Biomedical subjects

A Ikai

Publications and source records attributed to A Ikai.

At least 91 records · Page 5Linked to original sources

PubMed1989

Negative functional interference between two active centers is indicated in animal fatty acid synthetase.

When fatty acid synthetase of the Harderian gland of guinea-pig was treated with various amounts of phenylmethanesulfonyl fluoride, the overall activity of the enzyme showed a quadratic decrease with respect to the inhibition degree of the thioesterase activity which was the primary target of inhibition. Moreover, the overall activity per active center of a heterodimer, which was formed between the native monomer and the thioesterase-less monomer, was higher than that of the native enzyme. These results are consistent with the view that the two active centers of the native enzyme exhibit a negative interference to each other.

Animals↗

PubMed1989

Purification and characterization of proline-beta-naphthylamidase, a novel enzyme from pig intestinal mucosa.

An enzyme hydrolyzing proline-beta-naphthylamide was purified to apparent homogeneity from porcine intestinal mucosa. The purified enzyme appears to consist of three identical subunit polypeptides with a molecular weight of about 58,000 each, associated noncovalently. The enzyme is a glycoprotein, and the subunit polypeptide contains 3 residues each of mannose and N-acetylglucosamine. A wide variety of peptidase substrates were tested for the enzyme, and the results showed that it hydrolyzes only aminopeptidase substrates, such as proline-beta-naphthylamide, glycine-beta-naphthylamide, leucine-beta-naphthylamide, and alanine-beta-naphthylamide. Among these substrates, proline-beta-naphthylamide is most efficiently hydrolyzed as judged by the kcat/Km value. The optimum pH for this substrate is around 9. The enzyme also hydrolyzes efficiently the ester substrates of these amino acids. No hydrolytic activity was observed for the peptide and protein substrates tested. The proline-beta-naphthylamidase activity was drastically inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, indicating that the enzyme is a serine hydrolase, whereas it was slightly inhibited by aminopeptidase inhibitors, such as amastatin, bestatin, and puromycin. No significant homology was found for the NH2-terminal sequence of 27 amino acid residues with any known protein sequences. From these results we conclude that the enzyme is a protein which has not been described before.

Amino Acids↗

PubMed1989

Inhibition of inflammatory proteinase, medullasin, by alpha 2-macroglobulin and ovomacroglobulin.

The in vitro activity of inflammatory proteinase, medullasin, was stoichiometrically inhibited by a serum proteinase inhibitor, alpha 2-macroglobulin, and its homolog, chicken ovomacroglobulin. The two inhibitors were cleaved by medullasin only in the bait region. The effectiveness of alpha 2-macroglobulin to inhibit medullasin in competition with alpha -1-proteinase inhibitor was measured under a simulated in vivo condition and an estimation was made that about 60-70% medullasin is inhibited by alpha-1-inhibitor and 30-40% by alpha 2-macroglobulin.

Binding, Competitive↗

PubMed1989

alpha 2-macroglobulin traps a proteinase in the midregion of its arms. An immunoelectron microscopic study.

alpha 2-Macroglobulin, one of the major plasma proteinase inhibitors with Mr = 720,000, is known to inhibit proteinases of all four classes through the "trap mechanism" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724), but the proteinase binding site of alpha 2-macroglobulin has not been identified precisely. We localized bound proteinase molecules on the electron microscopic images of alpha 2-macroglobulin, using anti-proteinase IgG. Serratial Mr = 56,000 proteinase produced by Serratia marcescens was chosen as the antigenic probe in this study because its affinity to specific antibodies was retained in its bound state to alpha 2-macroglobulin. Dimers of alpha 2-macroglobulin/Mr = 56,000 proteinase complexes cross-linked with anti-Mr = 56,000 proteinase IgG were prepared and subjected to electron microscopic observations. The electron microscopic image of alpha 2-macroglobulin complexed with Mr = 56,000 proteinase had four straight arms with an overall shape looking like the character "H." From the way anti-Mr = 56,000 proteinase IgG linked two alpha 2-macroglobulins, it was concluded that the proteinase existed in the midregion of one of the arms. This result helps us to form a more concrete view of the trap mechanism in that one of the arms of alpha 2-macroglobulin wraps the trapped proteinase and holds it isolated from high molecular weight substrates in the surrounding medium.

