (49% identity). This indicates that progastricsin diverged from pepsinogen A in the early phase of the evolution of gastric aspartyl proteinases.     

The complete amino acid sequence of proapolipoprotein A-I of chicken high density lipoproteins

The complete amino acid sequence of proapolipoprotein (proapo) A-I of chicken high density lipoproteins was determined by sequencing overlapping peptides produced by trypsin, S. aureus V8 protease, and cyanogen bromide cleavage. There are 240 amino acid residues in mature chicken apoA-I. By direct sequence analysis of a cyanogen bromide peptide, we also determined the sequence of a 6-amino-acid prosegment which is present at approx. 10% the molar amount of the mature peptide in chicken plasma. Sequence comparison among apoA-I from chicken, human, rabbit, dog and rat, and secondary structure analysis indicate that while the degree of sequence homology is only moderate (less than 50% between chicken and man), there is good conservation of apoA-I secondary structure, especially in the N-terminal two-thirds of the protein in these widely separated species.     

Rat embryonic fibroblast tropomyosin 1. cDNA and complete primary amino acid sequence

A cDNA expression library of approximately 80,000 members was prepared from rat embryonic fibroblast mRNA using the plasmid expression vectors pUC8 and pUC9. Using an immunological screening procedure and 32P-labeled cDNA probes, clones encoding rat embryonic fibroblast tropomyosin 1 (TM-1) were identified and isolated. DNA sequence analysis was carried out to determine the amino acid sequence of the protein. Rat embryonic fibroblast TM-1 was found to contain 284 amino acids and is most homologous to smooth muscle alpha-tropomyosin compared with skeletal muscle alpha- and beta-tropomyosins and platelet beta-tropomyosin. Among the various tropomyosins, two regions where the greatest sequence divergence is evident are between amino acids 185 and 216 and amino acids 258 and 284. Rat embryonic fibroblast TM-1 and chicken smooth muscle alpha-tropomyosin are most closely related from amino acids 185 and 216 compared with skeletal muscle and platelet tropomyosins. In contrast, rat embryonic fibroblast TM-1, smooth muscle alpha-tropomyosin, and platelet tropomyosin are most homologous from amino acids 258 and 284 compared with skeletal muscle tropomyosins. These differences in sequences at the carboxyl-terminal region of the various tropomyosins are discussed in relation to differences in their binding to skeletal muscle troponin and its T1 fragment.