ABSTRACT
Guaiacol peroxidases [GP] are haem-containing enzymes participating in many physiological processes in plants. Theexpression pattern of these enzymes is organ-specific and developmentally regulated. The presence of GP activity in extract samples, prepared from Crocus sativus L. corms that were either dormant or rooting for 3, 6 and 10 days, was investigated. Kinetic studies revealed a significant similarity among GP activities detectable in the corm at different stages of development: in all extract samples, the activity was maximal at pH 7.5 and after preincubation at 30-40°C. When guaiacol was used as the varying substrate, Michaelis-Menten kinetics behavior was observed in all extract samples and resulted in similar KM values; catalytic efficiencies were also very similar. The corm GP activity was inhibited by cyanide, azide and ascorbate. The GP activities from different extract samples had the same sensitivities to azide, cyanide and ascorbate and the type of inhibition by azide and cyanide was competitive and uncompetitive, respectively, while ascorbate inhibited the GP activity non-competitively. Corm extract samples from different stages of rooting similarly responded to temperature treatment and a biphasic Arrhenius plot resulted for each extract sample studied. When dormant, 3-, 6- and 10-days-rooting corm extracts were submitted to non-denaturing polyacrylamide gel electrophoresis, the GP-specific activity staining revealed one band on the gel, with the same migrating distances. This finding in combination with kinetic studies demonstrated that at least one form of GP, with an apparent molecular weight of 68 kDa, was expressed during development of Crocus sativus L. corm
ABSTRACT
The effect of Triton X-100, Na cholate and Tween 80 on the solubilization of integral membrane proteins in intact cells, spheroplasts and inner-membrane fragments of Salmonella typhimurium was studied. The detergents were used in various concentrations [1.6 to 64 mM] and cytochromes b and d were used as marker to monitor the solubilization of membrane-bound proteins. Results showed that no inner-membrane protein solubilization was detected after the treatment of intact cells with detergents. The effect of Na cholate and Tween 80 on spheroplasts and inner-membrane fragments was also negligible in comparison to Triton X-100. Triton X-100 solubilized cytochromes from inner-membrane fragments more efficiently than from spheroplasts. The ratio of total protein solubilization to solubilize cytochromes showed that in spheroplasts this ratio was maximum at 1.6 mM Triton X-100 while it was maximum at 16-32 mM Triton X-100 in inner-membrane fragments. This difference between spheroplasts and inner-membrane fragments may be due to the orientation of the inner- membrane in spheroplasts [right side out] and inner-membrane fragments [in-side out as well as right side out], and to the presence of peripheral proteins attached to cytoplasmic membrane in spheroplasts