(Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties

Two metallothioneins (MTs) from bovine fetal liver were purified by a combination of gel filtration and ion-exchange chromatography. The primary structures of the isoproteins MT-1 and MT-2 were elucidated by peptide and amino acid sequence analysis. The amino-terminal part was deduced from automated Edman degradations of the pyridylethylated CNBr-cleaved derivatives. The remaining part of the sequence was established by a comparison of the carboxamidomethylated tryptic peptides to those from equine liver MT-1A and MT-2B. Peptides differing in either amino acid composition or retention time from high pressure liquid chromatography were further subjected to manual Edman degradations or carboxypeptidase Y digestion. The two isoproteins consist of 61 amino acids and show a sequence identity of 90%. When compared with the primary structures of other mammalian MTs, the 20 cysteinyl residues are totally conserved, in agreement with their function as metal ligands. The two isoproteins contain Cu and Zn at a ratio of 3:4. Spectroscopic data reveal absorption properties typical for both Cu- and Zn-thiolate transitions. The marked differences of MT-1 and MT-2 in the Cu-thiolate CD features can be attributed to the six amino acid substitutions occurring exclusively in the amino-terminal parts of the molecules. It is proposed that in bovine fetal MTs also the three copper ions are preferentially bound to the first 9 cysteinyl residues (cluster B) and the four zinc ions to the remaining 11 cysteinyl residues (cluster A) suggested previously by 113Cd NMR spectroscopy of calf liver MTs (Briggs, R. W., and Armitage, I. M. (1982) J. Biol. Chem. 257, 1259-1262).     

Sequence analysis of rat hypothalamic corticotropin-releasing factor with the o-phthalaldehyde strategy

Sequence analysis was performed on a 41-residue polypeptide that has been identified as the predominant form of high intrinsic corticotropin-releasing activity of rat hypothalamus. The sequence of residues 1-39 of this corticotropin-releasing factor (CRF) was determined by Edman degradation of a partially purified peptide in a highly sensitive spinning cup sequencer after selective blocking of CRF or its main contaminant with o-phthalaldehyde. This approach was validated by peptide mapping of CRF of a highly purified preparation. Peptide mapping was accomplished with reverse-phase high-pressure liquid chromatography of CRF fragments obtained by digestion with clostripain. The identities of the fragments cleaved from CRF were established by chromatographic comparison with synthetic peptides, amino acid analysis, and Edman degradation. On the basis of these experiments, the primary structure of rat hypothalamic CRF was established to be H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu- Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn - Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. It is expected that the o-phthalaldehyde strategy will facilitate the sequence analysis of partially purified peptides containing proline residues.     

Amino acid sequence of bovine osteoinductive factor

The complete amino acid sequence of bovine osteoinductive factor (OIF) was determined by automated Edman degradation of S-pyridylethylated bovine OIF and selected fragments. Cleavage with endoproteinase Lys-C, endoproteinase Glu-C, or endoproteinase Asp-N established all fragments in an unambiguous sequence. Bovine OIF contains 105 residues with a calculated molecular weight of 12,055. It is a single chain polypeptide containing two intramolecularly linked cysteines at residues 62 and 95. Two asparagine-linked glycosylation sites at positions 52 and 65 were found by comparing sequence data and peptide profiles of native and deglycosylated OIF fragments. The amino acid sequence of OIF has no homology to other reported proteins.     

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.     

Primary structure of sheep prostaglandin endoperoxide synthase deduced from cDNA sequence

The complete amino acid sequence of prostaglandin endoperoxide synthase from sheep vesicular gland has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA. The results were confirmed by digestion of the enzyme with carboxypeptidase Y and by automated Edman degradation of the intact enzyme polypeptide and peptide fragments obtained by limited proteolysis of the enzyme with Achromobacter proteinase I. Mature sheep prostaglandin endoperoxide synthase is shown to be composed of 576 amino acids with an Mr of 66,175. The precursor peptide is predicted to contain a 24-residue signal peptide. The serine residue susceptible to acetylation by aspirin is found to be located near the C-terminus of the enzyme polypeptide.     

