Amino acid sequence of the Anthopleura xanthogrammica heart stimulant, anthopleurin-B

Anthopleurin-B, the most potent peptide heart stimulant from the sea anemone Anthopleura xanthogrammica, was shown to exist as a single polypeptide chain consisting of 49 amino acid residues. The sequence of the peptide was shown to be: Gly-Val-Pro-Cys-Leu-Cys-Asp-Ser-Asp-Gly- Pro-Arg-Pro-Arg-Gly-Asn-Thr-Leu-Ser-Gly-Ile-Leu-Trp-Phe-Tyr-Pro-Ser- Gly-Cys-Pro-Ser-Gly-Trp-His-Asn-Cys-Lys-Ala-His-Gly-Pro-Asn-Ile-Gly- Trp-Cys-Cys-Lys-Lys. The carboxymethylcysteine derivative, tryptic and chymotryptic peptides (obtained from the derivative and separated by high performance liquid chromatography) were sequenced by manual Edman degradation. Although six carboxymethylcysteine residues were formed by reduction and alkylation of the polypeptide, no cysteine residues were detectable in the native protein, indicating that there are three cystine residues in anthopleurin-B. The amino acid sequence differs in 7 places from anthopleurin-A: at residues 3 (Pro for Ser), 12 (Arg for Ser), 13 (Pro for Val), 21 (Ile for Thr), 24 (Phe for Leu), 42 (Asn for Thr), and 49 (Lys for Gln). These differences are important since anthopleurin-B is about a 12.5-fold better heart stimulant than anthopleurin-A from A. xanthogrammica, anthopleurin-C from Anthopleura elegantissima, and toxin II from Anemonia sulcata.     

Locust collagen: morphological and biochemical characterization

Natural-abundance 13C NMR spectra (at 15.04 MHz) of the polypeptide toxin II from the sea anemone Anemonia sulcata have been analysed and compared with corresponding spectra reported recently for a closely related polypeptide anthopleurin A. The spectra contain many resolved one-carbon and two-carbon resonances from carbonyl, aromatic and methyl carbons, many of which have been assigned to individual carbons in the molecule on the basis of their chemical shifts, including their pH dependence, and by comparison with the 13C NMR spectrum of anthopleurin A. Analysis of the effects of pH on the spectrum yields estimates for the pKa values of a number of functional groups in the molecule, as follows: side-chain carboxylates of the two aspartic acid residues 2 and 3.1; COOH-terminal carboxylic acid, 3.5; imidazolium moieties of the two histidine residues, 6.7 and 7.6 NH2-terminal ammonium, 8. The similarity between the pKa values of these functional groups in toxin II and those of corresponding groups in anthopleurin A, together with the close agreement between chemical shifts of conserved carbons, indicates that many local interactions are nearly identical in the two molecules, and thus supports the thesis that their overall conformations in solution are similar. However, the local interactions involving one of the aspartic acid residues are altered in toxin II. Together with other data, this leads to a proposal for the site in these two molecules which is responsible for their cardiac stimulatory activity.     

An amino acid sequence in the active site of lipoamide dehydrogenase from pig heart

1. The two cysteine residues forming the disulphide bridge that comprises part of the active site of lipoamide dehydrogenase from pig heart were specifically labelled with iodo[2-(14)C]acetic acid. 2. A tryptic peptide containing these carboxymethylcysteine residues was isolated from digests of reduced and S-carboxymethylated lipoamide dehydrogenase and its amino acid sequence of 23 residues was determined. 3. The sequence is highly homologous with a similar sequence containing the active-site disulphide bridge of lipoamide dehydrogenase derived from the 2-oxoglutarate dehydrogenase complex of Escherichia coli (Crookes strain) and it is probable that, as in the bacterial enzyme, the disulphide bridge forms an intrachain loop containing six residues. The results indicate that the bacterial and mammalian proteins have a common genetic origin. 4. Amino acid sequences containing six other unique carboxymethylcysteine residues were also partly determined. 5. The analysis of the primary structure thus far is consistent with the view that the enzyme (mol.wt. approx. 110000) is composed of two identical polypeptide chains.     

Amino-acid sequence of toxin I from Anemonia sulcata.

