Table 4

Autophosphorylation of R1 precedes the phosphotransfer to amylopectin

SampleAddition 43 min radioactivity, cpm
0 min30 min33 min
AR1 + [β-33P]ATP+ Nonlabeled ATP+ Amylopectin58, 63
+ Buffer28, 34
BR1+ [β-33P]ATP, + nonlabeled ATP+ Amylopectin4, 12
+ Buffer7, 0
  • R1 (5 μg) was preincubated with 0.15 μCi [β-33P]ATP (nonlabeled ATP was absent) in buffer C for 30 min in a total volume of 110 μl. Subsequently 10 μl of 0.1 M nonlabeled ATP was added and the suspension was incubated for a further 3 min. Fifty microliters each were then withdrawn and mixed with 5 mg of amylopectin dissolved in 450 μl of buffer C or in buffer C alone (A). Following incubation for 10 min the reaction was terminated by heating at 95°C for 15 min. The sample was divided and 230 μl each were filtered through microcon filter units (see Materials and Methods) and the radioactivity retained on the filter was counted. Another sample was treated identically; however, [β-33P]ATP was omitted during the preincubation and was instead simultaneously applied together with nonlabeled ATP (B). The background counts (scintillation fluid alone, 43 ± 7) were subtracted.