Biomedical subjects

F Sakiyama

Publications and source records attributed to F Sakiyama.

At least 19 recordsLinked to original sources

PubMed1992

A simple, rapid method for purification of epsilon-subunit, coupling factor 6, subunit d, and subunit e from rat liver H(+)-ATP synthase and determination of the complete amino acid sequence of epsilon-subunit.

The rat liver mitochondrial epsilon-subunit, coupling factor 6, subunit d, and subunit e of H(+)-ATP synthase, which are all extra subunits with no counterparts in Escherichia coli, were purified by reverse-phase high performance liquid chromatography. The complete amino acid sequence of the rat epsilon-subunit was determined by automated Edman degradation of the whole protein and derived peptides. The protein contains 50 amino acids and has a molecular mass of 5635 kDa. It is a basic hydrophilic protein with an isoelectric point of 10.5. The sequence of the rat epsilon-subunit is highly homologous with that of the epsilon-subunit of bovine heart and slightly similar to those of the epsilon-subunit of the yeast and sweet potato mitochondria. However, it has no homology with any subunit of bacterial or chloroplast H(+)-ATP synthase.

Amino Acid Sequence↗

PubMed1992

Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins.

Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV-40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import.

Amino Acid Sequence↗

PubMed1992

Deblocking and subsequent microsequence analysis of N alpha-blocked proteins electroblotted onto PVDF membrane.

A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.

Acetylation↗

PubMed1992

The primary structure of porcine aminoacylase 1 deduced from cDNA sequence.

A cDNA encoding the complete amino acid sequence of aminoacylase 1 (N-acylamino acid aminohydrolase, ACY-1) [EC 3.5.1.14], a dimeric metalloprotein having two Zn2+ in the molecule, which catalyzes the deacylation of N-acylated L-amino acids except L-aspartic acid, has been isolated from porcine kidney lambda gt10 cDNA library and sequenced. From sequence analysis of the cDNA and the N- and C-terminal amino acid analyses of the purified protein, it is deduced that porcine kidney ACY-1 consists of two identical subunits (M(r) 45,260), each of which consists of a single chain of 406 amino acids with acetylalanine at the N-terminus. A cDNA encoding porcine liver ACY-1 was also cloned. The amino acid sequence deduced from the nucleotide sequence of the cDNA from porcine liver was identical to that deduced for porcine kidney ACY-1. Northern blot analysis suggested that ACY-1 is more highly expressed in kidney than in liver. Comparison of the amino acid sequence of porcine ACY-1 with those of other Zn2+-binding metalloenzymes showed no significant homologies in either the overall sequence or the consensus sequences for the metal binding sites. This indicates that ACY-1 is a new type of metalloprotein.

Amidohydrolases↗

PubMed1992

Characterization of saccharide moiety in the electroplax sodium channel.

Carbohydrate chains on the large peptide of the voltage-sensitive sodium channel from Electrophorus electricus electroplax have been partially characterized by the lectin-blotting technique combined with digestion using three glucosidases: neuraminidase, endo-beta-N-acetylglucosaminidase H, and peptide: N-glycosidase F. The results show that both N-linked oligosaccharides and O-linked (mucin-type) oligosaccharides are present. In N-linked oligosaccharides, the results suggest the presence of complex- and hybrid-type oligosaccharides which contain bisecting N-acetylglucosamine(s), as well as the complex-type oligosaccharides with the alpha-Fuc-GlcNAc-(Asn) residue(s). In O-linked oligosaccharides, they must carry Gal beta1----3GalNAc- moieties which contain NeuNAc residues in the terminal.

Animals↗

PubMed1992

Inhibition of Achromobacter protease I by lysinal derivatives.