Toxin I from Anemonia sulcata, a major component of the sea anemone venom, consists of 46 amino acid residues which are linked by three disulfide bridges. The [14C]carboxymethylated polypeptide was sequenced to position 29 by automated Edman degradation. The remaining sequence was determined from cyanogen bromide peptides and from tryptic peptides of the citraconylated [14C]carboxymethylated toxin. Toxin I is homologous to toxin II from Anemonia sulcata and to anthopleurin A, a toxin from the sea anemone Anthopleura xanthogrammica. These toxins constitute a new class of polypeptide toxins. No significant homologies exist with toxin III from Anemonia sulcata nor with known sequences of neurotoxins or cardiotoxins of various origin.     

Molecular organization of the sex steroid-binding protein (SBP) of human plasma

Several years ago this laboratory presented evidence that SBP is a dimer composed of two subunits having similar molecular weights. The question of whether or not these subunits are identical and therefore products of a single gene remained unanswered. We now report that the two polypeptide chains are identical and that SBP is a homodimer. The experimental approach was to reduce and [14C]alkylate cystine residues in human SBP, digest the product with trypsin or cyanogen bromide and determine the number of unique amino acid sequences around each [14C]carboxymethylcysteine residue. Only four unique sequences were found when all the radioactive peptides were analyzed. Since there are eight half-cystine residues per dimer, the results support a homodimeric structure.     

Cloning and expression of wild-type and mutant forms of the cardiotonic polypeptide anthopleurin B.

Venom of the sea anemone Anthopleura xanthogrammica contains a minimum of three polypeptide toxins capable of prolonging the repolarization phase of the action potential. A synthetic gene for the most toxic of the Anthopleura toxins, anthopleurin B (ApB), has been designed, synthesized, and expressed as a fusion protein with the gene 9 product of bacteriophage T7 in Escherichia coli. The fusion protein has been purified and its disulfide bonds reoxidized using glutathione redox couples. Upon cleavage with staphylococcal protease, this protocol yields approximately 1 mg of native ApB/liter of original culture. The recombinant protein has been shown to be identical to natural ApB with respect to amino acid composition, amino-terminal sequence, secondary structure, high pressure liquid chromatographic mobility, and biological activity. A second form of ApB containing additional residues of glycine and arginine at its amino terminus has also been characterized. This protein, termed GR-ApB, is identical in specific activity to the wild-type form. This work lays the groundwork for a detailed analysis of ApB structure and action by site-directed mutagenesis.     

Effect of poly(ADP-ribose) formation on DNA synthesis in chick-embryo-liver nuclei. Possible mechanism of template activation.

The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions.     

Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence of protein SCMBK-IIIB3

The complete amino acid sequence of wool protein SCMKB-IIIB3 was determined. The peptides used for the sequence work were obtained by peptic and thermolysin digestions and were fractionated by chromatography on DEAE-cellulose, paper chromatography and electrophoresis. The peptides were analysed by dansyl-Edman degradation, mass spectrometry and tritium-labelling of C-terminal residues. The protein consists of 98 residues and has acetylalanine as N-terminal residue and carboxymethylcysteine as C-terminus. It is homologous with protein SCMKB-IIIB2 (Haylett & Swart, 1969). A salient feature of the sequence of protein SCMKB-IIIB3 is three consecutive cysteine residues.     

Haloacetyl groups as reversible protection of the amino function: cleavage with 2-aminothiophenol.

Haloacetylamino acids and haloacetyl peptides react rapidly with 2-aminothiophenol in weakly alkaline media to yield 2-aminothiophenoxyacetyl derivatives. These intermediates are subject to acidolysis under mild conditions with release of free amino acids or peptides. With this mild method for removal of the haloacetyl group N-haloacetoxysuccinimide derivatives, which rapidly and specifically acylate amino groups of polypeptides in aqueous solutions, become promising reagents for the reversible protection of amino groups. The chloroacetylation of amino groups in lima bean trypsin inhibitor and the quantitative removal of the chloroacetyl groups demonstrate the applicability of the method for polypeptides. The haloacetyl group also serves an analytical function in that treatment of a completely or partially haloacetylated polypeptide with cysteine forms one carboxymethylcysteine residue per haloacetyl group in the polypeptide derivative. Carboxymethylcysteine is readily measured by amino acid analysis of acid hydrolysates. Approaches to further improvement of conditions for removal of haloacetyl groups are discussed and potential applications of the general chemistry of 2-haloacids to modern polypeptide chemistry are outlined.     

