RESUMO
The energy bandgap of an insulator is large enough to prevent electron excitation and electrical conduction. But in addition to charge, an electron also has spin, and the collective motion of spin can propagate-and so transfer a signal-in some insulators. This motion is called a spin wave and is usually excited using magnetic fields. Here we show that a spin wave in an insulator can be generated and detected using spin-Hall effects, which enable the direct conversion of an electric signal into a spin wave, and its subsequent transmission through (and recovery from) an insulator over macroscopic distances. First, we show evidence for the transfer of spin angular momentum between an insulator magnet Y(3)Fe(5)O(12) and a platinum film. This transfer allows direct conversion of an electric current in the platinum film to a spin wave in the Y(3)Fe(5)O(12) via spin-Hall effects. Second, making use of the transfer in a Pt/Y(3)Fe(5)O(12)/Pt system, we demonstrate that an electric current in one metal film induces voltage in the other, far distant, metal film. Specifically, the applied electric current is converted into spin angular momentum owing to the spin-Hall effect in the first platinum film; the angular momentum is then carried by a spin wave in the insulating Y(3)Fe(5)O(12) layer; at the distant platinum film, the spin angular momentum of the spin wave is converted back to an electric voltage. This effect can be switched on and off using a magnetic field. Weak spin damping in Y(3)Fe(5)O(12) is responsible for its transparency for the transmission of spin angular momentum. This hybrid electrical transmission method potentially offers a means of innovative signal delivery in electrical circuits and devices.
RESUMO
Plastic surgeons require experience in supermicroscopic vascular anastomosis. Herein, we report a simple, rapid, and cost-effective training method using chicken wings and colored water. The avian ventral metacarpal artery was selected for dissection and anastomosis to mimic supermicrosurgery. Over 14 weeks (one anastomosis per day), the ulnar artery in 100 chicken wings was exposed by dissection, cut proximally, and injected with blue food dye-colored water by an inexperienced surgeon. After ligating the artery branches, it was cut and subjected to end-to-end anastomosis. Next, colored water was injected into the ulnar artery to check for suture sufficiency. The vessel was re-dissected to inspect the lumen and sutures qualitatively. Of the 100 wings, the first and last 20 wings' ventral metacarpal artery dissection, anastomosis times, and leakage frequency were compared. Avian ventral metacarpal artery diameter was recorded, and the cumulative anastomosis time where individual anastomosis times started decreasing was determined. Leakage rates before and after this point were compared. The avian ventral metacarpal artery diameter was 0.7-0.8 mm. The last 20 wings had significantly shorter median dissection times (12:27 vs. 17:45 min), anastomosis times (9:02 vs. 12:29 min), and leakage rates (15% vs. 70%); more even stitching and parallel ligature points; and less vessel layer inversion than the first 20 wings. After a cumulative anastomosis time of 10 h 26 min, individual times sharply decreased, and the leakage rate decreased significantly (58.3% vs. 23.8%). The proposed method significantly improved supermicrosurgical anastomosis. Thus, we believe that this method will help surgeons improve their supermicrosurgical skills.
Assuntos
Galinhas , Procedimentos Neurocirúrgicos , Animais , Procedimentos Neurocirúrgicos/métodos , Asas de Animais/irrigação sanguínea , Artéria Ulnar , Anastomose Cirúrgica/métodos , Microcirurgia/métodosRESUMO
Thermoelectric generation is an essential function in future energy-saving technologies. However, it has so far been an exclusive feature of electric conductors, a situation which limits its application; conduction electrons are often problematic in the thermal design of devices. Here we report electric voltage generation from heat flowing in an insulator. We reveal that, despite the absence of conduction electrons, the magnetic insulator LaY(2)Fe(5)O(12) can convert a heat flow into a spin voltage. Attached Pt films can then transform this spin voltage into an electric voltage as a result of the inverse spin Hall effect. The experimental results require us to introduce a thermally activated interface spin exchange between LaY(2)Fe(5)O(12) and Pt. Our findings extend the range of potential materials for thermoelectric applications and provide a crucial piece of information for understanding the physics of the spin Seebeck effect.
