Author(s): Miles, M. A ; Lanham, Sheila M ; Souza, Adelson Alcimar Almeida de ; P?voa, Marinete Marins
Date: 2016
Origin: Oasisbr
Subject(s): Trypanosoma cruzi / classifica??o; Trypanosoma cruzi / enzimologia; Trypanosoma cruzi / isolamento & purifica??o
Minist?rio da Sa?de. Funda??o Servi?os de Sa?de P?blica. Instituto Evandro Chagas. Bel?m, PA, Brasil / London School of Hygiene and Tropical Medicine. Departament of Medical Protozoology. Keppel Street, London.
London School of Hygiene and Tropical Medicine. Departament of Medical Protozoology. Keppel Street, London.
Minist?rio da Sa?de. Funda??o Servi?os de Sa?de P?blica. Instituto Evandro Chagas. Bel?m, PA, Brasil
Minist?rio da Sa?de. Funda??o Servi?os de Sa?de P?blica. Instituto Evandro Chagas. Bel?m, PA, Brasil
Starch-gel electrophoresis of 38 enzymes was attempted with extracts of Trypanosoma cruzi culture forms. 18 of the enzymes that gave discrete electrophoretic bands were selected for routine characterization of T. cruzi stocks; the enzymes were: aspartate aminotransferase (E.C.2.6.1.1, ASAT); alanine aminotransferase (E.C.2.6.1.2, ALAT); phosphoglucomutase (E.C.2.7.5.1, PGM); glucosephosphate isomerase (E.C.5.3.1.9, GPI); malate dehydrogenase (oxaloacetate decarboxylating) (NADP+) (E.C.l.l.l.40, ME); glucose 6- phosphate dehydrogenase (E.C.l.l.l.49, G6PD); malate dehydrogenase (E.C.l.l.l.37, MDH); aconitate hydratase (E.C.4.2.1.3, ACON); isocitrate dehydrogenase (NADP+) (E.C.l.l.l.42, ICD); alcohol dehydrogenase (NADP+) (E.C.l.l.l.2, ADH); lactate dehydrogenase (E.C.l.l.l.27,LDH); aminopeptidase (cytosol) (E.C.3.4.11.1, PEP); pyruvate kinase (E.C.2.7.1.40, PK); phosphoglycerate kinase (E.C.2.7.2.3, PGK); enolase (E.C.4.2.1.11, ENO); hexokinase (E.C.2.7.1.1, HK); mannosephosphate isomerase (E.C.5.3.1.8, MPI); and glutamate dehydrogenase (E.C.l.4.1.2, GD). ADH (NADP+) in the genus Trypanosoma, and PGK, MPI and ENO, in T. cruzi, were apparently demonstrated for the first time. Between six and 18 enzymes were used to characterize more than 250 T. cruzi stocks, newly isolated from a wide range of sources in northern and central Brazil. Ali stocks were identified as belonging to T. cruzi zymodemes 1, 2 or 3, as originally defined-that is, by combination of electrophoretic patterns of ASAT, ALAT, PGM, GPI, ME and G6PD. The composite range of results with ali enzymes confirmed the presence of three principal T. cruzi zymodemes, but some enzymic characters overlapped between zymodemes and others suggested subgroups within individual zymodemes. Seven (MDH, ACON, LDH, PK, PGK, ENO, HK) of the 18 enzymes did not distinguish the three zymodemes; tive (ASA T, PGM, GPI, ICD, PEP) distinguished ali three zymodemes; 10 (ASAT, ALAT, PGM, GPI, ME, G6PD, ICD, ADH, PEP, GD) distinguished zymodemes 1 and 2, of which seven plus MPI and eight plus MPI separated zymodemes 1 from 3 and 2 from 3 respectively. T. cruzi stocks were taken from a smali area of the natural species distribution; the fuli range of enzymic characters within the species T. cruzi is expected to be far more complex. The epidemiological distribution of the zymodemes continued to accord with local transmission cycles and supported the hypothesis that distinct T. cruzi strains might be responsible for the enigmatic distribution of chronic Chagas's disease. Some of the difficulties in the empirical selection of new electrophoretic methods and the interpretation of results were presented, and the present and prospective significance of T. cruzi enzymic characters was discussed. Until the stability and genetic basis of T. cruzi enzymic characters are better understood it is recommended that isoenzymic profiles be confirmed routinely, botIl before and after stocks are used experimentally, as representative of a given zymodeme. A multiple biochemical approach to T. cruzi strain identification is recommended, using characters suitable for a numerical taxonomy.