Binding Sites↗

PubMed1989

Transducin activation by molecular species of rhodopsin other than metarhodopsin II.

Decay of metarhodopsin II was accelerated by hydroxylamine treatment or dark incubation of metarhodopsin II at 30 degrees C. The products thus obtained after decay of metarhodopsin II induced GTPase activity on transducin as well as metarhodopsin II suggesting that rhodopsin could activate transducin after the decay of metarhodopsin II intermediate. After urea-treated bovine rod outer segment membrane was completely bleached, rhodopsin in the membrane was regenerated by the addition of 11-cis retinal at various temperatures between 0 and 37 degrees C. The capacity to induce GTPase activity on transducin and phosphate incorporating capacity catalyzed by rhodopsin kinase were measured on such rhodopsins. The results showed that: (1) Regeneration of alpha band of rhodopsin was complete regardless of regeneration temperature; (2) When regenerated at temperatures below 10 degrees C, rhodopsins induced a GTPase activity on transducin in the dark even after treatment with hydroxylamine, whereas rhodopsins after regeneration at temperatures above 13 degrees C did not; (3) When regenerated at 0 degrees C, rhodopsin was phosphorylated if incubated with rhodopsin kinase and ATP in the dark, whereas the spectrally regenerated rhodopsin at 30 degrees C was not. The complete quenching of functions of photoactivated rhodopsin was achieved by recombination with 11-cis retinal at temperatures above 13 degrees C but not below 10 degrees C suggesting the existence of a low temperature intermediate upon regeneration.

Animals↗

PubMed1988

Proteasomes (multi-protease complexes) as 20 S ring-shaped particles in a variety of eukaryotic cells.

Latent multicatalytic protease complexes, named proteasomes, were purified to apparent homogeneity from various eukaryotic sources, such as human, rat, and chicken liver, Xenopus laevis ovary, and yeast (Saccharomyces cerevisiae), and their functional and structural properties were compared. They showed latency in breakdown of [methyl-3H]casein, but were greatly activated in various ways, such as by addition of polylysine. They all degraded three types of fluorogenic oligopeptides at the carboxyl side of basic, neutral, and acidic amino acids, and the three cleavage reactions showed different spectra for inhibition, suggesting that they had three distinct active sites. The proteasomes all seemed to be seryl endopeptidases with similar pH optima in the weakly alkaline region. Their physiochemical properties, such as their sedimentation coefficients (19 S to 22 S), diffusion coefficients (2.0-2.6 X 10(-7) cm2 s-1), molecular masses (700-900 kDa), and circular dichroic spectra, were similar. Their amino acid compositions were also very similar. Electron microscopy showed that they had similar well-defined symmetrical morphology, appearing to be ring-shaped particles with a small hole in the center. All the proteasomes seemed to be multisubunit complexes consisting of 15-20 polypeptides with molecular masses of 22-33 kDa and isoelectric points of pH 3-10, but they showed species-specific differences in subunit multiplicity. Moreover, they differed immunologically, as shown by Ouchterlony tests and immunoblotting analyses, although cross-immunoreactivities of some subunits or domains were observed. These results indicate that the sizes and shapes of these proteasomes have been highly conserved during evolution, but that they show species-specific differences in immunoreactivities and subunit structures. Thus proteasomes with similar structure and function seem to be ubiquitously distributed in eukaryotic organisms ranging from man to yeast. This distribution implies the general importance of these proteasomes for proteolysis.

Amino Acids↗

PubMed1988

Molecular organization of a high molecular weight multi-protease complex from rat liver.