RESUMO
To elucidate the metabolic abnormality of musclar dystrophy, 27 kinds of enzyme activity in various organs of control and dystrophic mice were examined. The organs examined included muscle, bone, heart, testis, uterus, spleen, thymus, submaxillary gland, stomach, pancreas, liver, kidney, brain, and lung. The activities of 14 different aminopeptidases, 5 endopeptidases, 4 glycosidases, phosphatase, esterase, and ribonuclease were measured. Most of the enzyme activities were significantly elevated in muscles and bones of dystrophic mice. These organs were similar in their patterns of enzyme abnormality. Among the 14 kinds of aminopeptidase activity studied, the degree of increased activity was greater for the aminopeptidases (AP):Ala-AP, Leu-AP, Met-AP, Phe-AP, Trp-AP, Gly-Pro-Leu-AP. In addition to aminopeptidases, there were significant increases in activities of chymotrypsinlike enzyme, cathepsin C, cathepsin D, several glycosidases and neutral ribonuclease in the muscles of dystrophic mice. Similarly increased enzyme activity was also observed in organs other than muscle and bone. Furthermore, protein content in most organs was higher in dystrophic mice than in those of control mice. These abnormalities were seen in both males and females. The present results suggest that there are extensive abnormalities in the protein metabolism in dystrophic mice. It seems therefore that the therapeutic approach to muscular dystrophy should be studies not only from the well-known abnormality of intramuscular endopeptidases, but from other aspects as well.
Assuntos
Hidrolases/metabolismo , Músculos/enzimologia , Distrofia Muscular Animal/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Osso e Ossos/enzimologia , Esterases/metabolismo , Glicosídeo Hidrolases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monoéster Fosfórico Hidrolases/metabolismo , Ribonucleases/metabolismoRESUMO
Three benzenoid ansamycin antibiotics (herbimycin, macbecin, and geldanamycin) were found to reduce the intracellular phosphorylation of p60src at a permissive temperature (33 degrees C) in a rat kidney cell line infected with a temperature-sensitive mutant of Rous sarcoma virus. This effect was accompanied by morphological changes from the transformed to the normal phenotype. The filamentous staining pattern of actin fibers was observed in the cells treated with these antibiotics at 33 degrees C. Removal of the antibiotics allowed the cells to revert to the transformed morphology. Ansamitocin, another benzenoid ansamycin, and naphthalenoid ansamycins such as streptovaricin and rifamycins did not show this effect. Pulse-labeling of the antibiotic-treated cultures with 32Pi showed a marked reduction of 32P radioactivity incorporated into p60src. A parallel experiment with [35S]methionine showed that synthesis of p60src was slightly inhibited. The immune complex prepared by mixing the herbimycin-treated cell extracts with antibody against p60src was inactive in vitro in phosphorylating the complex itself. On the contrary, the immune complex derived from untreated cells was active in vitro even in the presence of the antibiotics. These results suggest that benzoquinonoid ansamycins have no direct effect on src kinase but destroy its intracellular environment, resulting in an irreversible alteration of p60src and loss of catalytic activity.
Assuntos
Antibacterianos/farmacologia , Transformação Celular Viral/efeitos dos fármacos , Proteínas dos Retroviridae/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Vírus do Sarcoma Aviário , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Lactamas Macrocíclicas , Proteína Oncogênica pp60(v-src) , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
The effect of six protease inhibitors, isolated from various species of actinomycetes, on focus formation by murine sarcoma virus was examined. Pepstatin was the only inhibitor. The treatment of cells with pepstatin at various times possibly retards the early stage of infection with murine sarcoma virus.