Electroforese em gel de amido de 38 enzimas foi tentada com extratos de formas de cultura de T. cruzi. Dezoito das enzimas, as quais deram discretas manchas electrofor?ticas, foram selecionadas para carcateriza??o de rotina dos stocks de T. cruzi; as enzimas foram asparato aminotransferase (E.C. 2.6.1.1, ASAT); alanina aminotransferase (E.C. 2.6.1.2, ALAT); fosfoglucomutase (E.C.2.7.5.1, PGM); glicosefosfato isomerase (E.C.5.3.1.9, GPI); malato dehidrogenase (oxaloacetato descarboxilando (NADP+) (E.C.l.l.l.40, ME); glicose 6-fosfato dehidrogenase (E.C.l.l.l.49, G6PD); malate dehidrogenase (E.C.l.l.l.37, MDH); aconitato hidratase (E.C.4.2.1.3, ACON); isocitrato dehidrogenase (NADP+) (E.C.l.l.l.42, ICD); alcool dehidrogenase (NADP+) (E.C.l.l.l.2, ADH); lactato dehidrogenase (E.C.l.l.l.27, LDH); aminopeptidase (citosol) (E.C.3.4.11.1, PEP); piruvato quinase (E.C.2.7.1.40, PK); fosfoglicerato quinase (E.C.2.7.2.3, PGK); enolase (E.C.4.2.1.11, ENO); hexoquinase (E.C.2.7.1.1, HK); manosefosfato isomerase (E.C.5.3.1.8, MPI); e glutamato dehidrogenase (E.C.I.4.1.2, GD). ADH (NADP+), no genero Trypanosoma e MPI, PGK e ENO, em T. cruzi, foram demonstradas pela primeira vez. Entre 6 e 18 enzimas foram usadas para caracterizar mais de 250 stocks de T. cruzi, recentemente isoladas de varias origens nas partes norte e central do Brasil. Todos os stocks foram identificados como pertencentes aos zymodemes de T. cruzi I, 2 ou 3, como originalmente definidosisto ?, pela combina??o de padr?es electrofor?ticos de ASAT, ALAT, PGM, GPI, ME e G6PD. A variabilidade de resultados combinados com todas as enzimas confumaram a presen?a de 3 zymodemes principais de T. cruzi mas alguns caracteres enzimaticos sobrepostos entre os zymodemes, e outros sugeriram subgrupos dentro de zymodemes individuais. Sete (MDH, ACON, LDH, PK, PGK, ENO, HK) das 18 enzimas n?o distinguiram os 3 zymodemes; 5 (ASAT, PGM, GPI, ICD, PEP) distinguiram todos os 3 zymodemes; 10 (ASA T , ALAT, PGM, GPI, ME, G6PD, ICD, ADH, PEP, GD) distinguiram zymodemes 1 e 2; dos quais 7 mais MPI e 8 mais MPI separaram zymodemes 1 de 3 e 2 de 3 respectivamente. Stocks de T. cruzi foram trazidos de uma pequena ?rea de distribui??o natural da esp?cie; ? considerado que a total variedade dos caracteres enzim?ticos dentro das esp?cies de T. cruzi sera muito mais complexa. A distribui??o epidemiol6gica dos zymodemes continuou em acordo com os ciclos de transmiss?o local e refor?ou a hipotese que amostras distintas de T. cruzi poderiam ser responsaveis pela distribui??o enigm?tica de Doen?a de Chagas cr?nica. Algumas dificuldades na sele??o empirica de novos m?todos eletrofor?ticos e a interpreta??o dos resultados foram descritos, e o valor, presente e prospectivo, de caracteres enzim?ticos de T. cruzi foi discutido. A stabilidade e base gen?tica de caracteres enzim?ticos de T. cruzi n?o s?o completemente entendidos ent?o ? recomendade que perfis isoenzimaticos sejam confirmados rotineiramente, ambos antes e depois dos stocks serem usados experimentalmente, como representativo de um dado zymodeme. Uma multipla proximidade bioquimica para identifica??o de amostras de T. cruzi ? recomendada, usando caracteres apropriados para uma taxonomia num?rica.
