Assays for the activities of polyamine biosynthetic enzymes using intact tissues

• Published in: Plant physiology and biochemistry 1999-07, Vol.37 (7-8), p.597-603
• Main Authors: Minocha, Rakesh, Long, Stephanie, Maki, Hisae, Minocha, Subhash C.
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADC; Agronomy. Soil science and plant productions; ARBOLES FORESTALES; ARBRE FORESTIER; ARGININA; ARGININE; Biological and medical sciences; BIOSINTESIS; BIOSYNTHESE; BIOSYNTHESIS; carrot; CELL CULTURE; CULTIVO DE CELULAS; CULTURE DE CELLULE; DAUCUS CAROTA; Economic plant physiology; enzyme assays; Enzymes; ENZYMIC ACTIVITY; FOREST TREES; Fundamental and applied biological sciences. Psychology; General aspects, methods; HORTALIZAS (PLANTAS); LIASAS; LYASE; LYASES; METHIONINE; METIONINA; Nutrition. Photosynthesis. Respiration. Metabolism; ODC; ORNITHINE; ORNITINA; PICEA RUBENS; Plant physiology and development; PLANTE LEGUMIERE; POLIAMINAS; POLYAMINE; POLYAMINES; POPULUS NIGRA; SAMDC; trees; VEGETABLE CROPS
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/S0981-9428(00)80112-6

Traditionally, most enzyme assays utilize homogenized cell extracts with or without dialysis. Homogenization and centrifugation of large numbers of samples for screening of mutants and transgenic cell lines is quite cumbersome and generally requires sufficiently large amounts (hundreds of milligrams) of tissue. However, in situations where the tissue is available in small quantities, or one needs to study changes in enzyme activities during development (e.g. somatic embryogenesis), it is desirable to have rapid and reproducible assay methods that utilize only a few milligrams of tissue and can be conducted without homogenization. Here, we report a procedure for the measurement of enzyme activities of the three key decarboxylases involved in polyamine biosynthesis utilizing small quantities of plant tissue without the homogenization and centrifugation steps. Suspension cultures of red spruce (Picea rubens (Sarg.)), hybrid poplar (Populus nigra × maximowiczii), and wild carrot (Daucus carota) were used directly to measure decarboxylation of ornithine, arginine and S-adenosylmethionine. Our results demonstrate that this procedure can be used to quantify the activities of arginine decarboxylase (EC 4.1.1.19), ornithine decarboxylase (EC 4.1.1.17) and S-adenosylmethionine decarboxylase (EC 4.1.1.50) in a manner quite comparable to the traditional assays for these enzymes that involve laborious steps of homogenization and centrifugation.