Assuntos
Transformação Celular Neoplásica , Gammaretrovirus/efeitos dos fármacos , Inibidores de Proteases , Vírus do Sarcoma Murino/efeitos dos fármacos , Animais , Carbamatos/farmacologia , Divisão Celular/efeitos dos fármacos , Inibição de Contato/efeitos dos fármacos , Dipeptídeos/farmacologia , Leupeptinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Oligopeptídeos/farmacologia , Compostos Organofosforados/farmacologia , Elastase Pancreática/farmacologia , Pepstatinas/farmacologia , Termolisina/antagonistas & inibidoresRESUMO
The effect of a microbial protease inhibitor, leupeptin, on the induction of urinary bladder tumors in W rats by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was examined. Three groups of animals were given 0.01% BBN in their drinking water for 12 weeks. A basal powder diet supplemented with 0.1% leupeptin was given to group A throughout the experiment and to group B when BBN administration was stopped. Group C was given the basal diet without leupeptin throughout the study. The total preservation period was 40 weeks. Results clearly showed that when leupeptin was given during the promotion step of bladder carcinogenesis (as in group B), it increased the size of tumors and the incidences of cancer and invasion. When leupeptin was given throughout the experiments, its effect was counteracted (as in group A).
Assuntos
Butilidroxibutilnitrosamina , Leupeptinas/farmacologia , Nitrosaminas , Oligopeptídeos/farmacologia , Neoplasias da Bexiga Urinária/induzido quimicamente , Animais , Butilidroxibutilnitrosamina/administração & dosagem , Interações Medicamentosas , Leupeptinas/administração & dosagem , Masculino , Neoplasias Experimentais/induzido quimicamente , Nitrosaminas/administração & dosagem , Inibidores de Proteases , Ratos , Neoplasias da Bexiga Urinária/ultraestruturaRESUMO
The 14C activity of [14C]bleomycin bound to DNA in bleomycin-sensitive rat ascites hepatoma cells (AH-66) was 8.7 times higher than in resistant cells (AH-66F) when the cells were incubated with [14C]bleomycin. The difference in permeability to bleomycin was not significant; uptake of [14C]bleomycin by the sensitive cells was only 1.2 times larger than that by the resistant cells, and the radioactivity incorporated into the nuclei of sensitive cells was only 1.3-fold greater. The bleomycin-inactivating enzyme level in the resistant cells was 3.5 times higher than in the sensitive cells, indicating that the antibiotic incorporated into the resistent cells was reduced in DNA-binding activity to a large extent. The level of protein-free thiol compound in the sensitive cells was 1.8-fold higher than in the resistant cells, suggesting a possible enhancement of bleomycin action by intracellular thiol compound as is found in vitro. These factors probably affect the DNA strand scission and the sensitivity of cells to this antibiotic. Binding of [14C]bleomycin to DNA in vitro was studied in the presence and the absence of dithiothreitol. A large portion of the radioactivity bound in the presence of dithiothreitol was unstable to acid, but the acid-resistant binding was also enhanced by this thiol compound.
