Spinach plastocyanin and turnip cytochrome f have been covalently linked by using a water-soluble carbodiimide to yield an adduct of the two proteins. The redox potential of cytochrome f in the adduct was shifted by -20 mV relative to that of free cytochrome f, while the redox potential of plastocyanin in the adduct was the same as that of free plastocyanin. Solvent perturbation studies showed the degree of heme exposure in the adduct to be less than in free cytochrome f, indicating that plastocyanin was linked in such a way as to bury the exposed heme edge. Small changes were also observed when the resonance Raman spectrum of the adduct was compared to that of free cytochrome f. The adduct was incapable of interacting with or donating electrons to photosystem I. Peptide mapping and sequencing studies revealed two sites of linkage between the two proteins. In one site of linkage, Asp-44 of plastocyanin is covalently linked to Lys-187 of cytochrome f. This represents the first identification of a group on cytochrome f that is involved in the interaction with plastocyanin. The other site of linkage involves Glu-59 and/or Glu-60 of plastocyanin to as yet unidentified amino groups on cytochrome f. Euglena cytochrome c-552 could also be covalently linked to turnip cytochrome f, although with a lower efficiency than spinach plastocyanin. In contrast, a variety of cyanobacterial cytochrome c-553's and a cyanobacterial plastocyanin could not be covalently linked to turnip cytochrome f.
The enzymatic activities of sn-glycerol-3-phosphate acyltransferase and lysophosphatidate acyltransferase were investigated in microsomal fractions prepared from MAC-T cells from bovine mammary gland and from FTO-2B cells from rat liver. In both cell lines, sn-glycerol-3-phosphate acyltransferase exhibited similar rates of palmitate and oleate incorporation. However, lysophosphatidate acyltransferase activity in MAC-T cells had a 2.8-fold greater rate of palmitate incorporation than of oleate incorporation. In FTO-2B cells, there was a 1.4-fold greater rate of oleate incorporation than of palmitate incorporation. FTO-2B and MAC-T cells displayed acyltransferase activities that were consistent with liver and mammary tissues, respectively. The acyltransferases were examined from FTO-2B and MAC-T cells that were treated with insulin and prolactin. Insulin suppressed both acyltransferase activities in FTO-2B cells, and prolactin had a stimulatory effect; however, these effects were very small. In contrast, insulin and prolactin had a stimulatory effect on both acyltransferase activities in MAC-T cells; prolactin elicited the largest effect. Treatment of MAC-T cells with cycloheximide inhibited the stimulatory effect of prolactin on acyltransferase activities.
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