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K T Yasunobu

Publications and source records attributed to K T Yasunobu.

At least 19 recordsLinked to original sources
PubMed1985

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.

Amino Acid Sequence↗
PubMed1985

Human uropepsinogen is a new human pepsinogen proisozyme.

The first 20 amino acid residues of human uropepsinogen has been determined to be H2N-Ile-Met-Tyr-Lys-Val-Pro-Leu-Pro-Ile-Lys-Lys-Ile-Leu-Val Val-Pro-Leu-Ile-Val-Tyr-Met. Comparison with the sequence of human stomach pepsinogen shows that these proteins are isozymes. Comparative sequence data are presented and discussed.

Amino Acid Sequence↗
PubMed1983

ESR analysis of the FAD in bovine liver monoamine oxidase.

Two hydrazine spin labels, 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carbonyl ethyl hydrazine and 1-oxyl-2,2,6,6-tetramethylpiperidino-4-hydrazine, were synthesized as probes of the FAD binding site of monoamine oxidase. The reporter nitroxide moiety showed an ESR spectrum classified as partially immobilized which is indicative of FAD near the surface of the enzyme. Attempts to pick up flavin semiquinone or free radical intermediates during substrate oxidation with the spin traps 5,5-dimethyl-1-pyrroline-1-oxidase and phenyl-t-butylnitrone were not successful.

Animals↗
PubMed1983

Heme binding and substrate-protected cysteine residues in P-450cam.

Reactions of the Pseudomonas putida cytochrome P-450-substrate complex or enzyme alone with 14C-labeled iodoacetic acid have been investigated at pH 7.0. After subsequent conversion of all of the cysteine residues to S-beta-carboxymethylcysteinyl residues, tryptic peptides of the derivative were separated by either high performance liquid chromatography or two dimensional electrophoresis, and their amino acid compositions and partial sequences were determined. All but cysteine residue-134 reacted to some extent. This result implicated residue-134 as the thiol group which is the axial heme ligand since the heme was intact in all of the derivatives made. Reaction of the enzyme-substrate complex with "cold" iodoacetic acid followed by substrate removal and reaction with 14C-labeled iodoacetic acid resulted in radiolabeling of mainly cysteine-240. This suggested cysteine-240 to be an active site cysteine residue.

Binding Sites↗
PubMed1983

Structure of oxidized flavodoxin from Anacystis nidulans.

The structure of oxidized flavodoxin from the cyanobacterium Anacystis nidulans has been determined at 2.5 A resolution with phases calculated from ethylmercury phosphate and dimercuriacetate derivatives. The determination of partial sequences, including a total of 85 residues, has assisted in the interpretation of the electron density. Preliminary refinement of a partial model (1072 atoms) has reduced R to 0.349 for the 10.997 reflections between 2.0 and 5.0 A with 1 greater than 2 sigma. The polypeptide backbone, which comprises 167 residues in the current model, adopts the familiar beta-alpha-beta conformation found in other flavodoxins and in the nucleotide-binding domains of the pyridine-nucleotide dehydrogenases, with five parallel strands in the central sheet. Comparison with flavodoxin from Clostridium MP (138 residues) shows that extra residues of A. nidulans flavodoxin are accommodated in a major insertion about 20 residues in length, which forms a lobe adjacent to the fifth strand of parallel sheet, and in additions to several external segments. Residues added between the fourth sheet strand and the start of the third helix alter the environment of the pyrimidine end of the flavin mononucleotide ring. The flavin mononucleotide phosphate binds to the start of helix 1, interacting with hydroxyamino acids and with main-chain amide groups. Two hydrophobic residues, both tentatively identified as Trp, enclose the isoalloxazine ring; the solvent-exposed Trp is nearly parallel to the flavin ring. The hydrophobic environment provided by these residues must be partly responsible for the pronounced vibrational resolution of the flavin spectrum near 450 nm. The flavin ring is tilted relative to its orientation in Clostridium MP flavodoxin. In addition, atoms N-3 and O-2 alpha of the isoalloxazine appear to form hydrogen bonds to the backbone at CO97 and NH99 in a conformation entirely different from that found in Clostridium MP flavodoxin but structurally analogous to Desulfovibrio vulgaris flavodoxin.

Amino Acid Sequence↗
PubMed1983

Amino acid sequence of bovine heart cytochrome oxidase subunit IV (Albany).

The preliminary data on the amino acid sequence of subunit IV from bovine heart cytochrome oxidase (Albany) is presented. The subunit consists of 97 amino acids linked together in a single polypeptide chain. The sequence was established by the isolation, purification and sequencing of some of the tryptic, chymotryptic and thermolytic and Staphylococcus aureus protease peptides. This subunit is present in all cytochrome oxidase preparations. It corresponds to polypeptide VIa in cytochrome oxidase (Aachen) and subunit a in cytochrome oxidase (Eugene).

Amino Acid Sequence↗
PubMed1982

Amino acid sequence of the Pseudomonas putida cytochrome P-450. I. Sequences of tryptic and clostripain peptides.

In order to elucidate the complete amino acid sequence of Pseudomonas putida cytochrome P-450, tryptic digestion was performed on the S-carboxymethylated enzyme. Although cleavage did not occur at every lysyl and arginyl bond, 31 tryptic peptides ranging in size from 1 to 55 residues were isolated. These were sequenced by manual Edman degradation and carboxypeptidase digestion. Overlaps of some od these tryptic peptides were obtained by data obtained from partial Edman degradation and amino acid composition of the clostripain cleavage products. These results, together with data from the cyanogen bromide and acid cleavage peptides reported in the accompanying paper, established the complete amino acid sequence of P. putida cytochrome P-450.