Z-Val-, Z-Pro-, Z-Leu-Leu-, and Z-Leu-Pro-lysinals and BZ-DL-lysinal were chemically synthesized and tested as novel inhibitors for Achromobacter protease I (API), a lysine-specific serine protease. Among the lysinal derivatives tested, Z-Val-lysinal was the most potent competitive inhibitor, its Ki being estimated as 6.5 nM in an esterolytic assay with Tos-Lys-OMe. In an amidolytic assay, Z-Leu-Leu-lysinal was the most potent inhibitor and the apparent mode of inhibition was non-competitive. The Kis of the other lysinal derivatives in both esterolytic and amidolytic assays were more than 10(3) times lower than that of leupeptin. Z-Val-lysinol, lacking the aldehyde group, was a poor competitive inhibitor. These results suggest that acyl-, acylaminoacyl-, and acylpeptidyllysinals function as a transition-state inhibitor for Achromobacter protease I.

Alcaligenes↗

PubMed1991

S-farnesylation and methyl esterification of C-terminal domain of yeast RAS2 protein prior to fatty acid acylation.

Posttranslational processing/modification is required for membrane localization and activation of ras proteins. In the case of yeast RAS2 protein, we have reported that the process starts with the removal of the initiator methionine followed by polyisoprenylation, removal of 3 amino acid residues from the C terminus, methyl esterification, and fatty acid acylation (Fujiyama, A., and Tamanoi, F. (1990) J. Biol. Chem. 265, 3362-3368). In this study, we demonstrate that polyisoprenylation and methyl esterification of the cysteine residue in the C-terminal domain of the RAS2 protein are involved in the conversion process from precursor form to intermediate form. The polyisoprenoid moiety attached to the RAS2 protein was identified as a 15-carbon farnesyl group through two independent experiments: the release of S-farnesylcysteine with carboxypeptidase Y from the RAS2 protein, and the recovery of radioactive farnesol through methyliodide treatment of the RAS2 protein purified from yeast cells labeled with [3H]mevalonic acid. The farnesyl group attached to the RAS2 protein was detected predominantly in the C-terminal peptide, SGSGGCC, both in the intermediate and in the fatty acid acylated RAS2 protein. The C-terminal cysteine of the intermediate protein is also modified by methyl esterification in a nearly stoichiometric manner.

Acylation↗

PubMed1991

Primary structure of nuclease P1 from Penicillium citrinum.

The primary structure of nuclease P1, which cleaves both RNA and single-stranded DNA, from Penicillium citrinum was elucidated. The complete amino acid sequence consisting of 270 residues was determined by analysis of peptides obtained by digestion with Achromobacter protease I of the reduced and S-aminoethylated protein and by digestion with Staphylococcus aureus V8 protease of the reduced and S-carboxymethylated protein. Four half-cystine residues were assigned to Cys72-Cys217 and Cys80-Cys85. N-Glycosylated asparagine residues were identified at positions 92, 138, 184 and 197. Fast-atom-bombardment and laser-ionization MS were successfully used to confirm the determined amino acid sequences of peptides and to estimate the molecular mass of this glycoprotein having heterogenous sugar moieties, respectively. Comparison of the amino acid sequence of nuclease P1 with other nucleases revealed that the protein has a high degree of sequence identity (50%) with nuclease S1 from Aspergillus oryzae. The His-Phe-Xaa-Asp-Ala sequence (positions 60-64) is similar to the sequence (His-Phe-Asp-Ala) involving the active-site His119 of bovine pancreatic RNase A, and the Pro-Leu-His sequence (positions 124-126) is identical with the sequence involving the active-site His134 of porcine pancreatic DNase I.

Amino Acid Sequence↗

PubMed1991

Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution.

P1 nuclease from Penicillium citrinum is a zinc dependent glyco-enzyme consisting of 270 amino acid residues which cleaves single-stranded RNA and DNA into 5'-mononucleotides. The X-ray structure of a tetragonal crystal form of the enzyme with two molecules per asymmetric unit has been solved at 3.3 and refined at 2.8 A resolution to a crystallographic R-factor of 21.6%. The current model consists of 269 amino acid residues, three Zn ions and two N-acetyl glucosamines per subunit. The enzyme is folded very similarly to phospholipase C from Bacillus cereus, with 56% of the structure displaying an alpha-helical conformation. The three Zn ions are located at the bottom of a cleft and appear to be rather inaccessible for any phosphate group in double-stranded RNA or DNA substrates. A crystal soaking experiment with a dinucleotide gives clear evidence for two mononucleotide binding sites separated by approximately 20 A. One site shows binding of the phosphate group to one of the zinc ions. At both sites there is a hydrophobic binding pocket for the base, but no direct interaction between the protein and the deoxyribose. A cleavage mechanism is proposed involving nucleophilic attack by a Zn activated water molecule.

