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Biomedical subjects

H Kihara

Publications and source records attributed to H Kihara.

At least 127 records · Page 7Linked to original sources
PubMed1987

[Hemerythrin].

Explore the source record for details and available documents.

Animals↗
PubMed1987

A standardized format for sequence data exchange.

At present there is no agreement upon a standard format for the presentation of sequence data; each of the major sequence databases has adopted their own format. As a result, efforts to pool these data and to develop software to manipulate the data have been hampered. A significant amount of software development time must be invested to handle the incompatibilities among these formats before software to solve biologically interesting problems can be implemented. In principle, the development of a standard format by the database distributors would be the best solution. However, because the databases have invested years of effort in the development of procedures specifically tailored to their own format, they are reluctant to change. Insisting that they convert to a new format would place an extreme burden on the already overtaxed resources of these groups. Furthermore, for certain specialized applications it is more efficient to present the data in nonstandard formats. An alternative solution is presented here. Rather than develop a single standard format for all sequence data, a standardized exchange format has been developed. This format was designed to serve as a common interface between the major formats currently in use. Data can be easily converted to and from it without significant loss of information. This alleviates difficulties inherent in dealing with multiple formats while preserving the local formats of the various databases.

Amino Acid Sequence↗
PubMed1986

Attenuated activities and structural alterations of arylsulfatase A in tissues from subjects with pseudo arylsulfatase A deficiency.

It had been shown previously that arylsulfatase A activity was attenuated in pseudo arylsulfatase A deficiency fibroblasts and that subunits of the enzyme were smaller than subunits of the enzyme in normal fibroblasts. Attenuated enzyme activity has now been affirmed in other tissues. Subunits of the enzyme from these sources were also found to be smaller with apparent molecular size 59 and 56 kdaltons. Subunits of enzyme in corresponding control tissues were larger and there was heterogeneity in apparent molecular size as follows: fibroblasts, 63 and 59 kdaltons; liver, 63 and 59 kdaltons; kidney, 63 and 58 kdaltons; spleen, 63 and 58 kdaltons; placenta, 62 and 58 kdaltons; and urine, 61 and 57 kdaltons. Attenuated enzyme activity and structurally altered enzyme in pseudo arylsulfatase A deficiency appears to be systemic. However, the reason for reduced amounts of structurally altered enzyme with normal catalytic activity is unresolved.

Cerebroside-Sulfatase↗
PubMed1986

Late-onset Krabbe disease initially diagnosed as cerebroside sulfatase activator deficiency.

Clinical and biochemical findings in a male subject with progressive encephalopathy and peripheral neuropathy are presented. Early development was normal. At age 3.5 years, he had seizures associated with fever. Subsequently, there was progressive neurologic deterioration. A CT brain scan at age 4 years, 2 months demonstrated multiple areas of variable density in the white matter. There was mild slowing of nerve conduction velocities and a sural nerve biopsy revealed segmental demyelinative neuropathy. Metachromatic leukodystrophy was suspected, but arylsulfatase A activity in leukocytes and fibroblasts was in the normal range. The cerebroside sulfate loading test on intact cultured fibroblasts showed attenuated hydrolysis leading to a tentative diagnosis of cerebroside sulfatase activator deficiency. However, the attenuated response of proband fibroblasts was not normalized by supplementation with activator in a reproducible manner, and urine showed hyperexcretion rather than deficiency of activator. Ultimately, an assay for galactosylceramide beta-galactosidase activity established a deficiency of this enzyme leading to the diagnosis of late-onset Krabbe disease.

Cerebroside-Sulfatase↗
PubMed1986

Identification of the tryptophan residue located in the calcium binding site of Trimeresurus flavoviridis phospholipase A2.

When phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased linearly with increase in the extent of oxidation of tryptophan residues. Oxidation of two of the four tryptophan residues caused an apparent loss of activity. The accessibilities of the tryptophan residues were analyzed with differently oxidized phospholipase A2 preparations and were determined to be in the following order: Trp-3 approximately Trp-30 greater than Trp-68 greater than Trp-108. The magnitude of the difference spectrum with a negative peak at 292 nm which is produced upon the binding of Ca2+ in the vicinity of tryptophan residue(s) decreased in a concave manner with increase in the extent of oxidation of tryptophan residues and was greatly diminished when 2 mol of tryptophan residues were oxidized. The activity and Ca2+-induced difference spectrum are thus related to either Trp-3 or Trp-30 or both. Des-octapeptide(1-8)-phospholipase A2 (L-fragment) is 14% as active as phospholipase A2 and is able to give a Ca2+-induced difference spectrum which is smaller than, but similar to, that of phospholipase A2. Its activity and the magnitude of the Ca2+-induced difference spectrum decreased along similar paths with increase in the amount of tryptophan residues oxidized, but in a manner indicating that two tryptophan residues are apparently responsible for the activity and the Ca2+-induced difference spectrum. The order of accessibility of the tryptophan residues of L-fragment was Trp-30 approximately Trp-108 greater than Trp-68. Trp-108, however, could be excluded from the residues located in the active site by reference to the tertiary structure of homologous Crotalus atrox phospholipase A2. Thus, Trp-30 is located in the Ca2+ binding site and is responsible for the activity of L-fragment. It is thus concluded that in phospholipase A2 Trp-30 is located in the Ca2+ binding site. From the concave decrease of relative magnitude of the Ca2+-induced difference spectrum and the linear decrease of relative activity upon oxidation of phospholipase A2, it may be assumed that both Trp-3 and Trp-30 are required to produce the Ca2+-induced difference spectrum, while only Trp-30 need be intact for activity. Anomalous binding of Ca2+ was observed for oxidized phospholipase A2.