A latent multifunctional protease with a molecular weight of 722,000 to 760,000 purified from rat liver cytosol has been reported. This paper reports on the structure and subunit composition of the enzyme. Electron microscopy showed that the enzyme was a ring-shaped particle of 160(+/- 7) A diameter and 110(+/- 10) A height with a small hole of 10 to 30 A diameter (1 A = 0.1 nm). Small-angle X-ray scattering analysis indicated that the enzyme had a prolate ellipsoidal structure with an ellipsoid cavity in the center. The maximum dimension of the enzyme was estimated to be 210 A from a pair-distance distribution function. The radius of gyration obtained from a Guinier plot and the Stokes radius based on the ellipsoidal model were 66 A and 76 A, respectively. On two-dimensional gel electrophoresis, the purified enzyme separated into 13 to 15 characteristic components with molecular weights of 22,000 to 33,000 and isoelectric points of 4 to 9. These multiple components were not artifacts produced by limited proteolysis during purification of the enzyme, because the cell-free translation products in a reticulocyte lysate with poly(A)-mRNA of rat liver consisted of multiple components of similar sizes, and because peptide mapping analyses with lysylendopeptidase and V8 protease demonstrated clear differences in the primary structures of these components. The 13 main components were isolated from the purified enzyme by reverse-phase high performance liquid chromatography and shown to be non-identical. A model of the enzyme is proposed on the basis of these observations and previous physicochemical studies. Interestingly, the morphology of this protease is similar to that of the 16 to 22 S ring-shaped particles found in a variety of eukaryotic organisms. The structural similarity between this multi-protease complex and various reported subcellular particles is discussed.

Animals↗

PubMed1988

Microtubule-assembly inhibitor protein. Its distribution, localization and physicochemical properties.

Microtubule-assembly inhibitor protein (MIP) is an acidic protein with Mr 33,000 which inhibits microtubule assembly in vitro [Kotani, S., Murofushi, H., Nishida, E. & Sakai, H. (1984) J. Biochem. (Tokyo) 96, 959-969]. Anti-MIP antibody was affinity-purified from rabbit anti-MIP sera raised against chemically modified MIP. MIP was localized in the nucleus in interphase culture cells as revealed by immunofluorescent light microscopy. Immunoblotting experiments showed that MIP exists in a variety of mammalian cells and tissues. Kidney appeared to be a better source of MIP than brain, the original source. Kidney MIP was isolated by the same procedure as for brain MIP and proved to be indistinguishable from brain MIP in the inhibitory activity of microtubule assembly, molecular mass, immunoreactivity, and one-dimensional peptide mapping. Physico-chemical characteristics of MIP were studied using the kidney protein. It contained 20% aspartic acid and 25% glutamic acid, accounting for its acidic nature. Hydrodynamically, MIP was a monomer with S20,w = 1.9 S and Mr = 30,000. The frictional ratio, f/fo = 1.7, indicated that MIP is not a globular molecule but has either an elongated or an expanded structure. Circular dichroic results showed a low content of alpha-helix or beta-sheet structure for MIP. Proton nuclear magnetic resonance analysis provided evidence that MIP consists mainly of very flexible structures (random-coil-like structures), but still contains a hydrophobic core structure below 60 degrees C.

Amino Acids↗

PubMed1988

Structure of fatty acid synthetase from the Harderian gland of guinea pig. Proteolytic dissection and electron microscopic studies.

Limited proteolysis and electron microscopic observation of fatty acid synthetase from the Harderian gland of guinea pig was performed to elucidate the higher-order structures of this multifunctional protein. Staphylococcus aureus V8 protease dissected the 250,000 Mr subunit of fatty acid synthetase into 120,000, 70,000, 35,000 and 30,000 Mr fragments, which were aligned in this order from the NH2 terminus. Some of the protease-resistant fragments produced with elastase, trypsin and lysyl endopeptidase were purified and fragment-specific antibodies (A40L, A33E and A25T) were prepared. A25T and A33F specifically bound the 35,000 and 30,000 Mr fragments, and A40L recognized the region between the 120,000 and 70,000 Mr fragments. Electron microscopic studies employing rotary shadowing, unidirectional shadowing and negative staining revealed that the overall dimension of the enzyme was 22 nm x 15 nm x 7 nm, and that two elongated subunits mainly composed of three subregions were in contact with each other at a few, three at most, points with two holes between them. The outer two attachment sites were often not in contact, indicating a certain flexibility of subunits at their ends. Immunocomplexes composed of fatty acid synthetase and fragment-specific antibodies were isolated and observed under the electron microscope. The attachment sites of A40L and A33E were located at the end of the minor and the major axes of the ellipsoidal contour of the molecule, respectively. Based on these results, the three-dimensional structure of animal fatty acid synthetase is discussed.