Acetylglucosamine↗

PubMed1991

Tryptophan 65 is essential for hemolytic activity of the thermostable direct hemolysin from Vibrio parahaemolyticus.

The effect of modification of aromatic and ionizable amino acid residues on the hemolytic activity of a thermostable direct hemolysin from Vibrio parahaemolyticus was examined. Tryptophan 65, one of the two tryptophan residues per subunit, was specifically modified with N-bromosuccinimide, resulting in complete loss of hemolytic activity. However, neither nitration with tetranitromethane of one of the nine tyrosine residues nor Nlm-ethoxyformylation of two of the four histidine residues caused any change in hemolytic activity. The hemolysin was fully active upon amidation of two reactive carboxyl group. On the other hand, acetylation of amino groups and the modification of one of the three arginine residues with 1,2-cyclohexanedione resulted in a partial loss of the hemolytic activity. The results suggest that Trp65 is essential for the hemolytic activity of V. parahaemolyticus hemolysin.

Acetic Anhydrides↗

PubMed1991

Amino acid sequence of nuclease S1 from Aspergillus oryzae.

The amino acid sequence of nuclease S1, a nuclease which cleaves both single-stranded DNA and RNA, from Aspergillus oryzae was determined. Reduced and S-carboxymethylated or S-aminoethylated nuclease S1 was digested with Achromobacter protease I, Staphylococcus aureus V8 protease, or endoproteinase Asp-N. Peptides thus obtained were purified by reverse-phase high-performance liquid chromatography and sequenced, and the complete primary structure was established. Nuclease S1 consists of a single peptide chain of 267 amino acid residues bearing N-glycosylated Asns 92 and 228. Five half-cystine residues are present at positions 25, 72, 80, 85, and 216, and the latter four residues are implicated in the formation of disulfide bonds by analogy with those in nuclease P1. Two short stretches of sequences involving His 60 and His 125 are shown to be identical with those involving active site His 119 in bovine ribonuclease A and active-site His 134 in porcine deoxyribonuclease I, respectively.

Amino Acid Sequence↗

PubMed1991

Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of [delta 1-13C]Trp 62-lysozyme.

The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed dehydration. [delta 1-13C]Trp 62-lysozyme in which Trp 62 is labeled with 90% 13C has the same chemical and enzymatic properties as the native protein. The reverted lysozyme gave a single 13C-NMR signal at 125 ppm. pH-titration of the 13C signal indicated a transition at pH 3.9 for the free enzyme. In the presence of (GlcNAc)3, the resonance signals were shifted 0.5-1 ppm upfield, and the transitions in the titration curve were observed at pH 3.9 and 6.5. Asp 52 and Glu 35 were assigned to the groups with pKas of 3.9 and 6.5, respectively. In [2-13C]AHT 62-lysozyme, which has 3-(2-amino-3-hydroxy-3H-[2-13C]indol-3-yl)alanine (AHT) at position 62, AHT 62 behaved quite differently from Trp 62 on pH-titration of the 13C-label. These results suggest that a conformational change around Trp 62 is induced upon ionization of the catalytic residue and that the structural flexibility of the side chain of this aromatic residue in the substrate binding site is closely related to the function of lysozyme.

Amino Acids↗

PubMed1991

Crystal structure of Streptomyces erythraeus trypsin at 2.7 A resolution.