C-Terminal Sequence Determination of Modified Peptides by MALDI MS

Peptides, cleaved by a mixture of carboxypeptidases CPP and CPY, can be detected by MALDI MS and the amino acid sequence thereby determined by calculation of the differences between consecutive peaks. In the present study we have used derivatizations of Lys and Cys to facilitate identification of these residues. Since the mass values do not readily distinguish Lys from Gln, we have converted Lys to homoarginine by guanidination, allowing simple detection of Lys. To identify the Cys positions in peptides that contain cystine, cysteic acid, or carboxymethylcysteine is not possible using CPY and CPP because of the lack of proteolytic cleavage. Instead we find that identification of Cys residues within the sequence can be achieved after conversion to a basic derivative, 4-thialaminine (Thi), by trimethylaminoethylation.     

Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence of protein SCMKB-IIIA3

The complete amino acid sequences of wool protein SCMKB-IIIA3 (131 residues) and a minor component SCMKB-IIIA3A (130 residues) have been determined. The proteins are mutually homologous and have free threonine as the N-terminal residue and carboxymethylcysteine as the C-terminus. The peptides used for the sequence work were obtained by trypsin, thermolysin, pepsin and chymotrypsin digestions and were fractionated by chromatography on DEAE-cellulose, gel filtration on Sephadex G-25 and G-50, paper chromatography and electrophoresis. The Edman degradation method (employing both the Beckman Sequencer and a non-automatic procedure) was used to obtain the sequences of the peptides.     

海葵神经毒素基因的克隆和序列分析

根据已发现的海葵神经毒素蛋白的两端保守氨基酸序列 ,设计简并引物 .用RT PCR方法 ,从侧花海葵 (Anthopleurasp .)触手总RNA中分离出多个神经毒素新基因 .它们分别编码 3个长度都是 4 7个氨基酸的毒素蛋白 ,它们的氨基酸序列与海葵神经毒素C(Ap C)最为相似 .新基因的发现为进一步研究其生物活性及应用打下了基础 .     

A new neuraminic acid derivative and three types of glycopeptides isolated from the Cuvierian tubules of the sea cucumber Holothuria forskali

The Cuvierian tubules of Holothuria forskali Della Chiaje, a sea cucumber found in the Adriatic Sea, were investigated with regard to their carbohydrate moieties. From a Pronase digest of these tubules three types of carbohydrate units were isolated and characterized. 1. A high-molecular-weight glycopeptide fraction was shown to contain sulphated polyfucose, galactosamine, a uronic acid and a previously unknown neuraminic acid derivative. The sulphate was shown by i.r. analysis to be present as an O-ester. The carbohydrate unit was linked O-glycosidically to threonine and serine residues in the polypeptide chain. The hitherto unknown neuraminic acid derivative (Hf-neuraminic acid) was resistant to enzymic cleavage by neuraminidase, even after mild alkaline hydrolysis for the removal of O-acyl residues. However, the glycosidic linkage of this compound to the other part of the carbohydrate moiety was readily cleaved by mild acid hydrolysis. Its chromatographic properties distinguished Hf-neuraminic acid from other known neuraminic acid derivatives (N-acetyl-, NO-diacetyl-, NOO-triacetyl- and N-glycollyl-neuraminic acid). Further, this acidic sugar was shown to possess neuraminic acid as its basic structure. Thus, an as yet unknown substituent lends the distinct properties to Hf-neuraminic acid. 2. The carbohydrate composition of a second glycopeptide fraction consisting of a derivative of neuraminic acid, galactose, mannose and glucosamine was similar to that of the well-known carbohydrate groups of the globular glycoproteins. 3. The third fraction contained two glycopeptides containing the disaccharide, glucosylgalactose, which was shown to be linked to the hydroxyl group of hydroxylysine residues of a collagen-like protein. Approximately half of these residues were glycosylated. In addition to these glycopeptides, a small amount of a third glycopeptide that carried only a galactosyl residue was detected. The amino acid sequence of the two major compounds were found to be Gly-Ala-Hyl*-Gly-Ser and Gly-Pro-Hyl*-Gly-Asp, where Hyl* represents a glycosylated amino acid residue.     

Conformational studies of peptide heart stimulant anthopleurin A. Laser Raman, circular dichroism, fluorescence spectral studies, and Chou-Fasman calculations.

