CHEMICAL MODIFICATION OF THE DICYCLOHEXYLCARBODIIMIDE REACTIVE M(R) 8000 SUBUNIT OF THE ENERGY COUPLING COMPLEX IN CHLOROPLASTS AS A PROBE FOR INTRAMEMBRANE PROTON MOVEMENT

NORMAN EDWARD TANDY, Purdue University

Abstract

The reaction of a protein from the chloroplast thylakoid membrane with acetic anhydride was studied. When illuminated the thylakoid membranes decreased in reactivity with acetic anhydride by about 20% or 14 nmol per mg of protein. One protein that decreased in reactivity to acetic anhydride had a molecular weight of about 8000. This protein was shown to be the M(,r) 8000 dicyclohexylcarbodiimide (DCCD) reactive subunit of the energy coupling complex, the subunit that is believed to be involved in proton translocation. This thesis is directed towards identifying, purifying and characterizing the M(,r) 8000 protein which shows the decrease in reactivity. The M(,r) 8000 subunit was isolated by reversed phase HPLC on Whatman Partisil 10 ODS. The protein thus purified was shown by {('14)C} DCCD labeling, amino acid composition and partial amino acid sequence to be the M(,r) 8000 CF(,o) subunit. The purified protein, from chloroplasts treated with {('3)H} acetic anhydride, was shown to have a light dependent decrease in anhydride derivatization of approximately 20%. Using these techniques and chromatography on Sephadex G-50, the acetic anhydride labeling was shown to occur only at lysine 48 of the M(,r) 8000 subunit. The fragments from CNBr cleaving the purified, {('3)H} acetyl labeled M(,r) 8000 CF(,o) subunit were separated on Sephadex G-50. The labeled fragment had only one reactive residue, lys 48. The complete inhibition of phosphorylation by DCCD requires only one out of six M(,r) 8000 subunits to be labeled at Glu 65. Kinetic and binding evidence suggested this one subunit was unique in its reactivity to DCCD. To determine the relationship between this DCCD inhibition site and the decrease in reactivity to acetic anhydride, chloroplasts were labeled with {('3)H} acetic anhydride in the light and dark, and then with {('14)C} DCCD at the minimum level for complete inhibition. The DCCD labeled subunits were separated from those that were not DCCD labeled and the location of the decrease in reactivity determined. The conclusion was reached that the light-dependent decrease in acetic anhydride reactivity occurs in the M(,r) 8000 CF(,o) subunits with the unique DCCD binding site, but it could not be determined if the decrease was only in these subunits.

Degree

Ph.D.

Subject Area

Biochemistry

COinS