Amino Acids↗
PubMed1986

Interactions of Trimeresurus flavoviridis phospholipase A2 and its N-terminal octapeptide-removed and p-bromophenacylated derivatives with acridine and anilinonaphthalene dyes.

Interactions of dimeric Trimeresurus flavoviridis (the Habu snake) phospholipase A2 (PLA2), des-octapeptide(1-8)-PLA2 (L-fragment) (14% of PLA2 activity), and p-bromophenacyl bromide (BPB)-inactivated PLA2 (BP-PLA2) with dyes, namely, proflavine, 1-anilinonaphthalene-8-sulfonate (Ans), and 2-toluidinylnaphthalene-6-sulfonate (Tns), were investigated. All dyes were bound in a 1:1 molar ratio to the subunit of the proteins. Proflavine was bound most strongly to PLA2 and Ans and Tns were bound to the three proteins with comparable affinities. Capabilities of the dyes for inhibiting alkylation of His-47 of PLA2 with BPB were in the following order: Ans greater than proflavine greater than Tns. Fluorescences of Ans and Tns that were increased in the presence of PLA2 were further greatly enhanced upon the addition of Ca2+, with concomitant formation of the ternary complexes. Ca2+, however, inhibited, competitively or noncompetitively, the bindings of the dyes to PLA2. All dyes were bound to the active site of PLA2 but with different orientations. Inactivation of L-fragment with BPB was inhibited by the dyes in the following order: Tns greater than proflavine approximately Ans. Addition of Ca2+ to the binary complexes formed from L-fragment and Ans or Tns caused no additional enhancement of fluorescence in spite of the formation of the ternary complexes. The active site structures are different between PLA2 and L-fragment, and the N-terminal octapeptide moiety of PLA2 possibly plays a role in maintaining the optimally arranged active site structure of the molecule. Comparison of the data suggests that the N-terminal moieties of PLA2S from snakes of an elapid family and from mammalian pancreas are essential for catalysis of a micellar substrate, whereas those of PLA2S from snakes of a viperid family, such as T. flavoviridis, are not. BP-PLA2 bound Ca2+ and was similar to L-fragment in terms of the fluorescence measurements. It appears that the active site of PLA2 has a space large enough to accommodate p-bromophenacyl, Ans or Tns, and Ca2+ together. Comparison of the emission maxima of Ans and Tns complexed with the three proteins indicated that Tns could be a useful fluorescent probe informing us of the state (disorder) of the active site of PLA2.

Acetophenones↗
PubMed1986

Amino acid sequence of Trimeresurus flavoviridis phospholipase A2.

The amino acid sequence of phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was determined. The enzyme subunit has a molecular weight of 13,764 and consists of a single polypeptide chain of 122 amino acids and seven disulfide bonds. The fragmentation was conducted by digesting the reduced and S-carboxymethylated derivative of the protein with Achromobacter protease I, chymotrypsin, and trypsin, respectively. Achromobacter protease I peptides were used for alignment and to establish overlaps over chymotryptic and tryptic peptides. The automated Edman degradation of the S-carboxymethylated protein, which was extended to the N-terminal 30 amino acid residues, supplemented the deletions found with the enzymatic peptides alone. T. flavoviridis phospholipase A2 was found to be highly (65-67%) homologous in sequence to the enzymes from T. okinavensis, Crotalus adamanteus, and Crotalus atrox (viperid family) and less (35-44%) homologous to those from elapid snakes and mammalian pancreas. The T. flavoviridis enzyme appears to be similar in secondary structure composition to the C. atrox enzyme.

Amino Acid Sequence↗
PubMed1985

Activator protein required for the enzymatic hydrolysis of cerebroside sulfate. Deficiency in urine of patients affected with cerebroside sulfatase activator deficiency and identity with activators for the enzymatic hydrolysis of GM1 ganglioside and globotriaosylceramide.

