Kinetic and proteolytic identification of heat-induced conformational changes in the urea herbicide binding site of isolated Phaseolus vulgaris chloroplast thylakoids |
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Authors: | Steven C Wiest |
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Affiliation: | Dept of Horticulture, Kansas State Univ., Manhattan, KS 66506, USA. |
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Abstract: | Kinetic parameters of 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU)-induced inhibition of electron transport in chloroplast thylakoids isolated from Phaseolus vulgaris L. cv. Oregon 1604 were determined from analysis of a convergent, parallel electrical circuit. Through this analogue, the apparent affinity of the purported binding site for DCMU (K1) and the relative amount of DCMU-insensitive electron transport (vmax1/vo) were obtained using a reiterative non-linear least squares curve-fitting procedure. Exposure of thylakoids to heat caused a gradual increase in K1 (or decrease in the affinity of the thylakoid for DCMU) with an apparent activation energy of 134 kJ mol?1. Tryptic susceptibility of a protein region regulating K1 also decreased gradually with exposure to 45°C, suggesting that the heat-induced increase in K1 might be due to a protein conformational change. On the other hand, thylakoid exposure to 45°C resulted in a rapid (<5 min) irreversible increase in vmaxI/vo, which was also the apparent result of a conformational change in a region of the protein which regulates this function. These results are suggestive of the existence of differential thermal sensitivities of proteins within the thylakoids and, perhaps, of different regions within a single membrane protein. |
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Keywords: | DCMU binding site high temperature injury membrane protein conformation trypsin |
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