Human skeletal-muscle aldolase: N-terminal sequence analysis of CNBr- and o-iodosobenzoic acid-cleavage fragments

Fructose-1,6-bisphosphate aldolase was purified from human skeletal-muscle by affinity elution chromatography. Four CNBr-cleavage fragments were purified by gel filtration, and their N-terminal amino acid sequences were determined. Cleavage with o-iodosobenzoic acid at the three tryptophan residues also yielded fragments suitable for N-terminal sequence analysis. Thus, the sequence of 272 of the 363 residues was established. These sequence results allow many of the discrepancies between the two published rabbit skeletal-muscle aldolase sequences to be resolved. The human aldolase sequence reported here is 96% identical to a "consensus" rabbit aldolase sequence. A comparison with a partial sequence of Drosophila aldolase (103 residues) shows 80% identity. The determination of the amino acid sequence of human aldolase is important for the interpretation of the crystal structure of this enzyme.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. III) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus. Hydroxylamine-, o-iodosobenzoic acid- and tryptic-fragments. The complete amino-acid sequence

Based on the partial sequence of the cyanogen bromide fragments [Tratschin, J.D., Wirz, B., Frank, G. and Zuber, H. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 879-892], the amino-acid sequence of thermophilic lactate dehydrogenase from B. stearothermophilus was completed by the preparation and sequencing (sequenator, carboxypeptidase A and Y) of further overlapping fragments. Suitable peptide fragments were obtained by lactate dehydrogenase cleavage with hydroxylamine, o-iodosobenzoic acid and trypsin. The polypeptide chain of thermophilic lactate dehydrogenase from B. stearothermophilus consists of 317 amino-acid residues. While sequence homology with mesophilic lactate dehydrogenase of higher organisms reaches 35%, it is substantially higher with this mesophilic enzyme of bacillae (greater than 60%, B. megaterium, B. subtilis). The secondary structure elements and amino-acid residues of the active site of thermophilic lactate dehydrogenase deducted from primary structure data were compared with those from the mesophilic enzyme, the same was done for the internal sequence homology at the nucleotide-binding units. A comparative structure analysis (matrix system) based on the primary structure data of thermophilic enzyme should provide insight into the characteristic structure differences between thermophilic and mesophilic lactate dehydrogenase.     

The complete amino acid sequence of chicken skeletal-muscle enolase.

The complete amino acid sequence of chicken skeletal-muscle enolase, comprising 433 residues, was determined. The sequence was deduced by automated sequencing of hydroxylamine-cleavage, CNBr-cleavage, o-iodosobenzoic acid-cleavage, clostripain-digest and staphylococcal-proteinase-digest fragments. The presence of several acid-labile peptide bonds and the tenacious aggregation of most CNBr-cleavage fragments meant that a commonly used sequencing strategy involving initial CNBr cleavage was unproductive. Cleavage at the single Asn-Gly peptide bond with hydroxylamine proved to be particularly useful. Comparison of the sequence of chicken enolase with the two yeast enolase isoenzyme sequences shows that the enzyme is strongly conserved, with 60% of the residues identical. The histidine and arginine residues implicated as being important for the activity of yeast enolase are conserved in the chicken enzyme. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Russell (1986) Biochem. J. 236, 127-130].     

A glyceraldehyde-3-phosphate dehydrogenase with eubacterial features in the amitochondriate eukaryote,Trichomonas vaginalis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), localized in the cytosol of Trichomonas vaginalis, was partially purified. The enzyme is specific for NAD⁺ and is similar in most of its catalytic properties to glycolytic GAPDHs from other organisms. Its sensitivity to koningic acid is similar to levels observed in GAPDHs from eubacteria and two orders of magnitude lower than those observed for eukaryotic GAPDHs. The complete amino acid sequence of T. vaginalis GAPDH was derived from the N-terminal sequence of the purified protein and the deduced sequence of a cDNA clone. It showed great similarity to other eubacterial and eukaryotic GAPDH sequences. The sequence of the S-loop displayed a eubacterial signature. The overall sequence was more similar to eubacterial sequences than to cytosolic and glycosomal eukaryotic sequences. In phylogenetic trees obtained with distance matrix and parsimony methods T. vaginalis GAPDH clustered with its eubacterial homologs. GAPDHs of other amitochondriate protists, belonging to early branches of the eukaryotic lineage (Giardia lamblia and Entamoeba histolytica—Smith M.W. and Doolittle R.F., unpublished data in GenBank), showed typical eukaryotic signatures and clustered with other eukaryotic sequences, indicating that T. vaginalis GAPDH occupies an anomalous position, possibly due to horizontal gene transfer from a eubacterium. Correspondence to: M. Müller     