Assuntos
Bleomicina/metabolismo , Carcinoma Hepatocelular/metabolismo , DNA de Neoplasias/metabolismo , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Fracionamento Celular , Núcleo Celular/análise , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração , DNA de Neoplasias/análise , DNA de Neoplasias/isolamento & purificação , Ditiotreitol/farmacologia , Neoplasias Hepáticas , Microssomos/metabolismo , Mitocôndrias/metabolismo , Peso Molecular , Ratos , Compostos de Sulfidrila/análiseRESUMO
The mechanism of antitumor action of oxanosine was studied using a strain of rat kidney cells infected with a mutant Rous sarcoma virus, the src gene of which was temperature sensitive. Oxanosine inhibited cell growth in vitro, as well as nucleic acid synthesis in these cells, 10 times more strongly at a permissive temperature (33 degrees C) than at a non-permissive temperature (39 degrees C). Protein synthesis was inhibited only slightly at either temperature. The inhibition of cell growth and nucleic acid synthesis was reversed by guanosine, GMP, and to a lesser extent by adenosine and inosine. Oxanosine inhibited the conversion of [14C]hypoxanthine to guanine nucleotides in cells and again in the same temperature-related fashion. The conversion to adenine nucleotides was not inhibited. Oxanosine-5'-monophosphate was found to be a potent nearly competitive inhibitor, with respect to IMP, of IMP dehydrogenase (EC 1.2.1.14; IMP:NAD+ oxidoreductase) isolated from cells grown either at 33 degrees C or at 39 degrees C; with the former and the latter enzyme preparations, KmS for IMP were 6.0 X 10(-6) M and 5.3 X 10(-6) M, respectively, while Kis for oxanosine-5'-monophosphate were 1-3 X 10(-6) M and 5.2 X 10(-6) M, as well.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Genes Virais/efeitos dos fármacos , Oncogenes , Animais , Vírus do Sarcoma Aviário/genética , Células Cultivadas , DNA/biossíntese , IMP Desidrogenase/antagonistas & inibidores , Rim/efeitos dos fármacos , Purinas/farmacologia , RNA/biossíntese , Ratos , Ribonucleosídeos/farmacologiaRESUMO
Spergualin exhibited a strong antitumor effect against L1210(IMC), a tumor cell line which has been maintained in BALB/c X DBA/2 F1 (hereafter called CD2F1) mice in the Institute of Microbial Chemistry. Mice inoculated i.p. with 10(5) cells of L1210(IMC) survived more than 60 days by daily i.p. administration of spergualin for 9 days at 5 mg/kg/day, which was started 1 day after the tumor inoculation. These cured mice rejected a second inoculation of 10(6) cells of L1210(IMC), but they did not reject the inoculation of 10(2) P388 cells. In Winn's tumor neutralization assay and in the 51Cr release assay, the T-cell fraction prepared from the spleens of the cured mice had higher cytotoxic activity against L1210(IMC) than whole spleen cells. The cytotoxic activity of spleen cells was diminished by treatment with anti-Thy-1.2 or anti-Lyt-2.1 antibody and complement. Therefore, the effector cells involved in the immunological rejection should be regarded as cytotoxic T-lymphocytes. The cytotoxic activity of these T-lymphocytes was measured during and after the spergualin administration for 9 days, and high activity was observed from 1 day after the final spergualin administration. The antitumor effect of spergualin against L1210(IMC) was much lower in T-cell-deficient athymic mice. These results suggest that cytotoxic T-lymphocytes are involved in the antitumor action of spergualin against L1210(IMC) in vivo.
Assuntos
Leucemia L1210/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Ciclofosfamida/uso terapêutico , Guanidinas/uso terapêutico , Leucemia L1210/tratamento farmacológico , Leucemia P388/tratamento farmacológico , Leucemia P388/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Nimustina , Compostos de Nitrosoureia/uso terapêuticoRESUMO
Formycin B inhibited growth of L5178Y mouse leukemia cells in concentrations of less than twice the concentration that inhibits cell proliferation at 50% by cytostasis; at higher concentrations (more than twice the 50% concentration mentioned), the cells were killed. In cells treated with the concentration that inhibits cell proliferation at 50%, the average cell volume did not change. The formycin B inhibitory effect on cell proliferation was reduced by coincubation with nicotinamide adenine diphosphate or adenosine. The biosyntheses of DNA,RNA, and protein in whole cells were sensitively inhibited by formycin B as checked by incorporation studies with radioactive precursors. In addition, the formation of polyadenosine diphosphoribose was reduced even with higher sensitivity; in particular the extent of adenosine diphosphate ribosylation of histone subfraction H1 was reduced. Formycin B has been shown to be an inhibitor for the polyadenosine diphosphoribose polymerase, isolated from oviduct nuclei of quails. Both the chromatin-bound and the soluble enzyme are inhibited competitively; the relative affinity (Ki/Km) of formycin B to the polyadenosine diphosphoribose polymerase is 1.5.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Formicinas/farmacologia , Leucemia Experimental/metabolismo , Açúcares de Nucleosídeo Difosfato/biossíntese , Poli Adenosina Difosfato Ribose/biossíntese , Adenosina/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatina/metabolismo , DNA de Neoplasias/biossíntese , Relação Dose-Resposta a Droga , Histonas/metabolismo , Camundongos , NAD/farmacologia , NAD+ Nucleosidase/metabolismo , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/biossínteseRESUMO
