Properties of enzyme activities involved in protein phosphorylation-dephosphorylation of thyroid plasma membranes.
Bovine thyroid tissue exhibited cAMP-dependent and Ca2+-dependent protein kinase activities as well as a basal (cAMP- and Ca2+-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kinase 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 Km values of cAMP and Ca2+ for the membrane-bound protein kinase were 5 . 10(-8) M and 8.3 . 10(-4) M in the presence of 1 Mm EGTA), respectively. The apparent Km values of Mg2+ were 7.10-4M (without (in the cAMP and Ca2+), 5 . 10(-4) M (with cAMP) and 1.3 . 10(-3) M (with Ca2+), and those of ATP were 3.5 . 10(-5)M (with or without cAMP) and 8.5 . 10(-5) M (with Ca2+). The Ca2+-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 Ca2+, but was stimulated by a rather broad range (5-25 mM) of Mg2+ and Mn2+. 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 Ca2+-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.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013961:Thyroid Gland / Q000201:enzymology*
D008565:Membrane Proteins; D000242:Cyclic AMP; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D002118:Calcium