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Biomedical subjects

A Ikai

Publications and source records attributed to A Ikai.

At least 109 records · Page 6Linked to original sources
PubMed1987

The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. Homologies with mammalian and insect lectins.

The complete amino acid sequence of echinoidin, the proposed name for a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina, has been determined by sequencing the peptides obtained from tryptic, Staphylococcus aureus V8 protease, chymotryptic, and thermolysin digestions. Echinoidin is a multimeric protein (Giga, Y., Sutoh, K., and Ikai, A. (1985) Biochemistry 24, 4461-4467) whose subunit consists of a total of 147 amino acid residues and one carbohydrate chain attached to Ser38. The molecular weight of the polypeptide without carbohydrate was calculated to be 16,671. Each polypeptide chain contains seven half-cystines, and six of them form three disulfide bonds in the single polypeptide chain (Cys3-Cys14, Cys31-Cys141, and Cys116-Cys132), while Cys2 is involved in an interpolypeptide disulfide linkage. From secondary structure prediction by the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13, 211-222) the protein appears to be rich in beta-sheet and beta-turn structures and poor in alpha-helical structure. The sequence of the COOH-terminal half of echinoidin is highly homologous to those of the COOH-terminal carbohydrate recognition portions of rat liver mannose-binding protein and several other hepatic lectins. This COOH-terminal region of echinoidin is also homologous to the central portion of the lectin from the flesh fly Sarcophaga peregrina. Moreover, echinoidin contains an Arg-Gly-Asp sequence which has been proposed to be a basic functional unit in cellular recognition proteins.

Animals↗
PubMed1987

The fate of internalized alpha-2-macroglobulin: alpha-galactosidase conjugate in fibroblasts from Fabry's hemizygote.

In our previous report we described the endocytotic incorporation of coffee bean alpha-galactosidase conjugated to human alpha-2-macroglobulin (alpha-2-M) into cultured fibroblasts derived from a patient with Fabry's disease (1). The fate of internalized alpha-galactosidase according to the method described in the above report is now studied. Measurement of the enzyme activity of subcellular fractions showed that it was concentrated in the lysosomal-mitochondrial fraction. The half-life of internalized alpha-galactosidase was determined to be 2 h.

Cells, Cultured↗
PubMed1987

Endocytotic internalization of alpha-2-macroglobulin: alpha-galactosidase conjugate by cultured fibroblasts derived from Fabry hemizygote.

Endocytotic internalization of alpha-galactosidase by cultured fibroblasts derived from a patient with Fabry's disease was achieved via receptor-mediated endocytosis of alpha-2-macroglobulin (alpha-2-M). alpha-galactosidase of coffee beans was conjugated to alpha-2-M when the latter was treated with trypsin. Internalization of the conjugate resulted in an increase of alpha-galactosidase activity in the crude cell extracts. The observed internalization was blocked by the presence of bacitracin, an inhibitor of binding between alpha-2-M and its receptor on the cell surface. When the cells were incubated at 4 degrees C with the conjugate, internalization was also inhibited. The alpha-galactosidase activity in the cells was saturated when the concentration of the conjugate in the medium was 40 micrograms/ml. Since non-conjugated alpha-galactosidase was not effectively internalized, the observed internalization of the conjugate was mediated by recognition of alpha-2-M by its receptor. The effective internalization of alpha-galactosidase described in this paper has a potential use in the enzyme replacement therapy of Fabry's disease.

Cells, Cultured↗
PubMed1987

Active site organization of bacterial type I fatty acid synthetase.

Four kinds of active sites of bacterial fatty acid synthetase were mapped on distinct regions within a subunit. Active sites were specifically labeled with radioactive substrates and active-site-directed inhibitors. Labeled enzymes were cleaved with proteases, and the fragments thus produced were identified with respect to specific labels by SDS-polyacrylamide gel electrophoresis and a fluorographic technique. The linear alignment of such fragments in the original subunit was established and when the results were combined with those of our previous work, five active sites were located in three regions as follows. Starting from the N-terminal of the subunit, we located acetyl, malonyl and palmitoyl transferases in the first region, the acyl carrier site in the second region (Morishima & Ikai (1985) Biochim. Biophys. Acta 832, 297-307), and beta-ketoacyl synthetase in the third region. The observed order of active sites of bacterial fatty acid synthetase can be correlated with that of the yeast enzyme, which has two kinds of subunits.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1986

Proton nuclear magnetic resonance study of human plasma alpha-2-macroglobulin.