Purification of MEA, a mast cell growth-enhancing activity, to apparent homogeneity and its partial amino acid sequencing

A novel growth factor for bone marrow derived murine mucosal type mast cells has been isolated from the conditioned medium of the Mlsa-reactive mouse Th cell line MLS-4.2. In proliferation assays this growth factor synergizes, like IL-4, with IL-3 on established mast cell lines and was therefore termed MEA: mast cell growth enhancing activity. MEA was characterized as a glycoprotein with a Mr range between 37,000 and 43,000. Apparent homogeneity was obtained by using a four-step purification scheme including cation exchange chromatography, Procion red affinity chromatography, IEF, and gel filtration. Inasmuch as MEA was N-terminally blocked during automated Edman-degradation, peptide fragments after digestion with trypsin were used for partial amino acid sequence determination. All evaluable MEA peptide fragments showed complete sequence homology to a recently purified and cloned novel T cell growth factor (P40/TCGF III), the mouse homologue of human IL-9.     

The complete amino acid sequence of yeast phosphoglycerate kinase.

The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined. The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene. The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes. Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis. Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure. The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371). Both these regions contribute to the nucleoside phosphate-binding site. A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes. The yeast has strikingly fewer methionine, cysteine and tryptophan residues.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, V. The complete amino-acid sequence of the mesophilic L-lactate dehydrogenase from Bacillus megaterium

The complete amino-acid sequence of L-lactate-dehydrogenase from the mesophilic Bacillus megaterium was determined. 92% of the 318 amino acids were established by sequence analysis of the N-terminus, of four CNBr fragments and of one fragment obtained by cleavage with BNPS-skatole. The primary structure was completed by sequencing overlapping fragments obtained by further cleavage of suitable CNBr fragments and BNPS fragments with either trypsin, endoproteinase Lys-C, o-iodosobenzoic acid or hydroxylamine. The C-terminal amino acids were determined by degradation with carboxypeptidase A. The sequence homology between lactate dehydrogenases from B. megaterium and those from other Bacilli is 59-61% and 35-37% to those from higher organisms. The high sequence homology among lactate dehydrogenases from Bacilli, adapted to different temperatures, allows comparative studies of the structural basis of protein thermostability.     

Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus

The complete amino-acid sequence of subtilisin DY, an extracellular alkaline proteinase produced by Bacillus subtilis strain DY was determined. This included automated sequence analysis of the whole molecule and its large fragments such as tryptic peptides obtained from the inactivated enzyme, peptides generated by cyanogen bromide, by o-iodosobenzoic acid and by hydroxylamine. The peptides were isolated by gel filtration and by reversed-phase high performance liquid chromatography. The amino-acid sequence of subtilisin DY was determined by overlapping the isolated peptides. It consists of 274 amino-acid residues, like that of subtilisin Carlsberg. By comparison with the structures of the subtilisins Carlsberg, amylosacchariticus and BPN' 32, 80 and 82 amino-acid substitutions were found, which are caused by 37, 102 and 106 nucleotide mutations, respectively. It was found also that 62.5% of the amino-acid residues in the molecules of these four subtilisins are identical with respect to kind and position of the residue, which suggests that these molecules have had a common ancestral precursor. The amino-acid replacement analysis of the four subtilisins leads to the conclusion that they have evolved almost independently.     