Activities of various hydrolytic enzymes were determined in rat organ homogenates and on the surface of cells from various sources, i.e., tumor cell strains, primary cultured cells, normal cells, and their transformants. Alanine, leucine, methionine, phenylalanine, and glycyl-proline aminopeptidases and esterase showed relatively high activities in all these organs and cells. In the kidney homogenate the aminopeptidase A activity was higher in other organs; i.e., the aminopeptidase A activity was lower than that of aminopeptidase B. Normal cells derived from kidneys showed the kidney-type pattern of amino-peptidases A and B on the surface of cells, whereas tumor cells from various origins were of another organ type. When cultured mouse fibroblast strain C3H2K and rat fibroblast strain 3Y1 cells were transformed by SV40 or by a ts A mutant and maintained at permissive temperature, aminopeptidase A activity was drastically decreased, and the ratio of aminopeptidase A to aminopeptidase B was reduced to the levels of tumor cells. If the ts A mutant-transformed cells were grown at the restrictive temperature, the ratio approached that of normal cells. In normal cells, however, cultivation at high or low temperature did not cause any change of the activities.
Assuntos
Aminopeptidases/metabolismo , Membrana Celular/enzimologia , Transformação Celular Neoplásica , Neoplasias Experimentais/enzimologia , Animais , Linhagem Celular , Cães , Feminino , Rim/enzimologia , Pulmão/enzimologia , Camundongos , Mutação , Ratos , Vírus 40 dos Símios/genética , TemperaturaRESUMO
The effect of ascorbate in reducing Adriamycin toxicity has been examined in mice and guinea pigs. Ascorbate had no effect on the antitumor activity of Adriamycin in mice inoculated with leukemia L1210, but it significantly prolonged the life of mice and guinea pigs treated with Adriamycin. Adriamycin elevated lipid peroxide levels in serum and liver, and ascorbate prevented the elevation. The significant prevention of Adriamycin-induced cardiomyopathy by ascorbate was proved by means of electron microscopy. The earliest alterations of dilation of the sarcoplasmic reticulum and transverse tubular system and the appearance of a large number of cytoplasmic fat droplets, which were seen in cardiac tissue from guinea pigs receiving Adriamycin, were apparently reduced in animals that were treated with ascorbate.
Assuntos
Ácido Ascórbico/uso terapêutico , Doxorrubicina/toxicidade , Animais , Medula Óssea/patologia , Doxorrubicina/antagonistas & inibidores , Doxorrubicina/uso terapêutico , Cobaias , Leucemia L1210/tratamento farmacológico , Leucemia L1210/patologia , Peróxidos Lipídicos/metabolismo , Camundongos , Miocárdio/patologia , Especificidade da EspécieRESUMO
The effect of N-bromosuccinimide on the enzymatic activity and the conformation of a Bacillus cereus beta-lactamase (penicillin amido-beta-lactamase EC 3.5.2.6) was studied. Incubation with 10 muM N-bromosuccinimide caused over 95% decrease of the enzymatic activity within 15 min. Spectrophotometric titration with N-bromosuccinimide showed that the reaction proceeded in two steps. The half-inactivated enzyme was prepared by the reaction with N-bromosuccinimide and its properties examined. Amino acid analysis showed that the half-inactivated enzyme contained one residue of tryptophan less while other amino acid contents were similar. Neither the molecular weight nor the mobility in disc electrophoresis was changed. However, the affinity to a cephalexin-CH-Sepharose column was increased, and the Km value for cloxacillin was one-third that of the native enzyme, although that for benzylpenicillin was similar. These results indicate that a tryptophan residue sensitive to N-bromosuccinimide is essential for the maintenance of the rigid conformation and that its oxidation alters the enzyme in a manner such that a substrate with a bulky group in its side chain can form an enzyme-substrate complex more easily. In the native enzyme, the value of (f(a))(eff) (Lehrer, S.S. (1971) Biochemistry 10, 3254-3263), did not vary significantly in the absence or the presence of cloxacillin. In contrast, in the half-inactivated enzyme the presence of cloxacillin affected the conformation such that over two thirds of the tryptophyl fluorescence were accessible for quenching by KI, although about half was accessible in the absence of cloxacillin.
