Partial resolution of the enzymes catalyzing photophosphorylation. XV. Approaches to the active site of coupling factor I.
1. Prolonged treatment of coupling factor I (CF1) from spinach chloroplasts with trypsin free of chymotrypsin yielded an active ATPase. The isolated preparation showed only two polypeptide chains (mol wt 55,000 to 60,000) on acrylamide gels run in the presence of sodium dodecyl sulfate. The three smaller subunits of CF1 were not detectable. The preparation no longer served as a coupling factor for photophosphorylation in either EDTA- or silicotungstate-treated chloroplasts. 2. An antiserum prepared against coupling factor I from chloroplasts inhibited the ATPase activity of the trypsin-treated CF1. In contrast, antisera prepared against the two individual (denatured) subunits did not inhibit the ATPase activity when tested either alone or together, although each interacted with the trypsin-treated protein, forming precipitin lines in Ouchterlony plates. 3. The trypsin-treated enzyme was still cold-labile, showing that the three smaller subunits are not required for this property. However, the enzyme was no longer sensitive to the natural inhibitor protein which is one of its subunits (subunit epislon), but was still sensitive to inhibition by the flavonoid quercetin. 4. Two equivalents of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole were sufficient to inhibit about 80% of the ATPase activity of the coupling factor, irrespective of whether it contained two of five subunits. The inhibition was completely reversed by dithiothreitol. 5. Triated 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was prepared. Treatment of the coupling factor with this tritium-labeled inhibitor followed by electrophoresis on acrylamide gels revealed that most of the radioactivity was incorporated into the beta subunit of the enzyme (molecular weight 56,000).
D000251:Adenosine Triphosphatases / Q000276:immunology / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D002118:Calcium / Q000494:pharmacology; D002736:Chloroplasts / Q000032:analysis / Q000187:drug effects / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005779:Immunodiffusion; D008970:Molecular Weight; D010080:Oxazoles / Q000494:pharmacology; D010785:Photophosphorylation / Q000187:drug effects; D010940:Plant Proteins / Q000302:isolation & purification*; D010944:Plants; D011794:Quercetin / Q000494:pharmacology; D014357:Trypsin; D014475:Uncoupling Agents / Q000494:pharmacology
D000596:Amino Acids; D010080:Oxazoles; D010940:Plant Proteins; D014475:Uncoupling Agents; D004492:Edetic Acid; D011794:Quercetin; D014357:Trypsin; D000251:Adenosine Triphosphatases; D002118:Calcium; D004229:Dithiothreitol