Urine specimens from two sibs affected with cerebroside sulfatase activator deficiency were examined to ascertain whether the deficiency of the supplementary activator protein required for the enzymatic hydrolysis of cerebroside sulfate was also evident in urine. Material from chromatographic fractionations was examined for the activator activity to avoid ambiguities resulting from protein inhibition. There were substantial deficits in all chromatographic fractions corresponding to activator-containing fractions of control urines. Since patient urines contained elevated amounts of lactosylceramide, digalactosylceramide, and globotriaosylceramide and since similarities between activators for cerebroside sulfate and GM1 ganglioside hydrolyses had been noted previously, the chromatographic fractions were also examined for activators in other glycosphingolipid hydrolase systems. There was coincidence of activators for the GM1 ganglioside/beta-galactosidase and the globotriaosylceramide/alpha-galactosidase A reactions with the cerebroside sulfatase activator in control urine fractions, and the patients' urines were deficient in activator activities for the three reactions. Identity of the three activators was suggested and antiserum to purified GM1 ganglioside activator was used to test this possibility. There were depressed levels of cross-reacting material in fractions of patient urines by Ouchterlony double diffusion and in unfractionated urine by enzyme-linked immunosorbent assay. Purified activators for the cerebroside sulfate and GM1 ganglioside systems showed lines of identity with no spurring on Ouchterlony double diffusion, identical mobility on immunoelectrophoresis, and similar stimulatory activities toward hydrolysis of the three glycosphingolipid species by their respective enzymes. Finally, the three activator activities were retained by anti-GM1-activator IgG coupled to Sepharose 4B. The results suggest strongly that the same protein entity serves as activator for the enzymatic hydrolysis of cerebroside sulfate, GM1 ganglioside, and globotriaosylceramide.

Animals↗
PubMed1985

Fine structural and immunohistochemical studies of goat adenohypophysial cells.

The fine structure of each type of anterior pituitary cell in the male goat was studied through the application of a superimposition technique in which adjacent thick sections were used to identify individual cells beforehand by light-microscopic immunohistochemistry. A cone of the pars intermedia protrudes into the pars anterior, being surrounded by the narrow pituitary cleft; the immunohistochemical appearances of the cells forming the cone resemble those of the pars anterior. Several follicles appear in the pars anterior. Ultrastructurally GH cells resemble prolactin cells. The secretory granules of both types are spherical; the diameter of the former is about 340 nm, whereas that of the latter is about 440 nm. ACTH cells are polygonal in shape with secretory granules, about 180 nm in diameter, scattered throughout the cytoplasm. TSH cells, which are spherical in shape, contain the smallest secretory granules, 150 nm in diameter. The highly electron-dense LH cells contain numerous secretory granules about 210 nm in diameter. Their nuclei are irregular with incisures. Thus, the anterior pituitary cells of the goat are ultrastructurally characteristics and species-specific.

Adrenocorticotropic Hormone↗
PubMed1985

Studies on the charge isomers of arylsulfatase A.

Human liver arylsulfatase A was resolved into six fractions by narrow pH range preparative isoelectric focusing. Analytical isoelectric focusing revealed that most enzyme fractions were composed of two adjacent charge isomers. Nevertheless, there was considerable enrichment of charge species which allowed a comparative study of selected properties. Except for the most cationic fraction, neuraminidase treatment converted enzyme in all fractions to the three most cationic species. The most electronegative enzyme species had the highest molecular mass being made up of 64-kDa subunits. As electronegativity decreased, there was concomitant decrease in molecular mass and increase in complexity of subunit composition. Two subunits--61 and 55 kDa--prevailed with increasing proportions of the smaller unit with loss of electronegativity. There was also an increasing amount of a 26-kDa fraction which became a substantial component of the most cationic subfraction. Only enzyme in the two fractions containing the largest and most anionic species were taken up by cultured fibroblasts at higher efficiency than unfractionated enzyme. It is suggested that processing or maturation of arylsulfatase A incurs stepwise removal of charge groups and/or peptide segments leading to smaller, less-charged enzyme species.

Cerebroside-Sulfatase↗
PubMed1985

Stopped-flow X-ray scattering device with a slit-type mixer.

A slit-type mixer specially designed for stopped-flow X-ray scattering was constructed. It mixes two solutions at a time, allowing a large-sized window to be used immediately after mixing. Dead time of the mixer was 13 ms with 3.5 kg/cm2 of pressure at the piston head. It has been successfully used for studying the tetramer-dimer interconversion of phosphorylase a. The applicability of this mixer to other detection methods, far-UV circular dichroism, light scattering, EXAFS and fluorescence is also indicated.

2,6-Dichloroindophenol↗
PubMed1985

Selective separation of tryptophan-containing peptides via hydrophobic modification with 2-nitro-4-carboxyphenylsulfenyl chloride.

Selective separation of tryptophan-containing peptides has been attempted using 2-nitro-4-carboxyphenylsulfenyl chloride (NCps)-C1 as a reagent for hydrophobic modification of tryptophan. S-Carboxymethylated proteins were modified with NCps-C1 in 70% acetic acid and digested with TPCK-trypsin, and the digests were fractionated, directly or after partial fractionation on a Sephadex G-25 column, by high performance liquid chromatography using a reverse phase column. The tryptophan-containing peptides from the tryptic digests of S-carboxymethylated hen-egg white lysozyme and Trimeresurus flavoviridis phospholipase A2 were thus selectively separated and the amino acid sequences were determined, showing the validity of the method. The phenylthiohydantoin derivative of 2-(2'-nitro-4'-carboxyphenylthio)-L-tryptophan was synthesized and its spectroscopic and chromatographic properties determined, enabling us to identify the derivative on Edman sequencing.

Amino Acid Sequence↗
PubMed1985

Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom.

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.

Amino Acids↗