Properties of enzyme activities involved in protein phosphorylatino-dephosphorylation of the thyroid plasma membranes

Abstract

Bovine thyroid tissue exhibited cAMP-dependent and Ca 2+-dependent protien kinase activities as well as a basal (cAMP- and Ca 2+-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kiniase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent K m values of cAMP and Ca 2+ for the membrane-bound protein kinase were 5·10 −8 M and 8.3·10 −4M (in the presence of 1 mM EGTA), respectively. The apparent K m values of Mg 2+ were 7·10 −4 M (without cAMP and Ca 2+, 5·10 −4 M (with cAMP) and 1.3·10 −3 M (with Ca 2+), and those ATP were 3.5·10 −5 M (with or without cAMP) and 8.5·10 −5 M (with Ca 2+). The Ca 2+-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using 32P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca 2+, but was sitmulated by a rather broad range (5–25 mM) of Mg 2+ and Mn 2+. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca 2+-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.