A proton nuclear magnetic resonance (NMR) study is reported of human alpha-2-macroglobulin (alpha-2-M). It was observed that alpha-2-M, which consists of four identical subunits and has a molecular weight of 720,000, gives several sharp resonances. After cleavage of the "bait" region peptide with trypsin and subsequent removal of the peptide under a high salt condition, most of the sharp resonances disappeared, indicating that the sharp resonances observed in the native alpha-2-M originate from the amino acid residues in the bait region. Resonances due to the aromatic protons of the Tyr residue, which exists in the bait region, have been assigned on the basis of chemical shift. It was observed that the C3- and C5-H proton resonances for the Tyr residue are especially narrow, indicating that the side chain of the Tyr residue in the bait region is in a highly mobile state. Photochemically induced dynamic nuclear polarization experiments clearly show that the Tyr residue is actually exposed to the solvent. It was possible to identify resonances due to several His residues that are exposed to solvent. Other resonances, which probably originate from Arg residues in the bait region, were also observable in the conventional NMR spectra. On the basis of the present NMR data, we conclude that the bait region of the native alpha-2-M is highly flexible and exposed to solvent. On treatment of alpha-2-M with methylamine, no significant change has been detected in the NMR spectra observed in both the conventional and CIDNP mode.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Unusual properties of crocodilian ovomacroglobulin shown in its methylamine treatment and sulfhydryl titration.

The inhibitory activity of chicken and crocodilian ovomacroglobulins against trypsin was measured before and after their incubation with methylamine. The result for crocodilian ovomacroglobulin showed that methylamine treatment destroyed half of its activity, in unique contrast to human alpha 2-macroglobulin and chicken ovomacroglobulin for which methylamine either destroys the inhibitory activity of the former completely or does not affect that of the latter at all. Free sulfhydryl groups of chicken and crocodilian ovomacroglobulins were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) before and after incubation with trypsin. Prior to the incubation with trypsin the chicken and crocodilian proteins respectively had 0 and 1 titratable sulfhydryl per molecule of Mr 720,000. After treatment with trypsin the crocodilian protein had 3.5-4 titratable sulfhydryls, whereas there were no titratable sulfhydryls in the chicken protein. After denaturation of the crocodilian protein in sodium dodecyl sulfate at 100 degrees C the number of titratable sulfhydryls was 4. Chicken ovomacroglobulin again did not have an appreciable number of titratable sulfhydryls under similar denaturing conditions. Incubation of crocodilian protein with [14C]methylamine showed an incorporation of at least 2 mol of methylamine per molecule. The result indicated the presence of three intramolecular thiol ester bonds in crocodilian ovomacroglobulin with differential stability against external perturbations.

Alligators and Crocodiles↗
PubMed1985

Mapping of acyl carrier domain within the subunit of type I bacterial fatty acid synthetase.

A fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, was used to label the acyl carrier site of the bacterial fatty acid synthetase from Brevibacterium ammoniagenes. The reagent bound preferentially to the 4'-phosphopantetheine thiol group of the acyl carrier domain and irreversively inactivated the enzyme. The modified enzyme was cleaved by proteinases for the mapping of the labeled site. The fluorescent fragment was readily detected on a polyacrylamide gel after electrophoresis. The region of 45 kDa containing the 4'-phosphopantetheine was located on the polypeptide at around two-thirds of the full length from the N-terminal.

Acyl Carrier Protein↗
PubMed1985

Structural changes in alpha-2- and ovomacroglobulins studied by gel chromatography and electron microscopy.

The structural change that occurs in alpha-2-macroglobulin upon its interaction with methylamine or chymotrypsin was studied by high-performance gel chromatography and electron microscopy. The result enabled us to estimate the Stokes radius of the protein as 8.8 nm and 7.9 nm before and after binding with the proteinase, respectively. The methylamine-treated protein also had the Stokes radius of 7.9 nm. Similar studies on the chicken and crocodilian ovomacroglobulins showed that these homologues of alpha 2-macroglobulin had Stokes radii of 9.2-9.3 nm and 8.5-8.7 nm before and after binding with chymotrypsin. Their Stokes radii did not change as a result of the methylamine treatment. Electron micrographs of the native and altered forms of the three proteins are presented. This study introduces a simple and quantitative method to study the structural change of alpha 2-macroglobulin and its homologues.

Alligators and Crocodiles↗
PubMed1985

A new multimeric hemagglutinin from the coelomic fluid of the sea urchin Anthocidaris crassispina.