Amino Acid Sequence↗

PubMed1988

Purification and characterization of kinesin from bovine adrenal medulla.

Kinesin was purified from bovine adrenal medulla. The sedimentation coefficient was 8.8 S. Sedimentation equilibrium ultracentrifugation studies showed the molecular weight of kinesin to be 300,000. The calculated axial ratio was 1:16. The Stokes radius was estimated to be 8.9 nm by gel filtration. Circular dichroism showed the alpha-helix content to be about 50%. Purified kinesin preparation contained a major polypeptide with a molecular weight of 120,000 and minor ones with molecular weights of 71,000, 68,000, and 65,000. Bovine adrenal kinesin had an ATPase activity which was stimulated severalfold by microtubules to a specific activity of about 0.1 mumol/min.mg. Kinesin molecules adsorbed to a glass slide promoted the movement of microtubules on the glass surface at a rate of about 0.5 micron/s. Immunostaining of EBTr (bovine embryonic trachea fibroblast) cells and bovine adrenal chromaffin cells in interphase with an affinity-purified antibody against the major polypeptide of kinesin showed that some kinesin was located on microtubules and the rest distributed throughout the cytoplasm in a diffuse manner. EBTr cells in mitotic phase gave a staining pattern showing that kinesin was present throughout the cytoplasm with higher concentration in the region of mitotic apparatus.

Adenosine Triphosphatases↗

PubMed1988

Antibodies against viral proteins can be produced effectively in response to the increased uptake of alpha 2-macroglobulin:viral protein conjugate by macrophages.

We have previously shown that foreign antigens conjugated to alpha 2-macroglobulin (alpha 2M) were effectively taken up by macrophages, which was followed by a remarkable increase in the proliferation of immune T cells. We present in this report that the production of antibodies against viral proteins can also be effectively achieved when viral proteins were conjugated to alpha 2M and fed to the in vitro immune system. Proteins derived from Kirstein murine sarcoma virus (Ki-MSV) were partially purified by gel chromatography and conjugated to alpha 2M by the action of proteinases. Viral proteins conjugated to alpha 2M were taken up by thioglycolate-induced murine peritoneal exudate cells (PEC) more effectively than the free viral proteins. Murine spleen cells were then added to PECs fed with free viral proteins or with alpha 2M: viral protein conjugates, and the cell mixtures were incubated for five days. Each culture medium was then assayed for a specific antibody production by enzyme-linked immunosorbent assay (ELISA). Figures based on such assays revealed that the production of antibodies against viral proteins was ten times higher when the proteins were fed to macrophages in conjugated forms with alpha 2M.

Animals↗

PubMed1988

Purification method of bovine rhodopsin kinase using regeneration of rhodopsin.

We report a rapid and high-yield purification method of bovine retinal rhodopsin kinase. According to our method, 500 micrograms of rhodopsin kinase was purified from 100 bovine retinae within 12 h. Rhodopsin kinase bound to bleached rhodopsin was extracted effectively from rod outer segment membranes after regeneration of rhodopsin by the incubation with exogenous 11-cis-retinal. Subsequent DE52 column chromatography further purified the protein to homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified rhodopsin kinase had an apparent molecular weight of 68,000 and phosphorylated rhodopsin at the rate of 10 nmol phosphate/min/mg of the enzyme.

Animals↗

PubMed1988

Polymerization of turtle alpha-macroglobulin through newly exposed sulfhydryls reveals the location of ex-thiolester bonds.

