Specific cyanylation and cleavage at cysteine-104 human hemoglobin alpha-chain. A novel approach to the problem of the alpha-chain tryptic core in the study of haemoglobin variants by "fingerprinting" methods.

1. A new approach to the analysis, by "fingerprinting", of the tryptic core region of human haemoglobin alpha-chain is described. 2. The alpha-chain is cyanylated at its single cysteine residue (alpha104) and then split, by exposure to mild alkali, at the N-peptide bond of the resulting beta-thiocyanoalanine residue. 3. The two cleavage fragments, alpha1-103 and alpha104-141, are separated by gel filtration, and the fragment alpha104-141, which contains all the residues of the alpha-chain tryptic core, is digested with pepsin. 4. Preparative "fingerprints" of these peptic peptides yield eight major peptides, which provide complete sequence information for the whole region alpha104-141. 5. The utility of the method is demonstrated by repeating the determination of the substitution in haemoglobin Hopkins-2, a known alpha-chain core variant in which histidine-alpha112 (G19) is replaced by an aspartic acid residue.     

The amino acid sequence of glutamate decarboxylase from Escherichia coli. Evolutionary relationship between mammalian and bacterial enzymes.

The amino acid sequence of glutamate decarboxylase from Escherichia coli was solved by a combination of automated Edman degradation of peptide fragments derived by proteolytic and chemical cleavage and sequencing of DNA. Correct alignment of three peptides, for which no peptide overlaps were available, was achieved by sequencing a 1.1-kbp fragment of DNA produced by a polymerase-chain reaction using primers corresponding to sequences known to be in amino-terminal and carboxy-terminal regions of the protein. Sequence similarity (24% identity) with mammalian glutamate decarboxylase was found to be limited to a 55-residue sequence around the lysine residue that binds the coenzyme. Stronger similarity (38% identity), again confined to the same region, is seen with bacterial pyridoxal-phosphate-dependent histidine decarboxylase.     

Enzyme catalysed non-oxidative decarboxylation of aromatic acids. II. Identification of active site residues of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger

In order to understand the mechanism of decarboxylation by 2,3-dihydroxybenzoic acid decarboxylase, chemical modification studies were carried out. Specific modification of the amino acid residues with diethylpyrocarbonate, N-bromosuccinimide and N-ethylmaleiimide revealed that at least one residue each of histidine, tryptophan and cysteine were essential for the activity. Various substrate analogs which were potential inhibitors significantly protected the enzyme against inactivation. The modification of residues at low concentration of the reagents and the protection experiments suggested that these amino acid residues might be present at the active site. Studies also suggested that the carboxyl and ortho-hydroxyl groups of the substrate are essential for interaction with the enzyme.     

Complete amino acid sequence of the Aspergillus cytotoxin mitogillin

The complete amino acid sequence of the cytotoxin mitogillin has been determined by sequencing the intact chain and peptide fragments produced by cleavage at methionyl, arginyl, lysyl, and tryptophanyl residues and at one aspartic acid-proline bond. The protein consists of 149 amino acid residues with alanine at the NH2 terminus and histidine at the COOH terminus. The calculated Mr of the native mitogillin was 16 867. The native molecule presents two disulfide bridges, one between cysteine residues at positions 5 and 147 and another one between cysteine residues at positions 75 and 131. The amino acid sequence of mitogillin shows 86% homology with another cytotoxic protein called alpha-sarcin.     