A hemagglutinin was purified from the coelomic fluid of the sea urchin Anthocidaris crassispina by ion-exchange chromatography on DEAE-cellulose and affinity adsorption to glutaraldehyde-fixed ghosts of human erythrocytes, followed by elution with 10 mM EDTA. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it showed a single protein band with a molecular weight of 13 000 and 26 000 in the presence and absence of 2-mercaptoethanol, respectively. The molecular weight of the native protein with a hemagglutinating activity was determined to be 300 000 by sedimentation equilibrium analysis. Its sedimentation coefficient, S0(20),w, and Stokes radius were 13.7 S and 5.5 nm, respectively. The hemagglutinating activity of this protein required calcium ions. When calcium ions were depleted, no activity was observed and its sedimentation coefficient, S0(20),w, decreased to 11.4 S while its Stokes radius increased to 6.7 nm without a change in its molecular weight. The purified hemagglutinin agglutinated human erythrocytes regardless of their ABO and MN blood types. The hemagglutination reaction was not affected appreciably by various simple sugars but was inhibited by tryptic fragments released from human erythrocyte membranes. The results of alkaline borohydride treatment of the inhibitory tryptic fragments showed that the receptor sites for this hemagglutinin were mainly composed of alkali-labile carbohydrate chains with the structure AcNeu alpha 2----3Gal beta 1----3(AcNeu alpha 2----6)GalNAc----serine (or threonine).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Structure of apoproteins in insect lipophorin.

The two polypeptide chains of cockroach and locust lipophorins were separated and their amino acid compositions were determined. Circular dichroic spectra of the lipophorins and apolipophorin from 190 to 250 nm showed a single trough at 218 nm and a peak at 194 nm. Infrared spectra of the lipophorins in D2O showed a strong peak at 1625 cm-1 and a weak shoulder at 1693 cm-1 corresponding to v (pi, 0) and nu (0, pi) of antiparallel pleated sheet. The resonance frequency splitting delta nu = nu (0, pi) -nu (pi, 0) was 68 cm-1, which was larger than that of ordinary globular proteins containing antiparallel pleated sheet. From circular dichroic and infrared spectra it was concluded that lipophorins contained polypeptides rich in antiparallel pleated sheet with longer unbroken extensions than the case for ordinary globular proteins. Partial proteolytic digestion study of lipophorins with trypsin, chymotrypsin, and subtilisin showed that the larger apolipophorin (AL1) was exposed to the surface of the particle and the smaller apolipophorin (AL2) lay protected from the attack of the enzymes. Crosslinked products between AL1 and AL2 were readily obtained when dimethylsuberimidate or dimethyladipimidate was added to the lipophorin solution, without giving lipophorin dimers, suggesting that the two chains were located within 11 A from each other. Such structural features of insect lipoprotein were compared with other insect lipophorins and the human serum low-density lipoprotein (LDL). Similarities between lipophorins and LDL were found in the molecular weight, amino acid compositions, and the secondary structure of major apoproteins.

Amino Acids↗
PubMed1985

The quaternary structure and activity of newly purified fatty acid synthetase from the Harderian gland of guinea-pig.

Fatty acid synthetase was isolated from the Harderian gland of guinea-pig. The fatty acids synthesized by the purified enzyme were analyzed by mass fragmentography. The purified enzyme had an inherent capacity to utilize methylmalonyl-CoA and synthesize methyl-branched fatty acids. Physicochemical studies indicated that an active enzyme was a dimer, consisted of two subunits of Mr = 2.5 X 10(5). The negatively stained enzyme had an electron micrographic image of an ellipsoidal contour with a continuous middle cleft along the major axis. The major and minor axes were approximately equal to 220 and 150 A, respectively. In a dimer, the subunit had a rod-like structure about 220 A long and 50 A wide. The enzyme was inactivated and dissociated into subunits by incubation at 0 degree C. The inactivated enzyme was fully reactivated by raising the temperature of the solution. The relationship between the quaternary structure of the enzyme and the occurrence of enzymatic activity was studied by high-performance liquid chromatography. Neither active monomers nor inactive dimers were found in inactivation and reactivation processes. The initial velocity of reactivation was proportional to the enzyme concentration over a concentration range of 160-800 micrograms/ml, indicating that the rate-determining step in the reactivation reaction was unimolecular.

Amino Acids↗
PubMed1985

The oxygen affinity of mammalian hemoglobins in the absence of 2,3-diphosphoglycerate in relation to body weight.

We studied the oxygenation of mammalian hemoglobins: mouse (Mus musculus molossinus), rabbit (Oryctolagus cuniculus domesticus), Japanese monkey (Macaca fuscata), man (Homo sapiens), sheep (Ovis aries), llama (Lama glama), pig (Sus scrofa domesticus), cow (Bos taurus domesticus) and horse (Equus caballus), in the absence of 2,3-diphosphoglycerate (DPG) and compared their oxygen affinity in relation to the body weight. The negative correlation between body weight and the oxygen affinity of the whole blood, observed by Schmidt-Nielsen and Larimer (1958), was not observed in the absence of DPG. Our results indicated that an adaptive evolution proposed for hemoglobin in terms of its oxygen affinity vs body weight of the animal can only be appreciated with DPG.

2,3-Diphosphoglycerate↗
PubMed1985

Purification of the most abundant protein in the coelomic fluid of a sea urchin which immunologically cross reacts with 23S glycoprotein in the sea urchin eggs.