Green turtle alpha-macroglobulin, which has previously been shown to contain thiolester bonds, formed linear polymers after being treated with proteinases. Biochemical analyses showed that the polymerization proceeded through disulfide-bond formation between monomers. The only sulfhydryl groups available for such polymerization after proteinase treatment were those created as the product of thiolester hydrolysis. Electron micrographs of polymers revealed H-shaped monomeric units aligned lengthwise in linear polymers. The average length per monomeric unit in the polymer estimated from the discrete distribution of polymer lengths was approximately 80% of the average length of free monomers, indicating that monomers overlapped each other within a region of about 4 nm. From such observations we concluded that the newly produced sulfhydryl groups were located on the four arms of the H-shaped molecule. The location of sulfhydryls can be taken as the site of the exposure of thiolesters which were originally sequestered in the hydrophobic interior of the molecule. Since the structure of turtle alpha-macroglobulin is very similar to that of human serum alpha 2-macroglobulin the results predict a similar location of sulfhydryls in human alpha 2-macroglobulin after proteinase treatment. The observed polymerization property is unique to sea turtle alpha-macroglobulin and has not been observed with human alpha 2-macroglobulin or other homologous proteins.

Animals↗

PubMed1988

Purification and characterization of alpha-macroglobulin and ovomacroglobulin of the green turtle (Chelonia mydas japonica).

The plasma alpha-macroglobulin and egg white ovomacroglobulin were purified from the sea turtle, Chelonia mydas japonica, and their structural and functional properties were studied with the aim of clarifying the degree of evolutional divergence of two homologous proteins specific to different tissues of the same animal. The concentration of alpha-macroglobulin in green turtle plasma was about 4 mg/ml. The protein was purified from the plasma by precipitation with polyethylene glycol 6000, followed by zinc chelate chromatography and gel chromatography on Sepharose CL-6B. The concentration of ovomacroglobulin in green turtle egg white was about 0.4 mg/ml. Ovomacroglobulin was purified by gel chromatography on Sepharose CL-6B. The two proteins had similar molecular weights and amino acid compositions, and both inhibited proteinases such as trypsin, chymotrypsin, papain, and thermolysin. The amino terminal sequences of the two proteins were homologous to each other but higher homologies were found between the ovomacroglobulin of turtle and chicken, and between the serum macroglobulins of the same animals. The functional difference between turtle alpha-macroglobulin and ovomacroglobulin became clear when they were treated with methylamine, which is known to destroy the inhibitory activity of human alpha 2-macroglobulin by splitting internal thiolester bonds. The inhibitory activity of the turtle plasma protein was completely destroyed by methylamine but that of ovomacroglobulin was only partially affected. The number of sulfhydryl groups as titrated with 5,5'-dithiobis(2-nitrobenzoate) before and after treatment with proteinases or methylamine was different for the two proteins. The amount of radioactive methylamine that was incorporated was also different between the two proteins. The two proteins purified in this study had no immunological cross-reactivity.

Amino Acid Sequence↗

PubMed1988

Conformational changes of alpha-macroglobulin and ovomacroglobulin from the green turtle (Chelonia mydas japonica).

Green turtle plasma alpha-macroglobulin and ovomacroglobulin underwent conformational changes when they were treated with proteinases or methylamine. Their conformational changes were studied by HPLC gel chromatography, circular dichroism, and electron microscopy. The Stokes radii of native green turtle alpha-macroglobulin and ovomacroglobulin were estimated to be 84.3 +/- 0.5 A, and 93.0 +/- 0.5 A, respectively, by means of an HPLC experiment. After reaction with methylamine or proteinases, the Stokes radius of alpha-macroglobulin changed to 83.0 +/- 0.5 A or 85.4 +/- 0.5 A, respectively, and that of ovomacroglobulin to 93.0 +/- 0.5 A or 87.1 +/- 0.5 A. The circular dichroic spectra of native alpha-macroglobulin and ovomacroglobulin exhibited a negative band at around 215 nm, indicating the presence of beta-structure. Reaction of the two macroglobulins with methylamine resulted in a slight decrease in the ellipticity and reaction with proteinases led to a slight increase. The electron micrographic images of native alpha-macroglobulin and ovomacroglobulin can be described as deformed rings for the former and rugby balls for the latter. A common characteristic feature of the two molecules was that the central parts of the molecules were only thinly occupied by subunit. After reaction of macroglobulins with proteinases, the void spaces became partially filled and their overall shape more rectangular. Methylamine treatment caused a structural change only in alpha-macroglobulin but not in ovomacroglobulin. The difference in the susceptibility of the macroglobulins to methylamine was taken as an indication of evolutional divergence of the two homologous proteins within the last 300 million years.