The crystal structure of Streptomyces erythraeus trypsin (abbreviated as SET) has been determined in order to clarify the precise structure of the vicinity of the active site of serine protease and to understand its structure-function relationship. Crystals of SET were prepared at its active pH range (pH 5-10) without any inhibitors which might have affected the circumstances around the active sites. The structure model of SET was made based on the electron density map obtained by the multiple isomorphous replacement method at 3.5 A resolution, and refined by the restrained least-squares method. The current model yields a crystallographic R-factor of 0.272 for 4,968 reflections between 8 and 2.7 A resolution. Though the sequence homology among SET, Streptomyces griseus trypsin and bovine trypsin, 32-37%, is not so high, their overall structures are similar to each other. Comparison of the three molecular structures shows that: 1) the folding of the main chains of the three proteins is essentially the same though there are significant differences on the molecular surface; 2) the spatial arrangements of the catalytic triads in the three proteins are similar to each other; 3) in SET and S. griseus trypsin a short stretch of 3(10)-helix is found through Ala56 to Thr59; His57 in this segment is one important amino acid residue involved in the active sites.

Amino Acid Sequence↗

PubMed1990

The role of an invariant tryptophan residue in alpha-bungarotoxin and cobrotoxin. Investigation of active derivatives with the invariant tryptophan replaced by kynurenine.

Ozone oxidation converted the single, invariant, tryptophan residue to N2-formylkynurenine in alpha-bungarotoxin and cobrotoxin. Upon this modification, the lethal toxicity was significantly reduced in cobrotoxin but mostly retained in alpha-bungarotoxin. Each neurotoxin containing kynurenine instead of tryptophan retained the same antigenicity as the native toxin. Fluorescence and CD spectroscopy revealed that, although the environment and state of the kynurenine residue were similar, [Kyn29]cobrotoxin was much more sensitive to pH change than alpha-[Kyn28]bungarotoxin. In terms of lethal toxicity and conformational stability, the invariant tryptophan residue appears to play a more important role in cobrotoxin, imparting a higher lethal toxicity than that in alpha-bungarotoxin, which has a disulfide bond at Cys29-Cys33.

Animals↗

PubMed1990

cDNA cloning and sequencing for the import precursor of coupling factor 6 in H(+)-ATP synthase from rat liver mitochondria.

The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 458 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions of both the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame consisted of 108 and 76 amino acid residues with a molecular weight of 12,494 and 8,927, respectively. The presequence of 32 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.

Adenosine Triphosphatases↗

PubMed1990

Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b.

C5 convertase of the classical complement pathway is a trimolecular protein complex consisting of C4b, C2a, and C3b. In the complex there is an ester bond between C3b and C4b. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. The covalently linked C4b.C3b complex was purified from a detergent extract of the erythrocytes and digested with lysyl endopeptidase. An Mr 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. Two amino acids were obtained at each cycle and identified with those in the sequences of C3 and C4. The sequence derived from C3 corresponded to the thioester region. The sequence derived from C4 started at Ala-1186. Alkali treatment of the fragment yielded an Mr 7,000 peptide derived from C4, which thus appeared to span the region of C4 from Ala-1186 to Lys-1259. Therefore, the covalent C3b-binding site on C4b is located within a 74-residue region of the primary structure. This finding supports the notion that after cleavage of C3 by the C4b2a complex, the covalent binding of metastable C3b to C4b is a specific reaction to form a trimolecular complex with a defined quaternary structure.

Amino Acid Sequence↗

PubMed1990

Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae.

Ribonuclease (RNase) T2 from Aspergillus oryzae was modified by diethyl pyrocarbonate and iodoacetic acid. RNase T2 was rapidly inactivated by diethyl pyrocarbonate above pH 6.0 and by incorporation of a carboxymethyl group. No inactivation occurred in the presence of 3'AMP. 1H-NMR titration and photo-chemically induced dynamic nuclear polarization experiments demonstrated that two histidine residues were involved in the active site of RNase T2. Furthermore, analysis of inactive carboxymethylated RNase T2 showed that both His53 and His115 were partially modified to yield a total of one mole of N tau-carboxymethylhistidine/mole enzyme. The results indicate that the two histidine residues in the active site of RNase T2 are essential for catalysis and that modification of either His53 or His115 inactivates the enzyme.

Amino Acid Sequence↗

PubMed1990

New tools for protein sequence analysis.

Analysis of protein sequence is an important tool in studies of both native and recombinant proteins. Novel techniques and instrumentation which facilitate determination of protein primary structure have recently been developed.

Amino Acid Sequence↗