The covalent attachment of polyamines to proteins in plant mitochondria

Plant mitochondria from both potato end mung bean incorporated radioactivit y into acid insoluble material when incubated with labelled polyamines (spe rmine, spermidine and putrescine). Extensive washing of mitochondrial preci pitates with trichloroncetic acid and the excess of cold polyamine failed t o remove bound radioactivity. Addition of nonradioactive polyamine stopped further incorporation of radioactivity but did net release radioactivity al ready bound. The radioactivity is incorporated into the membrane fraction, The labelling process has all the features of an enzymatic reaction: it is long lasting with distinctive kinetics peculiar to each polyamine, it is te mperature dependent and is affected by N-ethylmaleimide, The latter inhibit s the incorporation of putrescine but stimulates the incorporation of sperm ine and spermidine. Treatment of prelabelled mitochondria with pepsin relea ses bound radioactivity thus indicating protein to be the ligand for the at tachment of polyamines. HPLC of mitochondrial hydrolysates revealed that th e radioactivity bound to mitochondria is polyamines; traces of acetyl polya mines were also found in some samples. On autoradiograms of SDS/PAGE gels s everal radioactive bands of proteins were detected. Protein sequencing of l abelled spots from a 2D gel gave a sequence which was 60% identical to cata lase. We suggest that the attachment of polyamines to mitochondrial protein s occurs cotranslationally possibly via transglutaminases.