Sea anemone contain a number of closely related peptide heart stimulants. In the present investigation, the conformation of anthropleurin A from Anthopleura xanthogrammica was investigated by laser Raman, circular dichroism, and fluorescence spectral methods and by the Chou-Fasman method using sequence data. The recent 13C NMR data of the peptide (Norton, R.S., and Norton, T.R. (1979) J. Biol. Chem., in press) provided useful information for the interpretation of the above-mentioned spectral data. The results from these spectral methods suggested that anthropleurin A and the related sea anemone peptides are roughly spherical in shape due to the presence of some beta-bends, possibly due to a beta-pleated sheet region and due to the 3 cystine residues in the peptide which exist in the gauche-gauche-gauche configuration. The sole tyrosine residue is exposed to the solvent, a finding which has now been confirmed by 13C NMR. The laser Raman and fluorescence spectral procedures showed that one or more of the tryptophan residues are buried. Interestingly, the reduction of the native protein with dithioerythritol did not change the spherical shape even in the presence of 5 M guanidine HCl and the carboxymethylcysteine derivative of the peptide was folded even in the presence of the denaturing agent, guanidine HCl.     

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.     

Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence of protein SCMKB-IIIB4

The complete amino acid sequence of protein SCMKB-IIIB4 is presented. It is closely related to the sequence of protein SCMKB-IIIB3 (Haylett, Swart & Parris, 1971) differing in only four positions. The peptic and thermolysin peptides of protein SCMKB-IIIB4 were analysed by the dansyl-Edman method (Gray, 1967) and by tritium-labelling of C-terminal residues (Matsuo, Fujimoto & Tatsuno, 1966). This protein is the third member of a group of high-sulphur wool proteins with molecular weight of about 11400. It consists of 98 residues and has acetylalanine and carboxymethylcysteine as N- and C-terminal residues respectively.     

Drug residue formation from ronidazole, a 5-nitroimidazole. VIII. Identification of the 2-methylene position as a site of protein alkylation.

Ronidazole protein-bound adducts were generated by the in vitro anaerobic incubation of [2-methylene-14C]ronidazole with microsomes from the livers of male rats. Acid hydrolysis of the protein adducts yielded an imidazole ring fragment bearing the radiolabel and an amino acid residue derived from the proteins. This fragment has been identified as carboxymethylcysteine by co-chromatography of the amino acid and its dansyl derivative with known standards under a variety of conditions. The carboxymethylcysteine was estimated to represent at least 15% of the radioactivity derived from the protein-bound adducts and provides unequivocal evidence that nucleophilic attack by protein cysteine thiols occurred at the 2-methylene position of ronidazole.     

Amino acid sequences around the cysteine residue of calf lens α-crystallin

1. Calf lens alpha-crystallin was carboxymethylated with radioactive sodium iodoacetate to label the thiol group. 2. The protein was then digested with trypsin or alternatively fractionated in urea to obtain the acidic (A) chains, which were then digested with trypsin. Either procedure gave two radioactive peptides containing carboxymethylcysteine. 3. These two peptides were closely related: the longer form contained 28 amino acid residues, and the shorter lacked two residues at the N-terminal end of the longer form. 4. The amino acid sequence of the peptides have been determined. 5. No evidence for the presence of more than one cysteine residue/chain was found. 6. The question of the molecular weight of the chains is discussed.     

Purification and characterization of human and bovine platelet factor 4.

Platelet antiheparin, platelet factor 4, was isolated from freeze-thaw lysates of fresh bovine and outdated human platelet concentrates by a single step affinity chromatographic procedure. The yields of PF4 were 93 microgram and 142 microgram/ml of human and bovine platelets respectively. Antiheparin activity of the products were 558 units/mg for the bovine isolate and 489 units/mg for the human material. The bovine product is a single chain polypeptide with an apparent molecular weight of 12,300. Amino acid composition indicates 107-109 residues compared to the smaller human product which has an apparent molecular weight of 8,000 for a 70 residue polypeptide. The intact polypeptide was resistant to enzymatic hydrolysis as opposed to the reduced-alkylated derivative which was susceptible to hydrolysis in the presence and absence of heparin.     

SsoII-like DNA-methyltransferase Ecl18kI: Interaction between regulatory and methylating functions

The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Δ(72–379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyl-transferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.