Proteolytic conversion of arginine-vasopressin and oxytocin by brain synaptic membranes. Characterization of formed peptides and mechanisms of proteolysis

This study concerned the fragmentation of the nonapeptides arginine-vasopressin (AVP-(1-9)) and oxytocin (OXT-(1-9)) by proteolytic enzymes present in a brain synaptic membrane preparation. The peptides formed during digestion of arginine-vasopressin and oxytocin were isolated by high pressure liquid chromatography and chemically characterized by amino acid composition, NH2-terminal amino acid residues, and the presence of 14C radioactivity in tyrosine-2 and glycinamide-9. The major peptide fragments of arginine-vasopressin were [Cyt6]-AVP-(2-9), [Cyt6]-AVP-(3-9), [less than Glu4, Cyt6]-AVP-(4-9), and a peptide having the AVP-(4-8) sequence. The characterized fragments of oxytocin were [Cyt6]-OXT-(2-9), [Cyt6]-OXT-(3-9), [Cyt6]-OXT-(4-9), [less than Glu4, Cyt6]-OXT-(4-9), and [Cyt6] OXT-(5-9). Employing differentially 14C-labeled arginine-vasopressin and oxytocin, the proteolysis of the two peptides into fragments was followed with time. The results showed the sequential formation of peptide fragments by proteolytic cleavage from the NH2 terminus onward, demonstrating the action of an aminopeptidase-like enzyme. Arginine-vasopressin was converted significantly more rapidly by the amino-peptidase activity than oxytocin. In contrast to known brain aminopeptidases, the synaptic membrane-associated activity cleaved the nonapeptides without prior reduction of the disulfide bridge. From the present data it is concluded that aminopeptidases predominate in the proteolytic mechanism by which brain synaptic membranes convert arginine-vasopressin and oxytocin. The role of the proteolytic events and the significance of formed peptide fragments is discussed in view of the concept that arginine-vasopressin and oxytocin are precursors for neuropeptides in brain.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, IV. The primary structure of the mesophilic lactate dehydrogenase from Bacillus subtilis

The complete amino-acid sequence of lactate dehydrogenase from the mesophilic Bacillus subtilis (B. X1) was determined. Approximately 70% of the sequence was obtained by sequence analysis of intact protein (N-terminal sequence) and of four CNBr fragments (CNBr3, CNBr4, CNBr5 and CNBr6). Sequences overlapping the CNBr fragments were determined from polypeptide fragments obtained by cleavage using o-iodosobenzoic acid (cleavage at Trp) or clostripain (cleavage at Arg). The C-terminal amino-acid residue (Asn) was detected by carboxypeptidase Y-degradation. Lactate dehydrogenase from B. subtilis shows a 69% sequence homology to that from the thermophilic strain B. stearothermophilus, and a 34% sequence homology to those from higher organism. The homology of these enzymes is particularly high at the active site regions (the coenzyme and substrate binding sites). The relatively high sequence conservation of the lactate dehydrogenases from B. subtilis and B. stearothermophilus (and from other bacilli) allows a structural comparison of this temperature variants.     

Expression in Escherichia coli and characterization of human growth-hormone-releasing factor

A chemically synthesized DNA sequence, coding for the 44 amino acid residues of human growth-hormone-releasing factor (GRF) preceded by a tryptophan codon, was cloned in frame with Escherichia coli trpE gene within a pBR322-derived plasmid. GRF was expressed in E. coli as a fused polypeptide chain (TrpE-GRF) and then the GRF amino acid sequence was released from the fused protein by specific chemical cleavage at the tryptophan residue using o-iodosobenzoic acid. The thioether group of the methionine residue of GRF was converted in the sulfonium salt derivative, in order to prevent irreversible oxidation of methionine to the sulfone derivative by the o-iodosobenzoic acid reagent. GRF was purified by HPLC and characterized in terms of amino acid composition after acid hydrolysis, protein sequencing and gel electrophoretic behaviour. These data clearly established that the biosynthetic GRF was identical to the natural one, except for the lack of amidation at the carboxyl-terminal amino acid. Far-ultraviolet circular dichroism measurements established that both biosynthetic and natural GRF are devoid of secondary structure in aqueous solution at neutral pH, whereas both peptide samples achieve a high percentage of helical structure in the presence of trifluoroethanol.     

Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl (Amphibia, Urodela). The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD(+). Only one GAPDH isoform (pI 7.57) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose. A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme--the first GAPDH from an amphibian of the Caudata group studied so far--with other GAPDHs of major vertebrate phyla.     

Antigenic structure of the cyanogen bromide peptide F-CB3 from fibrinogen alpha-chain.

The antigenic properties of the cyanogen bromide peptide F-CB3 from bovine fibrinogen alpha-chain were studied in radioimmune assays with rabbit antibodies to fibrinogen or to peptide F-CB3. Both fibrinogen and peptide F-CB3 were indistinguishable in inhibition and dissociation tests. Modification of the single disulfide bridge in peptide F-CB3 either by reduction or by cleavage with cyanide was not accompanied by loss of serologic activity. Inhibition studies with three individual fragments obtained after cyanide cleavage (molecular weight range 7000 to 23000) indicated the presence of at least three distinct antigenic determinants in peptide F-CB3. After trypsin digestion of peptide F-CB3 still 75% of its maximal inhibiting capacity was retained. Lack of change in antigenic activity of peptide F-CB3 after release from the fibrinogen molecule by cyanogen bromide and upon further fragmentation is presumably due to the presence of several sequential antigenic determinants but the presence of conformational determinants could not be entirely excluded. Since no cross-reaction was observed between bovine and human peptides F-B3 one may expect considerable variation in their amino acid sequence.     

Human adenine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme

We defined the amino acid sequence of adenine phosphoribosyltransferase isolated from human erythrocytes. Peptide fragments formed by cleavage at arginine, lysine, glutamic acid, and methionine were purified by high pressure liquid chromatography and sequenced by manual Edman degradation. The complete primary structure of human adenine phosphoribosyltransferase was established by sequence analysis of 19 peptide fragments. Presumed homology between the human and rodent enzymes was used to order fragments that had inadequate overlapping sequences. The enzyme has 179 residues with a calculated subunit molecular weight of 19,481. Mass spectrometry indicated that the NH2-terminal residue is acetylated. Human adenine phosphoribosyltransferase has sequence homology with xanthine-guanine phosphoribosyltransferase from Escherichia coli in 110-amino acid region encompassing the NH2-terminal section of the enzyme.     

Isolation of a cDNA coding for human galactosyltransferase

Human milk galactosyltransferase (EC 2.4.1.22) was purified to homogeneity using affinity chromatography. Edman degradation was used to determine the amino acid sequences of eight peptide fragments isolated from the purified enzyme. A 60-mer "optimal" oligonucleotide probe that corresponded to the amino acid sequence of one of the galactosyltransferase peptide fragments was constructed and used to screen a lambda gt10 cDNA library. Two hybridization-positive recombinant phages, each with a 1.7 Kbp insert, were detected among 3 X 10(6) recombinant lambda gt10 phages. Sequencing of one of the cDNA inserts revealed a 783 bp galactosyltransferase coding sequence. The remainder of the sequence corresponded to the 3'-region of the mRNA downstream from the termination codon.     

Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthase

The proposed mechanism of action of thymidylate synthase envisages the formation of a covalent ternary complex of the enzyme with the substrate dUMP and the cofactor 5,10-methylenetetrahydrofolate (CH2H4folate). The proposed structure of this adduct has been based by analogy on that of the covalent inhibitory ternary complex thymidylate synthase-FdUMP-CH2H4folate. Our recent success in using the protein precipitant trichloroacetic acid to trap the latter complex and covalent binary complexes of the enzyme with FdUMP, dUMP, and dTMP led to the use of this technique in attempts to trap the transient putative covalent catalytic ternary complex. Experiments performed with [2-14C]dUMP and [3',5',7,9-3H]CH2H4folate show that both the substrate and the cofactor remained bound to the protein after precipitation with trichloroacetic acid. The trapped putative covalent catalytic complex was subjected to CNBr fragmentation, and the resulting peptides were fractionated by reverse-phase high-pressure liquid chromatography. The isolated active site peptide was shown to retain the two ligands and was further characterized by a limited sequence analysis using the dansyl Edman procedure. The inhibitory ternary complex, which was formed with [14C]FdUMP and [3H]CH2H4folate, served as a control. The active site peptide isolated from the CNBr-treated inhibitory ternary complex was also subjected to sequence analysis. The two peptides exhibited identical sequences for the first four residues from the N-terminus, Ala-Leu-Pro-Pro, and the fifth amino acid residue was found to be associated with the labeled nucleotides and the cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dam methyltransferase from Escherichia coli: sequence of a peptide segment involved in S-adenosyl-methionine binding.