The complete primary structure of phospholipase A2 from human pancreas

The complete amino acid sequence of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human pancreas was determined. The protein consists of a single polypeptide chain of 125 amino acids and has a molecular weight of 14003. The chain is cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digestion of the reduced and thialaminated derivative of the protein with clostripain, yielding three fragments. The largest fragment (residues 7-100) was further degraded both with staphylococcal proteinase and chymotrypsin. The sequence was determined by automated Edman degradation of the intact protein and of several large peptide fragments. Phospholipase A2 from human pancreas contains the same number of amino acids (125) as the enzyme from horse, while the enzymes from pig and ox contain 124 and 123 residues, respectively. The enzymes show a high degree of homology; human phospholipase differs from the other enzymes by substitutions of 26 (porcine), 28 (bovine) and 32 (equine) residues, respectively.     

Antimicrobial peptides derived from goose egg white lysozyme

Peptide fragments possessing antimicrobial activity were obtained by protease digestion of goose egg white lysozyme. Digested peptide purified from RP-HPLC which showed no lysozyme activity exhibited bactericidal activity toward Gram-negative and Gram-positive bacteria. LC/MS–MS and automated Edman degradation revealed the amino acid sequence to be Thr-Ala-Lys-Pro-Glu-Gly-Leu-Ser-Tyr. This sequence corresponds to amino acid positions 20–28, located at the N-terminal outer part of goose lysozyme. The peptide acted on bacterial membrane as shown by scanning electron microscopy. The mechanism of action could be explained from a helical structure that may be formed by the centered Pro residue and the terminal Lys residue after the peptide attaches to a cell membrane. This is the first study to report that a peptide derived from the protease digests of G-type lysozyme possesses antimicrobial activity with broad spectrum activity. Our result is comparative to the previous reports of Chicken lysozyme and T4 phage lysozyme, which showed antimicrobial activity after digestion with protease. These results might contribute to the usage of antimicrobial peptides engineered by genetic or chemical synthesis.     

Amino acid sequence of the proteolipid subunit of the proton-translocating ATPase complex from the thermophilic bacterium PS-3

The proteolipid subunit of the ATPase complex was identified in whole membranes of the thermophilic bacterium PS-3 by means of a covalent modification with the 14C-labelled inhibitor dicyclohexylcarbodiimide. The proteolipid could be purified from the membrane in free and carbodiimide-modified form by extraction with chloroform/methanol and subsequent carboxymethylcellulose chromatography in mixtures of chloroform/methanol/water. The complete amino acid sequence of the 72-residue polypeptide could be determined by automated solid-phase Edman degradation of the whole protein, and of fragments obtained after cleavage with cyanogen bromide and N-bromosuccinimide. Chemical cleavages and separations of the resulting fragments by gel chromatography were performed in 80% formic acid. The amino acid sequence shows a concentration of hydrophobic amino acids in two segments of about 25 residues at the amino-terminal and carboxy-terminal ends. The polar residues are clustered in the middle of the polypeptide chain. The bound [14C]dicyclohexylcarbodiimide label is recovered exclusively at position 56, which is occupied by a glutamyl residue. The proteolipid from PS-3 exhibits homology to the corresponding ATPase subunit from mitochondria. The carbodiimide-reactive glutamyl residue occurs at the position as in the mitochondrial proteins.     

Primary structure of two forms of gonadotropin-releasing hormone from brains of the American alligator (Alligator mississippiensis)

Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brains of the American alligator, Alligator mississippiensis, using reverse-phase high-pressure liquid chromatography (HPLC). The concentration of total GnRH was 8.8 ng/g of frozen brain tissue or 21.1 ng per brain. The amino acid sequence of each form of GnRH was determined using automated Edman degradation. The presence of the N-terminal pGlu residue was established by digestion studies with bovine pyroglutamyl aminopeptidase and coelution with synthetic forms of the native peptide. The primary structure of alligator GnRH I is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2 and alligator GnRH II is pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2.     