Proteolytic processing of the alpha-chain of the lysosomal enzyme, beta-hexosaminidase, in normal human fibroblasts

The two subunits of beta-hexosaminidase undergo many post-translational modifications characteristic of lysosomal proteins, including limited proteolysis. To identify proteolytic cleavage sites in the alpha-chain, we have biosynthetically radiolabeled the transient forms, isolated these by immunoprecipitation, gel electrophoresis, and electroelution, and subjected them to automated Edman degradation. The position of the NH2-terminal amino acid was inferred from the elution cycle of the radioactive amino acid and the primary sequence encoded in the alpha-chain cDNA. The amino terminus of the precursor obtained by in vitro translation of SP6 alpha-chain mRNA in the presence of microsomes was leucine 23. The same amino terminus was found in precursor alpha-chain synthesized by normal human fibroblasts (IMR90) in a 1- or 3-h pulse or secreted by these cells in the presence of NH4Cl. The alpha-chain isolated after a 3-h pulse followed by a 5-h chase (intermediate form) included a mixture of molecular species of which the amino terminus was arginine 87 (most abundant), histidine 88, or leucine 90. After a 20-h chase (mature form) the latter species predominated. This mature form of the alpha-chain remained fully reactive with antibody raised against the carboxyl-terminal 15 amino acids, indicating little if any proteolysis at the carboxyl terminus. Thus synthesis and maturation of the alpha-chain of beta-hexosaminidase includes two major proteolytic cleavages: the first, between alanine 22 and leucine 23, removes the signal peptide to generate the precursor form, whereas the second occurs between the dibasic amino acids, lysine 86 and arginine 87. The second cleavage is followed by trimming of 3 additional amino acids to give the mature form of the alpha-chain.     

Isolation and characterization of an organic solvent soluble polypeptide component from photoreceptor complexes of Rhodospirillum rubrum.

An organic solvent soluble polypeptide has been isolated from photoreceptor complexes and chromatophores of Rhodospirillum rubrum. After extraction of the protein from lyophilized samples with 1:1 chloroform-methanol, it was purified by column chromatography. Its isoelectric point determined by isoelectric focusing was 7.10. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified polypeptide ran as a single band of an apparent molecular weight of 12 000. However, according to amino acid analysis, the minimal molecular weight based on one histidine residue per polypeptide is 19 000. The polypeptide contains no cysteine and no tyrosine. Amino acid analysis indicated that three methionines were present per histidine residue and cyanogen bromide cleavage gave four smaller peptides which were isolated by two-dimensional electrophoresis and chromatography. Spectroscopic analysis indicated the presence of three tryptophan residues per histidine and N-bromosuccinamide cleavage also gave four smaller peptides which could be isolated by two-dimensional electrophoresis and chromatography. The C-terminal amino acid was shown to be glycine by two methods, while the N-terminal amino acid appears to be blocked. The organic solvent soluble polypeptide accounts for approximately 50% of the chromatophore protein and seems to bind the antenna bacteriochlorophyll and carotenoid molecules. Using this procedure, organic solvent soluble polypeptides were isolated from several photosynthetic bacteria and were found to have substantially different amino acid contents.     

Location of the interchain disulfide bonds of the fourth component of human complement (C4): evidence based on the liberation of fragments secondary to thiol-disulfide interchange reactions

Treatment of human C4 with chemical denaturants and heat produces rapid, autolytic peptide bond cleavage of the alpha-chain. These alpha-chain fragments are linked to the parent C4 molecule through disulfide bonds. On more prolonged heating, however, there is liberation of several peptides, including the beta-chain, the gamma-chain, and a C-terminal alpha-chain fragment. This reaction is inhibited by iodoacetamide. By using a fluorescent thiol reagent and 14C-iodoacetamide, the thiol group present on each peptide was analyzed. The results suggest that the thiol residue exposed by cleavage of the thioester bond induces thiol-disulfide interchange reactions to liberate the peptides. Based on the identification of fragments liberated, the kinetics of their appearance, their sulfhydryl content, and the reported primary structure of human C4, a model of the interchain disulfide bonds is proposed in which the amino terminal portion of the alpha-chain is disulfide-linked to both the beta- and gamma-chains, whereas the carboxyl terminal portion of the alpha-chain is disulfide-linked to only the gamma-chain.     

The amino-acid sequence of troponin C from frog skeletal muscle.

