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MATERIALS AND METHODSTobacco (Nicotiana tabacum L. cv Turkish Samsun), spinach (Spinacia oleracea L. cv Northland), and pea (Pisum sativum L. cv Feltham First) were grown from seed in a soil-vermiculite mixture in a glass house. Thylakoid membrane preparation from leaves and assay of total thylakoid protein kinase activity were as previously described (18, 19), except that the amount of labeled ATP in the assay was increased to 10 uCi.Assessment of specific phosphorylation of the LHCP apoproteins was accomplished by halting the kinase assay by adding a final concentration of 3% (w/v) SDS. The samples, each containing 6 ,ug of Chl, were then electrophoretically fractionated on a 10% (w/v) acrylamide gel (15). Following electrophoresis, the gels were dried and the LHCP apoproteins, two closely migrating phosphoproteins of Mr approximately 28,000, were located by autoradiography. The 32P-labeled LHCP bands were excised and the amount of phosphate incorporated into them was measured from Cerenkov radiation in a scintillation counter. Assessment of phosphorylation of individual thylakoid polypeptides was carried out using the assay conditions described above, except that the Chl concentration in each 0.1 ml incubation volume was increased to 50 ,ug and each contained 200 gCi of [j-32p] ATP. Incubations were terminated after 3 min with 3% (w/v) SDS, and duplicate samples were electrophoretically fractionated on gels containing 7.5 or 12.5% (w/v) acrylamide and processed as above. Use of these two acrylamide concentrations facilitated fractionation of high and low mol wt polypeptides, respectively.

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