The most abundant glycoprotein in the coelomic fluid of sea urchin Anthocidaris crassispina was purified and its subunit structure, molecular form in the native state, amino acid composition, and electron micrographic image were studied. The results showed that the protein in its native state was basically a tetramer with a total molecular weight of about 700,000, which was in equilibrium with a high molecular weight form corresponding to an octamer. The electron micrograph of the tetramer showed two ellipsoidal units aligned in parallel with a wide gap in between. The subunits all had the same molecular weight of 180,000 +/- 10,000 and were disulfide bonded in pairs. The carbohydrate content was about 16% with mannose and fucose as the two most abundant sugars. Although this protein accounted for 70% of the total protein in the coelomic fluid, it did not take part in the known activities of the fluid, namely hemagglutination and coagulation. Despite its structural similarity to the mammalian alpha-2-macroglobulin or reptilian and avian ovomacroglobulins it did not interact with bovine trypsin or chymotrypsin. This protein showed immunological cross reactivity with 23S glycoprotein purified from sea urchin eggs which, we believe, corresponds to the previously described 22-27S protein particles in eggs.

Amino Acids↗
PubMed1985

Purification and physical chemical characterization of 23S glycoprotein from sea urchin (Anthocidaris crassispina) eggs.

A large glycoprotein with a sedimentation coefficient, S(0)20,w, of 23.3S was purified to homogeneity from sea urchin eggs (Anthocidaris crassispina) by gel filtration on Sepharose CL-4B and ion-exchange chromatography on DEAE-cellulose. The molecular weight of the protein was 700,000 as determined by sedimentation equilibrium. On polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS) it showed a single band with an apparent molecular weight of 180,000 or 360,000 in the presence or absence of 2-mercaptoethanol, respectively. The protein consisted of four polypeptides of equal molecular weight, which were disulfide bonded in pairs. Its carbohydrate content as determined by the phenol-sulfuric acid method was 20% of the total weight. The amino acid and carbohydrate compositions, circular dichroic spectrum and electron microscopic image are also presented. The protein showed many structural similarities with the previously purified major glycoprotein (MCP) in the coelomic fluid of the same animal in addition to being immunologically cross reactive with it. However, the two proteins were distinct glycoproteins. Their biological functions have not been identified.

Amino Acids↗
PubMed1985

Structural unit of the erythrocyte cytoskeleton. Isolation and electron microscopic examination.

We isolated a protein complex containing major cytoskeletal components from the Triton shell of bovine erythrocytes. This protein complex, which we called the 26-S complex, consisted of three major components, spectrin, band-4.1 protein and actin, and one minor component, band-4.9 protein. The molar ratio of spectrin heterodimer:band 4.1:actin was determined by sodium dodecyl sulfate (SDS) gel electrophoresis to be about 1:2:2, approximately the same as that for the Triton shell. By electron microscopic examinations of rotary-shadowed specimens, it was revealed that the 26-S complex had a "spider-like" morphology with a central core and several spectrin heterodimers radiating from it. The number of spectrin arms in the complex was not constant but was in the range between 3 and 6. The complexes with five spectrin heterodimers were the most numerous. The results showed that the 26-S complex contained on the average five spectrin heterodimers, ten band-4.1 polypeptides and ten actin monomers. As judged from the formation of oligomeric 26-S complexes through spectrin arms, the central core of the complex presumably contains band 4.1 and actin. Supporting this conclusion, the central core acted as a nucleus for actin polymerization when the 26-S complex was mixed with G-actin under an actin-polymerizing condition. The 26-S complex could form large aggregates under a certain condition that spectrin was promoted to associate from dimer to tetramer. We conclude that the 26-S complex is the structural unit of the erythrocyte cytoskeleton.

Actins↗
PubMed1984

Role of subunit interactions in the self-assembly of oligomeric proteins.

In oligomeric proteins, the native conformation and its functional properties depend on the interactions which exist between the different chains. The role of these subunit interactions can be studied using either the unfolded state or the native state as a starting point. During the folding process, the properties which appear following a bimolecular reaction are related to the formation of an association area. Similarly, the properties which are lost upon partial dissociation of the native state are related to the association area which is disrupted. Four examples are presented in this article: phosphofructokinase and aspartokinase-homoserine dehydrogenase from E. coli are studied through their folding process, and fatty acid synthetase from B. ammoniagenes and reptilian ovomacroglobulin are studied through their dissociated forms. In all cases, the function of the protein is a sensitive index of the formation of the subunit interactions, and can be more conveniently measured than other size/shape parameters. The extrapolation from the folding of small proteins to the assembly of large and complex structures can be reasonably achieved by admitting that subunit interactions are coupled to the subtle adjustments required by the protein to exert its biological function.

Animals↗