Assuntos
Bacillus cereus/enzimologia , Bromosuccinimida , Penicilinase , Penicilinase/metabolismo , Succinimidas , Sítios de Ligação , Bromosuccinimida/farmacologia , Cromatografia de Afinidade , Cloxacilina , Inibidores Enzimáticos/farmacologia , Iodoacetatos/farmacologia , Cinética , Penicilinase/isolamento & purificação , Ligação Proteica , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Fatores de TempoRESUMO
The effect of negamycin and its derivatives on protein synthesis in an Escherichia coli cell-free system was determined. (1) Unlike tetracycline and streptomycin, negamycin did not inhibit codon-specific binding of aminoacyl-tRNA to ribosomes. (2) The miscoding activity of the genamycin family compounds was not parallel with the activity inhibiting the termination reaction. (3) Although negamycin strongly inhibited the overall termination reaction, it inhibited only slightly the two substeps involved; the formation of releasing factor 1 - U-A-[3H]G - ribosome complex and the peptidyl-transferase action (release of formylmethionine from the initiation complex by puromycin). (4) The termination reaction performed with ribosomes from either streptomycin- or kanamycin-resistant E. coli cells was sensitive to negamycin. These results indicate that the inhbitory effect of negamycin on the termination reaction is specific to negamycin and distinct from that of tetracycline and streptomycin.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Sítios de Ligação , Sistema Livre de Células , Cloranfenicol/farmacologia , Códon , Escherichia coli/metabolismo , Metionina/metabolismo , Fatores de Alongamento de Peptídeos , Fatores de Terminação de Peptídeos , Peptidil Transferases/metabolismo , Fenilalanina , Puromicina/farmacologia , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Estreptomicina/farmacologia , Tetraciclina/farmacologiaRESUMO
A specific enzyme for the liberation of N-terminal N-formylmethionine from N-formylmethionyl peptides was purified 4750-fold from rat liver by successive applications of (NH4)SO4 precipitation, DEAE-cellulose, N-formylbestatin-AH-Sepharose 4B and AH-Sepharose 4B chromatography followed by Sepharose CL-6B gel filtration. The molecular weight was determined by gel filtration on Sepharose CL-6B as 290 000 +/- 5000. This was suggested to be a tetramer consisting of a subunit which was shown by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis to have a 72 000 +/- 2000 molecular weight. The optimum pH of the enzyme was 7.8. Cd2+ and Hg2+ were highly toxic to the enzyme. Michaelis constants of N-formylmethionyl leucine and N-formylmethionine beta-naphthylamide were 0.03 and 0.2 mM, respectively.