Studies of amino-acid sequence in dihydrofolate reductase from a human methotrexate-resistant cell line KB/6b. Structural and kinetic comparison with mouse L1210 enzyme

The partial amino acid sequence of dihydrofolate reductase (DHFR, EC 1.5.1.3) from human KB/6b cells has been determined by using 3.5 mg of protein. Peptides covering the entire polypeptide chain were recovered from preparative peptide maps generated by the combination of paper chromatography and electrophoresis at pH 4.4 Peptide maps from mouse L1210 DHFR were also generated for comparison. Amino acid sequence of 75% of the 186 amino acid residues in the polypeptide chain of human KB/6b DHFR was obtained from Edman degradations and the remaining sequence was deduced from the amino acid compositions, from electrophoretic mobilities of related peptides and from the sequence homologies with other known mammalian DHFR sequences. A comparison of the proposed human DHFR sequence with the previously known sequences of mouse enzyme [Stone, et al. (1979) J. Biol. Chem. 245, 480-488] indicates that 18 differences are located in the established sequence of 139 residues and that 5 additional differences are in the tentative sequence of the remaining 47 amino acids. Kinetic properties of human KB/6b and mouse L1210 DHFR, which were determined in parallel experiments, are also compared. The possible structural-functional relationships between human and mouse DHFR are discussed.     

Structural studies on rat prostatic binding protein. The primary structure of component C2 from subunit S

The amino acid sequence of component C2, the polypeptide specific for subunit S of prostatic binding protein, the major secretory glycoprotein of the rat ventral prostate, has been determined. Its structure was established using the manual Edman degradation on the most relevant fragments obtained by enzymatic digestion of the S-carboxamidomethylated component C2 and the native subunit S and by chemical cleavage of the remaining undigestible 'cores' with cyanogen bromide. Component C2 contains 92 amino acids corresponding to a molecular weight of 10619. It is a slightly acidic polypeptide in which the acidic and basic residues are unevenly distributed. The N terminus is blocked and three cysteine residues are almost evenly distributed over the peptide chain. A highly polar region is found in position 23-34 and two hydrophobic segments are located in the C-terminal part of the molecule. Component C2 is compared with component C1 of subunit F and their high sequence homology reveals an evolutionary relationship.     

The structure of ubiquitinated histone H2B.

Ubiquitinated histone H2B (uH2B) has been purified from both calf and pig thymus by exclusion chromatography in 7 M urea. Digestion of uH2B with Staphylococcus aureus V8 protease yielded the peptide 114-125 containing the ubiquitin moiety. Further digestion of this peptide with trypsin removed the ubiquitin and three H2B residues from the N-terminus. Edman degradations of both peptides established that ubiquitin is attached to the epsilon-amino group of lysine 120 in both calf and pig uH2B by an iso-peptide bond to the C-terminal glycine 76 of ubiquitin.     

Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the ATP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 80% formic acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 50% homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 gene in Aspergillus nidulans.     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

The covalent structure of calf skin type III collagen. II. The amino acid sequence of the cyanogen bromide peptide alpha 1(III)CB1,8,10,2(Positions 223--402).

The cyanogen bromide peptide alpha 1-(III)CB1,8,10,2 is 180 amino acid residues in length and occupies position 223 to 402 along the alpha 1(III) chain. In order to elucidate its amino acid sequence, alpha 1(III)CB1,8,10,2 was fragmented with hydroxylamine, protease from Staphylococcus aureus V8 and trypsin. Peptides necessary for sequence analysis with the automated Edman degradation were separated using molecular and ion exchange chromatography. Edman degradation of the hydroxylamine-derived fragments resulted in the elucidation of 80% of the entire sequence. The rest was completely established by sequence analysis of some protease V8 and trypsin-derived peptides.     

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.     