DNA adenine methyltransferase (Dam methylase) has been crosslinked with its cofactor S-adenosyl methionine (AdoMet) by UV irradiation. About 3% of the enzyme was radioactively labelled after the crosslinking reaction performed either with (methyl-3H)-AdoMet or with (carboxy-14C)-AdoMet. Radiolabelled peptides were purified after trypsinolysis by high performance liquid chromatography in two steps. They could not be sequenced due to radiolysis. Therefore we performed the same experiment using non-radioactive AdoMet and were able to identify the peptide modified by the crosslinking reaction by comparison of the separation profiles obtained from two analytical control experiments performed with 3H-AdoMet and Dam methylase without crosslink, respectively. This approach was possible due to the high reproducibility of the chromatography profiles. In these three experiments only one radioactively labelled peptide was present in the tryptic digestions of the crosslinked enzyme. Its sequence was found to be XA-GGK, corresponding to amino acids 10-14 of Dam methylase. The non-identified amino acid in the first sequence cycle should be a tryptophan, which is presumably modified by the crosslinking reaction. The importance of this region near the N-terminus for the structure and function of the enzyme was also demonstrated by proteolysis and site-directed mutagenesis experiments.     

The complete amino acid sequence of rabbit muscle phosphoglucomutase

The complete amino acid sequence of rabbit muscle phosphoglucomutase has been determined by isolating the 11 peptide fragments produced by the cyanogen bromide cleavage reaction and subjecting these to automated sequencing procedures. Products produced by treatment of some of these fragments with hydroxylamine, iodosobenzoic acid, mild acid, cyanogen bromide in formic and heptafluorobutyric acids, Staphylococcus aureus V8 protease, and trypsin (with or without blocking at lysine residues) were used to complete the sequence for each of the cyanogen bromide fragments. The cyanogen bromide fragments were ordered by isolating the four tryptic peptides produced by a limited tryptic digest of the native enzyme in the presence of its substrates and its bivalent metal ion activator, Mg2+, degrading these by means of trypsin, after blocking digestion at lysine residues, and isolating and identifying all fragments thus produced that contained 10 or more residues. The 561-residue sequence thus obtained is one of the longest that has been determined by chemical means. There is excellent agreement between this sequence and published compositions after appropriate normalization. The absorbance of the enzyme is about 7.0 at 278 nm for a 1% solution; this value is 9% lower than that previously used.     

Studies on the structure of rabbit muscle aldolase. 3. Primary structure of the BrCN peptide containing the active site

The four peptide segments obtained from rabbit muscle aldolase by cleavage with BrCN and separation with gel-filtration chromatography (1) have been redesignated according to their positions in the molecule, N-A-B-C. The primary structure of segment A, containing 66 amino acid residues, including the Schiff base-forming lysine at the active site, has been elucidated by isolation and sequence analyses of the proteolytic subfragments. Preliminary separation of tryptic peptides containing 7–25 residues was achieved by chromatography on Sephadex G-25 which facilitated subsequent purification. For the study of the tryptic peptide of 25 residues further fragmentation with pepsin then subtilisin (Nagarse) was employed. Edman degradation directly after subtilisin cleavage of a peptide was found useful in avoiding deamidation of a glutamine NH₂-terminus newly formed in the proteolysis. The sequence of 90 amino acids in the center region of the polypeptide chain of rabbit muscle aldolase has now been established.