) Fig. 3 Separation of membranes in a spinach leaf homogenate. Open circles (top curve)—chlorophyll absorption at 655 nm/mg dry weight; solid circles (top curve)—plastoquinone (labeled as Q254), mg/g dry weight; solid circles (bottom curve)—coenzyme Q (labeled as Q275), 10 mg/g dry weight. Open circles (bottom curve)—succinic dehydrogenase (S.D.) mmole × 100/min × mg dry weight. This experiment indicated that Q254 could function in photosynthesis. (After Crane 1959a) Further definition of a role in photosynthesis would wait for study of PQ oxidoreduction function in chloroplasts

since our focus in David Green’s laboratory at the Enzyme Institute in Madison, Wisconsin, was a study of energy conversion in heart. Our first functional studies involved testing if Q254 acted like coenzyme Q in mitochondrial NVP-HSP990 manufacturer electron transport. In these extraction studies, we used isooctane

as the solvent which was a mistake since we knew that it gave rather non-specific restoration of succinoxidase and induced a requirement for phospholipid and neutral lipids. After all, when Donaldson et al. (1958) reported tocopherol restoration of DPNH oxidase after isooctane extraction, I wrote to warn him that the effect was unspecific since beef serum albumin also worked. In the isooctane procedure, Q254 often gave some restoration of succinate oxidase. The complications of isooctane extraction are illustrated in Crane (1959b, 1960). We used isooctane because we could purchase a check details spectral pure grade chemical with no impurities to interfere with the UV Ureohydrolase spectrum. Amesz (1977) has

discussed the problems involved with solvent extraction. After switching to acetone extraction in which Q254 did not replace coenzyme Q (Ambe and Crane 1960), we concluded that Q254 did not belong in the coenzyme Q group, contrary to our earlier conclusion (Crane 1959b). To our delight, David Green was very tolerant of our further study of Q254 even after it was clear that it was not involved in mitochondrial energy coupling. One day I had a big separatory funnel full of spinach extract on my bench. David came in and said ‘Oh! Cytochrome oxidase’. When I said ‘no it is spinach lipids’, he turned and stomped out. I think he was quite happy when Q254 fitted into the general concept of quinones in energy coupling. Fortunately, studies of solvent extraction of chloroplasts were done with heptane or petroleum ether, and the re-addition was mostly done by the evaporation technique. Lynch and AR-13324 cell line French (1957) had earlier used this procedure to extract carotene which restored dye photoreduction when added back. Bishop (1958) took up this extraction approach and found that the extract restored activity but purified carotene was inactive. Instead, he found that Vitamins K3 and K5 were effective. Later examination of the extract showed that no Vitamin K was present even though biological assay showed as if Vitamin K was present.