Human spleen histone H1. Isolation and amino acid sequence of a main variant, H1b

The complete amino acid sequence of a main variant, H1b, of human spleen histone H1 was determined, following previous determinations of human spleen histones H2B, H2A, H3, and H4. High-performance liquid chromatography on C8 silica of the H1 fraction yielded the homogeneous H1b subfraction; this variant was estimated to account for 60% of the total of the four H1 variants. The sequence determination was performed with four main fragments, I to IV, obtained by limited chymotryptic digestion of H1b. Together with direct sequencing by automated Edman degradation of fragments II, III, and IV, fragment I, blocked at the N-terminal, and fragment IV, the C-terminal half the H1b molecule, were sequenced after further digestion with staphylococcal protease and others. The four fragments were aligned with three overlapping peptides each derived from chymotryptic partial fragments, I-II and I-II-III, and intact H1b. Carboxypeptidase digestion of intact H1b confirmed the C-terminal sequence of the molecule. Thus, the total sequence of H1b was completely determined; it consists of a total of 218 amino acid residues, has a molecular weight of 21,734 in the unmodified form, and is completely acetylated at the N-terminal serine residue and partially methylated at the lysine residue 25. This sequence is compared with two mammalian somatic H1 sequences.     

Primary structure of human lymphotoxin derived from 1788 lymphoblastoid cell line

The amino acid sequence of human lymphotoxin derived from a 1788 lymphoblastoid cell line was determined. Peptide fragments obtained by trypsin, lysine-C peptidase, cyanogen bromide, and acetic acid cleavage of the intact protein were purified by reverse-phase high performance liquid chromatography and analyzed by amino acid composition and by automated Edman degradation. The protein is 171 amino acids long with a molecular weight of 18,664. It contains one asparagine-linked glycosylation site and lacks cysteine. The salient features of the amino acid sequence of lymphotoxin are described.     

Isolation, characterization and N-terminal sequences of the CNBr-cleavage peptides from human complement Factor B. Localization of a free thiol group and a sequence defining the site cleaved by factor D.

Nine CNBr-cleavage peptides from Factor B (a component of the alternative pathway of complement) were isolated. Each was characterized by amino acid analysis and automated Edman degradation. One peptide contained a methionyl bond resistant to cleavage by CNBr. The number of CNBr-cleavage peptides is in agreement with the results of amino acid analysis of Factor B and the fragments Ba and Bb. A total of 358 unique residues were identified from the N-terminal sequences of the CNBr-cleavage peptides. These represent approx. 50% and 60% of the total residues of Factor B and fragment Bb respectively. Alignment of two CNBr-cleavage peptides (CB-VIc and CB-IV) provided a continuous segment of 140 residues. This sequence contained the site cleaved by Factor D to generate the Ba and Bb fragments during the activation of complement. Peptide CB-IV contained a free thiol group at a position corresponding to residue 33 of fragment Bb. Amino sugar analyses of Factor B and of fragments Bb and Ba indicated that all the carbohydrate structures of factor B are N-linked to asparagine through N-acetylglucosamine. The two carbohydrate-attachment sites of the Bb fragment were identified.     

Structural studies on equine glycoprotein hormones. Amino acid sequence of equine chorionic gonadotropin beta-subunit

The complete amino acid sequence of the beta-subunit of equine chorionic gonadotropin (eCG beta) has been established by both automated Edman and manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradations. Specific fragments were produced by cleavage with Staphylococcus aureus V8 protease, trypsin, or dilute HCl. For the sequence analyses of the heavily glycosylated COOH-terminal portion, a chemical deglycosylation procedure with trifluoromethanesulfonic acid was employed. The peptide chain of eCG beta consists of 149 amino acid residues. Five or more oligosaccharide chains are attached to the protein, 1 unit linked by an N-glycosidic bond to asparagine at residue 13 and four or more units linked by O-glycosidic bonds to serine or threonine at residues in the COOH-terminal portion. The carbohydrate-bearing hydroxy amino acids have not yet been rigorously established. As compared to the beta-subunits of the pituitary gonadotropin hormones, lutropin, follitropin, and thyrotropin, eCG beta possesses a glycosylated COOH-terminal extension of about 30 amino acid residues, as does the human chorionic gonadotropin beta-subunit (hCG beta). When the comparison is restricted inside the disulfide bond-containing core (residues 1-110), the beta-subunit of eCG is highly homologous to hCG beta (66%). On the other hand, although the overall structural features closely resemble each other, much less homology exists in the COOH-terminal extensions of eCG beta and hCG beta.