Differential scanning calorimetry of chloroplast membranes

Karen Ann Smith, Purdue University

Abstract

Differential Scanning Calorimetry (DCS) was employed to investigate chloroplast membrane structure. DSC of chloroplasts suspended in a high ionic strength phosphate-buffered medium gave rise to six endotherms between 40-90$\sp\circ$C, centered at the following temperatures: A (42.0), B (56.5), C (69.5), D (76.5), E (82.7), and F (88.7). When these chloroplasts were suspended in low ionic strength HEPES/sucrose buffer, the C transition was split into two separate transitions, C$\sb1$ and C$\sb2$, and the B transition was shifted to higher temperatures so that it appeared as a shoulder preceding the C$\sb1$ endotherm. In this case, the chloroplast transitions occurred at: A (42.5), B (60.6), C$\sb1$ (64.9), C$\sb2$ (69.6), D (75.8), E (84.3), and F (88.9). Each of these transitions arises from the denaturation of protein complexes in the thylakoid membrane. The sources of the B, C$\sb1$, and D transitions were determined and much experimental evidence was obtained to establish their identities. The B transition was found to originate from the denaturation of the photosystem II reaction center complex. The C$\sb1$ endotherm was demonstrated to derive from the soluble subunit complex of the chloroplast coupling factor. The light harvesting complex of photosystem II was determined to be the source of the D transition. Furthermore, preliminary evidence indicated that ribulose 1,5 bisphosphate carboxylase may be the origin of the C$\sb2$ transition. In an additional study, the effects of photosynthetic inhibitors on the calorimetric transitions of chloroplasts were analyzed. The inhibitors examined were hydroxylamine, allethrin, DCMU, atrazine, and DBMIB. It was found that these inhibitors were unspecific in their action on the thylakoid membrane in that they altered more than one membrane protein complex as seen by DSC.

Degree

Ph.D.

Advisors

Low, Purdue University.

Subject Area

Biochemistry

COinS