J. Biol. Chem., Vol. 265, Issue 4, 2173-2177, Feb, 1990
Differential proteolysis of the subunits of pyrophosphate-dependent 6- phosphofructo-1-phosphotransferase
HF Cheng and M Tao
Department of Biochemistry, University of Illinois College of Medicine, Chicago 60612.
Antibodies against the alpha (Mr 67,000) and beta (Mr 60,000) subunits of
wheat seedling Fru-2,6-P2-stimulated pyrophosphate-dependent 6-
phosphofructo-1-phosphotransferase (PFP) were used to probe the subunit
structures of several partially purified plant PFPs after tryptic
digestion. Antisera to the alpha and beta subunits of wheat seedling PFP
cross-reacted with the corresponding alpha and beta subunits of PFP
preparations from wheat germ, potato tubers, and lettuce leaves. With the
mung bean PFP, both antisera reacted with a protein band of Mr 60,000. A
protein band corresponding to the Mr 67,000 alpha subunit was not detected
in the mung bean PFP preparation. Tryptic digestion of wheat seedling and
potato tuber PFPs resulted in the preferential cleavage of the alpha
subunit. The trypsinized PFP retained most of its Fru-2,6-P2-stimulated
activity but not its basal activity. The proteolyzed enzyme also exhibited
a 2-fold increase in Ka for Fru-2,6- P2. Studies with the mung bean enzyme
revealed that the anti-alpha immunoreactive component was more sensitive to
trypsinization than the anti-beta immunoreactive component of the Mr 60,000
protein band. Thus, the Mr 60,000 protein band of the mung bean PFP appears
to be heterogeneous and contains both alpha and beta-like proteins. The
above observations indicate that the alpha and beta subunits of PFP are two
distinct polypeptides and that alpha acts as a regulatory protein in
regulating both the catalytic activity and the Fru-2,6-P2-binding affinity
of the beta subunit.
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