Measurement of Heme Efflux and Heme Content in Isolated Developing Chloroplasts

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Abstract
Hemes destined for cytosolic hemoproteins must originate in one of the cellular compartments which have the capacity for heme synthesis, namely the chloroplast or the mitochondria. Since developing chloroplasts from greening cucumber (Cucumis sativus, cv. Sumter) cotyledons are known to contain complete heme and chlorophyll biosynthetic pathways, they were tested for their capacity export hemes. Picomole quantities of heme were measured by reconstitution of the heme with apo-peroxidase and subsequent determination of peroxidase activity. The assay method was sensitive (as little as 0.7 picomole of heme could be detected in a volume of 100 microliters) and was linear with heme concentration. When intact plastids were incubated with apo-peroxidase, a steady-state rate of efflux between 0.12 and 0.45 picomole heme/minute/milligram plastid protein was measured. The efflux rate was not due to plastid breakage and could be enhanced by incubating with the heme precursor, delta-aminolevulinic acid. Cold acetone extraction removed 47 +/- 17 picomoles heme/milligram plastid protein from the total b-type heme pool in the chloroplasts (166 +/- 9 picomoles heme/milligram protein, by acid-acetone extraction). The reconstitution technique provided a similar estimate of readily exchangeable heme in the plastid, 37 +/- 8 picomoles heme/milligram protein (or 6 micromolar in the plastids). These values may be indicative of a ;free heme pool' which exists in the chloroplast.

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