The primary structure of the troponin C from skeletal muscle of the frog Rana esculenta has been determined. The amino acid sequence was deduced from amino acid determinations of peptides obtained after cleavage with cyanogen bromide. Overlapping peptides were isolated from tryptic digests of performic-acid-oxidized troponin C and phthalylated performic-acid-oxidized troponin C. All overlaps have been determined except for the Arg-Ile sequence at position 103--104, which has been obtained by comparison with homologous troponins C. Frog troponin C consists of one polypeptide chain containing 152 amino acids. The calculated molecular weight is 18299. There is a single cysteine residue at position 101 and a single tyrosine residue at position 112. No histidine or tryptophan residues are present. The amino-terminal amino acid is N-acetylated. The homology of frog troponin C with other skeletal and cardiac troponin C is briefly discussed.     

Cleavage of C3 by a neutral cysteine proteinase of Entamoeba histolytica

The major secreted proteinase of Entamoeba histolytica, a 56-kDa neutral cysteine proteinase, activates C by cleaving C3. The action of the proteinase is similar to C-derived C3 convertases because it produces a single cleavage of the alpha-chain in a dose- and time-dependent manner and cleaves C3 between residues 78 and 79, only one amino acid residue distal to the natural site acted on by the C3 convertases. C3a generation was detected by RIA. The 105-kDa fragment produced by the cleavage of the alpha-chain was structurally and functionally equivalent to the alpha'-chain of C3b, as demonstrated by susceptibility to the action of factors I and H and participation in the activation of the alternative pathway of C. Activation of C by the 56-kDa neutral cysteine proteinase may play a role in the early inflammatory response in amebic lesions and thus contribute to the pathogenesis of invasive amebiasis.     

Prenylated proteins: demonstration of a thioether linkage to cysteine of proteins

Prenylated amino acid fragments have been isolated from prenylated proteins of Chinese hamster ovary cells. Gel-exclusion chromatography indicates that these proteins are modified by two different prenyl groups. The prenyl-amino acid fragments are labeled by 35S from cysteine, and this bond is cleaved by Raney-Ni, proving that the prenyl residue is linked to protein via a thioether to cysteine. Hydrazinolysis has been used to demonstrate that the cysteine is carboxy terminal.     

Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase

The nucleotide sequence of the fabA gene encoding beta-hydroxydecanoyl thioester dehydrase, a key enzyme of the unsaturated fatty acid synthesis pathway of Escherichia coli, has been determined by the dideoxynucleotide sequencing technique. Most of the sequence was obtained by sequencing intragenic insertions of the transposon, Tn1000, isolated in vivo. A synthetic primer complementary to a portion of the inverted repeat sequences at the ends of the transposon was used to prime DNA synthesis into the flanking fabA sequences. The gene is composed of 516 nucleotides (171 amino acid residues) encoding a protein with a molecular weight of 18,800. Approximately half of the derived amino acid sequence was confirmed by automated Edman sequencing of peptides obtained by cyanogen bromide cleavage. The active site histidine residue (His-70) has been identified by analysis of the peptides labeled by reaction with 14C-labeled 3-decynoyl-N-acetylcysteamine, a specific mechanism-activated inhibitor. A cysteine residue (Cys-69) adjacent to the active site histidine may play the role in catalysis previously assigned to a tyrosine residue. We also report a simplified purification process for the dehydrase beginning with extracts of a brain which greatly overproduces the enzyme.     

Cytochrome c from Schizosaccharomyces pombe. 2. Amino-acid sequence.