Assuntos
Aminopeptidases/isolamento & purificação , Fígado/enzimologia , Animais , Cromatografia de Afinidade , Cinética , Masculino , Peso Molecular , RatosRESUMO
An enzyme catalyzing the reduction of leupeptin acid to leupeptin was partially purified from a cell extract of Streptomyces roseus MA839-A1, a leupeptin producer. The enzyme was tentatively named leupeptin acid reductase. The molecular weight was estimated to be 320,000 by chromatography on Sepharose 6B. The reductase eluted with leupeptin acid synthetase both in molecular sieve chromatography and in affinity chromatography. The main properties of the reductase were: (1) ATP and NADPH were required for activity. ATP could not be replaced by GTP, ADP or AMP. NADPH could not be replaced by NADH. (2) Michaelis constants for ATP and NaDPH were 4.2 . 10(-5) M and 1.3 10(-6) M, respectively. (3) The enzyme was inhibited by leupeptin, the reaction product, and antipain. Both inhibitors have an L-argininal residue at the C-terminal structure. (4) The enzyme did not catalyze the conversion of leupeptin to leupeptin acid. Leupeptin acid reductase and leupeptin acid synthetase were found in the 10,000 x g pellet of the cell homogenate. The reductase was not released as readily from the pellet as the synthetase either by washing or by repeated freeze-thawing. Synthesis of leupeptin from acetyl-CoA, L-lucine and L-arginine in vitro was accomplished by combining leucine acyltransferase and the enzyme complex consisting of leupeptin acid synthetase an leupeptin acid reductase.
Assuntos
Aldeído Oxirredutases/metabolismo , Leupeptinas/metabolismo , Oligopeptídeos/metabolismo , Trifosfato de Adenosina/farmacologia , Aldeído Oxirredutases/isolamento & purificação , Membrana Celular/enzimologia , Estabilidade de Medicamentos , Peso Molecular , NADP/farmacologia , Oxirredução , Peptídeo Sintases/metabolismo , Streptomyces/enzimologiaRESUMO
1. Amastatin, a specific inhibitor of aminopeptidase A (L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7), was linked to an agarose matrix and by this affinity chromatography aminopeptidase A of pig kidneys was purified as a single protein shown by acrylamide gel electrophoresis. 2. Aminopeptidase A which was purified 710-fold, hydrolyzed only acidic amino acid beta-naphthylamide. The optimum pH and the optimum temperature was 7.5 and 45-50 degrees C, respectively. 3. The molecular weight was approx. 300 000 as determined by Sephadex G-200 gel filtration. 4. The activity of aminopeptidase A was not affected by sulfhydryl agents, S-S dissociating agents and serine proteinase inhibitor, but was inhibited strongly by metal chelating agents, and enhanced by alkaline earth metals. 5. Amastatin inhibited aminopeptidase A in a competitive manner with L-glutamic acid beta-naphthylamide, and the Ki value was calculated to be 2.5 x 10(-7) M. The inhibitory effect of amastatin on aminopeptidase A was not reversed by addition of Ca2+.
Assuntos
Aminopeptidases/isolamento & purificação , Antibacterianos , Rim/enzimologia , Peptídeos , Aminopeptidases/antagonistas & inibidores , Animais , Cromatografia de Afinidade , Glutamil Aminopeptidase , Humanos , Oligopeptídeos , SuínosRESUMO
The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).
Assuntos
Encéfalo/enzimologia , Inibidores de Ciclo-Oxigenase , Ácidos Graxos/farmacologia , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Animais , Ácidos Graxos Insaturados/farmacologia , Técnicas In Vitro , Prostaglandina D2 , Prostaglandinas D/biossíntese , Prostaglandinas D/metabolismo , Ratos , SuínosRESUMO
Griseolutein acts as a bactericidal agent, its toxicity decreasing with increase in cell density. There is no evidence that griseolutein acts at a specific stage of cell cycle. Inhibition of incorporation of radioactive thymidine into acid-insoluble fraction of cells is marked and rapid, while inhibition of incorporation of uridine also takes place. Incorporation of 32Pi into the acid-insoluble fraction of cells is inhibited while the incorporation into the nucleotide pool is not. Concentration of any of the four deoxyribonucleoside triphosphates in griseolutein-treated cells are similar to or higher than those in untreated cells. No extensive degradation of cellualr DNA is caused by griseolutein. DNA synthesis in plasmolyzed cells in not inhibited by griseolutein.