RESUMO
The two enzymes required to synthesize glutathione de novo have been purified from human erythrocytes. Glutamylcysteine synthetase was purified 4300-fold and was approximately 80% pure based on polyacrylamide gel electrophoresis. The purified enzyme catalyzes the formation of 30.5 mumoles of gamma-glutamyl-cysteine per mg of protein per hr and is inhibited by sulfhydryl inhibitors. Glutathione synthetase was purified 6000-fold from erythrocytes to homogeneity as determined by polyacrylamide gel electrophoresis. The erythrocyte enzyme has a molecular weight of 150,000 and catalyzes the formation of 35.9 mumoles of glutathione per mg of protein per hr. Comparison of the amino acid composition and some kinetic parameters of yeast glutathione synthetase and the erythrocyte enzyme demonstrate similarities between these enzymes.
Assuntos
Eritrócitos/metabolismo , Glutationa/biossíntese , Ligases/isolamento & purificação , Acetamidas/farmacologia , Aminoácidos/análise , Antimetabólitos/farmacologia , Bioensaio , Isótopos de Carbono , Catálise , Fenômenos Químicos , Físico-Química , Cromatografia , Cromatografia DEAE-Celulose , Cisteína/metabolismo , Eletroforese Descontínua , Etilmaleimida/farmacologia , Glutamatos/metabolismo , Glicina/metabolismo , Humanos , Ligases/análise , Compostos de Sulfidrila/antagonistas & inibidores , Trítio , UltracentrifugaçãoRESUMO
The two enzymes required for de novo glutathione synthesis, glutamyl cysteine synthetase and glutathione synthetase, have been demonstrated in hemolysates of human erythrocytes. Glutamyl cysteine synthetase requires glutamic acid, cysteine, adenosine triphosphate (ATP), and magnesium ions to form gamma-glutamyl cysteine. The activity of this enzyme in hemolysates from 25 normal subjects was 0.43+/-0.04 mumole glutamyl cysteine formed per g hemoglobin per min. Glutathione synthetase requires gamma-glutamyl cysteine, glycine, ATP, and magnesium ions to form glutathione. The activity of this enzyme in hemolysates from 25 normal subjects was 0.19+/-0.03 mumole glutathione formed per g hemoglobin per min. Glutathione synthetase also catalyzes an exchange reaction between glycine and glutathione, but this reaction is not significant under the conditions used for assay of hemolysates. The capacity for erythrocytes to synthesize glutathione exceeds the rate of glutathione turnover by 150-fold, indicating that there is considerable reserve capacity for glutathione synthesis. A patient with erythrocyte glutathione synthetase deficiency has been described. The inability of patients' extracts to synthesize glutathione is corrected by the addition of pure glutathione synthetase, indicating that there is no inhibitor in the patients' erythrocytes.
Assuntos
Trifosfato de Adenosina/metabolismo , Cisteína/metabolismoAssuntos
Pão , Alimentos Fortificados , Ferro/metabolismo , Adulto , Pão/análise , Proteínas Alimentares , Difosfatos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Absorção Intestinal , Ferro/administração & dosagem , Ferro/análise , Ferro/sangue , Isótopos de Ferro , Masculino , Pessoa de Meia-Idade , Oxirredução , Tamanho da Partícula , Fosfatos/metabolismo , Sódio/metabolismo , Relação Estrutura-Atividade , Sulfatos/metabolismo , Transferrina/metabolismo , Triticum/metabolismoRESUMO
Histidine-requiring mutants of Salmonella typhimurium that can be reverted to prototrophy by a variety of mutagens were used mutagenic activity in the urine of patients receiving chemotherapeutic agents. Patients given cyclophosphamide and BCNU had detectable urinary mutagenic activity over a 24-hour period, with maximal levels occurring 12 to 21 hours after drug injection. Whereas native cyclophosphamide required the presence of a rat liver extract to be mutagenic in the test system, the cyclophosphamide metabolites in the urine were fully active in the absence of added liver extract. Mutagenic activity was detected in only the first voided urine specimen of patients receiving fluorouracil. Patients receiving Adriamycin, methotrexate, Mitomycin C, and low doses or oral melphalan did not have detectable mutagenic activity in their urines. One thousand and ten random urine speciments were screened for mutagenic activity. Only eight had greater than 26 revertant colonies per plate. Four of the eight had received metronidazole (Flagyl) for vaginitis while two others had received chemotherapeutic drugs. We were unable to detect increased mutagenic metabolites in the urine of 43 patients with known malignancies, using the standard assay conditions.