The amino acid sequence of Schizosaccharomyces pombe cytochrome c has been established by automatic degradation of the protein and by manual degradation of fragments obtained by cyanogen bromide cleavage and chymotryptic digestion. The chymotryptic peptides were aligned by homology with other known cytochrome c sequences. The protein is 108 residues long, with a four-residue amino-terminal tail. It has only one methionine residue and differs from other fungal cytochromes c in lacking the one-residue deletion at the C-terminal end. After a cyanogen bromide step, an unexpected cleavage of the peptide chain before a cysteine residue was observed. This is ascribed to formation of a dehydroalanyl residue during an incomplete S-carboxymethylation of the apoprotein, and subsequent cleavage under acidic conditions. Experimental evidence is presented in favour of the proposed mechanisms.     

Primary structure of the major β-chain of rat haemoglobins

The amino acid sequence of the major β-chain, ^IIβ, from rat haemoglobins was established with an automated sequencer. Amino acid heterogeneities were found that appear to result from allelic variation at particular residues. We applied several new or unusual techniques in determining the sequence: (1) reaction of the polypeptide with dansylaziridine for detection of cysteine; (2) blockage of the N-terminal residue and the ε-amino group of lysine residues with 1-fluoro-2-nitro-4-trimethylammoniobenzene iodide and subsequent identification of the modified lysine phenylthiohydantoin by absorbance at 420nm; (3) identification of histidine phenylthiohydantoin by its blue fluorescence under long-wave u.v. light; (4) cleavage of the chain into two or three fragments and subsequent sequencing without purification [a detailed statement giving the major phenylthiohydantoins assigned at each step for each sequence run before their alignment in individual sequences has been deposited as Supplementary Publication SUP 50084 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5]; (5) separation of fragments produced by CNBr cleavage by cation-exchange chromatography; (6) peptide sequencing after attachment of the peptide to cytochrome c. The amino acid sequence was confirmed by amino acid compositions of the complete chain, of CNBr fragments 1 and 3, and of 11 purified tryptic peptides.     

Partial sequence data for the L-(+)-lactate dehydrogenase from Streptococcus cremoris US3 including the amino acid sequences around the single cysteine residue and at the N-terminus

The following amino acid sequence information has been determined for the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus cremoris US3: the C-terminal amino acid, the N-terminal sequence of the first 20 amino acids and the sequence of a 53-residue tryptic peptide containing the only cysteine residue in the protein. The enzyme was cleaved by alkali at the cysteine residue following reaction first with 5,5'-dithiobis(2-nitrobenzoic acid) and then with K14CN. This treatment yielded two cleavage products as well as some higher polymers and some uncleaved enzyme. The radioactive cleavage product was purified and its size indicated that the cysteine residue is 80 residues from the C-terminus. Comparisons of the sequences determined for the S. cremoris enzyme with those already known for dogfish lactate dehydrogenase indicate that the two enzymes are only distantly related since the sequence homology between them is limited and of borderline statistical significance.     

Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.     

Amino-terminal processing of actins mutagenized at the Cys-1 residue.

Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.     

Structure of alpha-polymer from in vitro and in vivo highly cross-linked human fibrin.

Peptides derived from plasmic and cyanogen bromide (CNBr) cleavage of highly cross-linked fibrin were isolated and characterized by sodium dodecyl sulfate-gel electrophoresis, amino acid analyses, cyanoethylation, and NH2-terminal analyses. Extended plasmic digestions of human fibrin containing four epsilon-(gamma-glutamyl)lysine cross-links per molecule produced a peptide of alpha-chain origin (Mr congruent to 21,000) which was comprised of a small donor peptide cross-linked to the acceptor site peptide from the middle of the alpha-chain. CNBr cleavage of highly cross-linked in vitro fibrin or of fibrin from a spontaneously formed in vivo arterial embolus produced about three cross-linked species of molecular weights 30,000 to 40,000, each of which contained the largest CNBr fragment (Mr = 29,000) from the alpha-chain. The predominant cross-link-containing CNBr fragments derived their donor group from the near COOH-terminal region of the alpha-chain as judged by difference amino acid compositions and NH2-terminal analyses. Additionally, cross-linked fragments of molecular weights 68,000 to 70,000 which appeared to contain two acceptor site peptides (Mr = 29,000) were detected in minor amounts in the CNBr digests of fibrin formed from whole plasma or from purified, plasminogen-free fibrinogen. No larger polymeric cross-linked CNBr fragment was generated from any of the highly cross-linked fibrin preparations examined. A model for the predominant mode of alpha-chain polymerization is proposed.     