Animals↗

PubMed1988

Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties.

ilvE gene of Escherichia coli was inserted into the region downstream of the tac promotor. As a result, the branched-chain amino acid aminotransferase was overproduced by about a hundred-fold in E. coli W3110. The overproduced aminotransferase was purified from cell extracts about 40-fold to homogeneity. Chemical and physicochemical analyses confirmed that it was a product of the ilvE gene. The enzyme existed in a hexamer with a subunit molecular weight of 34,000; the double trimer model of the enzyme presumed by the previous chemical cross-linking experiments (Lee-Peng, F.-C. et al. (1979) J. bacteriol. 139, 339-345) was supported by electron micrographs. The circular dichroic (CD) spectrum of branch-chain amino acid aminotransferase had double negative maxima at 210 and 220 nm. The alpha-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm. The absorption spectrum of the enzyme showed two peaks at 330 and 410 nm. There was no pH-dependent spectral shift. The CD spectrum of the coenzyme, pyridoxal 5'-phosphate, had negative peaks at 330 and 410 nm. These spectral properties of branched-chain amino acid aminotransferase were quite different from those of E. coli aspartate aminotransferase. Each subunit bound approximately 1 mol of pyridoxal 5'-phosphate. A lysyl residue, which forms a Schiff base with the aldehyde group of the pyridoxal 5'-phosphate, was identified in the primary structure of the enzyme.

Amino Acid Sequence↗

PubMed1988

[The inhibitory effects of chicken ovomacroglobulin on collagenolytic activity in Bacteroides gingivalis culture supernatant, human PMN and human gingival crevicular fluid].

We examined in vitro the inhibitory effects of ovomacroglobulin on collagenolytic activity in Bacteroides gingivalis (B. gingivalis) culture supernatant, in human peripheral blood polymorphonuclear leucocytes (PMN), and in gingival crevicular fluid (GCF) from periodontitis patients. Measurement of collagenolytic activity was conducted with a CollagenoKit CLN-100 using FITC-conjugated type I collagen. The FITC-conjugated collagen was reacted with the sample in solution, and the residue was selectively degenerated at 35 degrees C and removed with ethanol. The fluorescence of the removed residue was then measured. The collagenolytic activity from B. gingivalis displayed dose dependent inhibition as high as 81.4% following addition of ovomacroglobulin at 224 micrograms/ml. The collagenolytic activity from human peripheral blood PMN showed, as a result of addition of 1,600 micrograms/ml of ovomacroglobulin, inhibition as high as 62.4%. The collagenolytic activity from human GCF, which was obtained from patients with different degrees of periodontal disease, exhibited as high as 71.0% inhibition after addition of 1,600 micrograms/ml ovomacroglobulin. Ovomacroglobulin showed almost the same level of inhibition obtained from alpha 2-macroglobulin, which was measured as a positive control. It was also recognized by SDS-PAGE that collagenolytic activity was inhibited after preincubation with added ovomacroglobulin. This collagenolytic activity, which dissolved the collagen substrate, was derived from B. gingivalis and human GCF. The above results demonstrate that ovomacroglobulin inhibits collagenolytic activity from B. gingivalis, human PMN, and human GCF.

Animals↗

PubMed1987

Murine T cell proliferation can be specifically augmented by macrophages fed with specific antigen: alpha-2-macroglobulin conjugate.

Foreign antigens conjugated to alpha-2-Macroglobulin (alpha-2-M) were effectively taken up by murine macrophages via alpha-2-M receptors. Such effective internalization of alpha-2-M:antigen conjugate by macrophages resulted in a remarkable increase in its ability to activate murine immune T cells under the following conditions. After macrophages were incubated with alpha-2-M:antigen conjugate or unconjugated antigen, they were cultured with immune T cells and antigen-stimulated tritiated thymidine incorporation by T cells was measured. The stimulation of T cell proliferative response by macrophages fed with the conjugate was sixteen times higher than what was observed with macrophages pretreated in the same concentration of unconjugated antigen. These findings suggest a physiological function of alpha-2-M and give us a new technique of immunization.

Animals↗