Amino Acid Sequence↗
PubMed1982

Bovine liver mitochondrial monoamine oxidase is not an iron-dependent enzyme.

It has been reported that rat liver monoamine oxidase is an iron-dependent enzyme. Calculations from reported data suggest that rat liver monoamine oxidase contains 2 g atoms of iron/mol of enzyme. However, our data over a 16-year period show that our purified bovine liver monoamine oxidase does not contain sufficient iron to justify being considered an iron protein. In order to ensure the correctness of the iron content, two microchemical analyses and atomic absorption spectroscopy were used to determine the iron content of wet ashed samples. The iron content was also determined during successive steps of enzyme purification and was found to decrease rather than increase. Monoamine oxidase, being a hydrophobic outer membrane enzyme, is very difficult to purify and detailed tests for homogeneity are necessary. Our preparations show a single band when examined by sodium dodecyl sulfate-polyacrylamide disc electrophoresis and behave as a pure protein by end group analysis. Thus, bovine liver monoamine oxidase is not an iron-dependent enzyme.

Animals↗
PubMed1981

The complete amino acid sequence of bovine heart cytochrome oxidase subunit VI.

The amino acid sequence of subunit VI derived from bovine heart cytochrome oxidase has been completed and is a single polypeptide chain consisting of 85 amino acid residues. The sequence is: acetyl (formula: see text). The NH2 terminus of subunit VI is blocked with an acetyl group and the molecular weight of the native protein including the acetyl group is 10,067. From the sequence of subunit VI, it is obvious that this subunit corresponds to polypeptide VII of Steffens et al. (Steffens, G. C. M., Steffens, G. J., and Buse, G. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1641-1650).

Amino Acid Sequence↗
PubMed1980

Electron spin resonance studies of bovine plasma amine oxidase. Probing of the environment about the substrate-liberated sulfhydryl groups in the active site.

A series of nitroxide spin-labeled reagents have been employed to explore the environment of the cysteine residues in bovine plasma amine oxidase. When the enzyme was reduced by substrate or phenylhydrazine, 1 essential sulfhydryl residue/subunit was liberated. This cysteine residue was reacted then with the spin label 3-(maleimido-methyl)-2,2,5,5-tetramethyl-1-pyrrolinyloxyl. The ESR spectra of the labeled enzyme derivatives suggested that this essential sulfhydryl residue is located in a pocket, whereas the nonessential sulfhydryl residues are probably located near the surface. By varying the length of the nitroxide spin-labeled N-ethylmaleimide derivatives, it was determined that the liberated essential cysteine residues are in a restricted environment. The ESR spectral data also suggested that the nitroxide radical and the essential copper in the enzyme do not interact with one another. The effect of ionic strength, pH, and urea denaturation on the environment of the essential sulfhydryl residue were also investigated.

Amine Oxidase (Copper-Containing)↗
PubMed1980

Bovine plasma amine oxidase interactions with concanavalin A in solution and with concanavalin A-Sepharose.

The reaction of bovine plasma amine oxidase, a glycoprotein, with Concanavalin A in 0.1 M potassium phosphate buffer, pH 7.0 at 25 degrees C were investigated by equilibrium and kinetic methods. A tentative mechanism for the reaction was derived. The Concanavalin A-enzyme interaction was used to show that the carbohydrate is not essential for activity and that the carbohydrate is covalently attached to the protein at a site distant from the active site. Concanavalin A-Sepharose 4B affinity chromatography of the enzyme was found to be useful for obtaining the pure enzyme. Chromatographic conditions which elute the enzyme are reported. The superiority of the Concanavalin A-Sepharose 4B over that of the substrate-affinity Sepharose affinity support is discussed. The phenylhydrazone derivative of the enzyme, which was inactive, was shown to bind to the concanavalin A-Sepharose column.

Animals↗
PubMed1979

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.

Amino Acid Sequence↗
PubMed1979

Amino acid sequence of subunit V of bovine heart cytochrome oxidase, the heme alpha-containing subunit.

The complete amino acid sequence of the heme alpha-containing subunit V of bovine heart cytochrome oxidase was determined to be: H2N-Ser-His-Gly-Ser-His-Glu-Thr-Asp-Glu-Glu-Phe-Asp-Ala-Arg-Trp-Val-Thr-Tyr-Phe-Asn-Lys-Pro-Asp-Ile-Asp-Ala-Trp-Glu-Leu-Arg-Lys-Gly-Met-Asn-Thr-Leu-Val-Gly-Tyr-Asp-Leu-Val-Pro-Glu-Pro-Lys-Ile-Ile-Asp-Ala-Ala-Leu-Arg-Ala-Cys-Arg-Arg-Leu-Asn-Asp-Phe-Ala-Ser-Ala-Val-Arg-Ile-Leu-Glu-Val-Val-Lys-Asp-Lys-Ala-Gly-Pro-His-Lys-Glu-Ile-Tyr-Pro-Tyr-Val-Ile-Gln-Glu-Leu-Arg-Pro-Thr-Leu-Asn-Glu-Leu-Gly-Ile-Ser-Thr-Pro-Glu-Glu-Leu-Gly-Leu-Asp-Lys-Val-COOH. The subunit V is a single polypeptide which consists of 109 amino acid residues. The protein contains 48.6% hydrophobic residues and 34.0% hydrophilic residues and it is an acidic protein having a net charge of -3 at neutral pH. The molecular weight of subunit V was calculated to be 12,436 and that for the heme alpha-containing polypeptide was 13,295.

Amino Acid Sequence↗