Limited tryptic proteolysis of pig kidney 3,4-dihydroxyphenylalanine decarboxylase

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase can be nicked by trypsin with complete loss of its catalytic activity. The original dimer of subunit molecular weight of about 52,000 yields fragments of Mr 38,000 and 14,000, as seen on sodium dodecyl sulfate-gel electrophoresis. Though inactive, the nicked protein retains its native molecular weight and its capacity to bind pyridoxal-5'-phosphate (pyridoxal-P), is recognized by an antiserum raised against the native enzyme, and forms Schiff's base intermediates with aromatic amino acids in L and D forms. Thus, the nicked protein appears to be in a conformation--closely resembling that of the original enzyme--which consists of a tight association of the two tryptic fragments. Dissociation and separation of the two fragments can be achieved under denaturing conditions on a reverse-phase HPLC column. The pyridoxal-P binding site is located on the larger fragment. No NH2-terminal residue is detected in either the intact enzyme or the larger fragment, whereas analysis of the smaller fragment yields a sequence of the first 50 amino acid residues. These data indicate that the smaller fragment is located at about one-third from the COOH terminus of Dopa decarboxylase, while the larger fragment constitutes the aminic portion of the molecule. The site of trypsin cleavage seems to be in a region of the enzyme particularly susceptible to proteolysis. The results of these studies contribute to a better understanding of the structural properties of pig kidney Dopa decarboxylase and may constitute an important step toward the elucidation of the enzyme's primary structure.     

Purification and properties of pyridoxal-5''-phosphate-dependent histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes.

Histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes were purified to homogeneity and compared with the histidine decarboxylase from Morganella morganii. All three enzymes required pyridoxal 5'-phosphate as a coenzyme, showed optimal activity at pH 6.5, decarboxylated only histidine among the amino acids derived from protein, and were tetramers or dimers of identical subunits. Amino-terminal sequences of the three enzymes showed up to 81% homology through residue 33, but the enzymes differed sufficiently in amino acid composition and sequence so that no cross-reaction occurred between the K. planticola or E. aerogenes enzymes and antibodies to the decarboxylase from M. morganii. All three enzymes were inhibited by carbonyl reagents; by amino-, carboxyl-, and some methyl-substituted histidines; and by alpha-fluoromethylhistidine. These decarboxylases, all from gram-negative organisms, differed greatly in subunit structure, biogenesis, and other properties from the pyruvoyl-dependent histidine decarboxylases from gram-positive organisms described previously.     

Chemical modification and amino terminal sequence of calotropin DI from Calotropis gigantea

The effect of chemical modification of histidine, lysine, arginine, tryptophan and methionine residues on the enzymatic activity of calotropin DI has been studied. 1,3-Dibromoacetone inhibited the enzyme completely, indicating that a single histidine residue and a cysteine residue are involved in its catalytic activity. Its second bistidine residue was modified with diethyl pyrocarbonate without loss of activity. Modification of seven of its 13 lysine residues with 2,4,6-trinitrobenzene sulphonic acid led to 90% loss of its activity, but no single lysine residue appears to be essential for its activity. Four of the 12 arginine residues by 1,2-cyclohexanedione can be modified with little loss of activity. Modification of a single tryptophan residue and two methionine residues did not inhibit enzymatic activity. The blocked amino-terminal amino acid residue of calotropin DI has been identified as pyroglutamic acid. Its amino-terminal amino acid sequence to residue 14 has been determined and compared with that of papain. They show an extensive homology in their amino-terminal amino acid sequences.