Purification and Characterization of a Chloroplast Outer-Envelope-Bound, ATP-Dependent Protein Kinase

An ATP-dependent protein kinase was partially purified from isolated outer envelope membranes of pea (Pisum sativum L., Progress No. 9) chloroplasts. The purified kinase had a molecular weight of 70 kilodaltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was of the cyclic nucleotide and Ca2+, calmodulin-independent type. The purification involved the detergent solubilization of purified outer envelopes by 0.5% cholate and 1% octylglycoside, followed by centrifugation on a linear 6 to 25% sucrose gradient. Active enzyme fractions were further purified by affinity chromatography on histone III-S Sepharose 4B and ion exchange chromatography on diethylaminoethyl cellulose. The protein kinase eluted at 100 millimolar and 50 millimolar NaCl, respectively. The protein kinase was essentially pure as judged by Western blot analysis. The enzyme has a KM of 450 micromolar for ATP and a Vmax of 25 picomoles of 32P incorporated into histone III-S per minute per microgram. Inhibition by ADP is competitive (Ki 150 micromolar)

Comparison of geranylgeranyl and phytyl substituted methylquinols in the tocopherol synthesis of spinach chloroplasts

Geranylgeranyl substituted methylquinols are shown to be precursors of tocopherol biosynthesis in spinach chloroplasts as well as phytyl substituted ones. The geranylgeranyl substituted quinols are methylated even to a greater extent than the phytyl substituted ones. The connection to the so far known biosynthetic origin of -tocopherol is probably -tocotrienol which is hydrogenated to γ-tocopherol and then further methylated to -tocopherol

Tocopherol and plastoquinone synthesis in spinach chloroplasts subfractions

Subfractions isolated from intact purified spinach chloroplasts are able to prenylate the aromatic moiety of -tocopherol and plastoquinone-9 precursors. The biosynthesis of -tocopherol and plastoquinone-9 is a compartmentalized process. The chloroplast envelope membranes are the only site of the enzymatic prenylation in -tocopherol synthesis whereas the thylakoid membrane is also involved in the prenylation and methylation sequence of plastoquinone-9 biosynthesis. A very active kinase which forms phytyl-PP is localized in the stroma. Phytol but not geranylgeraniol is the polyprenol precursor of the side chain of -tocopherol in spinach chloroplasts

Site of biosynthesis of a-tocopherol in spinach chloroplasts

The chloroplast envelope is a continuous boundary of two osmiophilic membranes and has an important role in galactolipid synthesis [ 11. It was interesting to determine whether the envelope is the site of synthesi