Interaction of the 56,000-dalton phosphoprotein phosphatase from reticulocytes with regulin and inhibitor 2.
The interaction of divalent metal ions with a homogeneous 56,000-dalton phosphoprotein phosphatase isolated from rabbit reticulocytes was studied. The effects of the ions on enzymatic activity and on fluorescence from a 3-(4-maleimidylphenyl)-4-methyl-7-(diethylamino)coumarin derivative of the protein were compared. Enzymatic activity is dependent on Mn2+. The apparent association constant for Mn2+ is about 0.5 mM-1 as judged from enzymatic activity and from changes in fluorescence caused by binding of the metal ion; Ca2+ and Mg2+ do not affect enzymatic activity and appear not to bind tightly to the enzyme; however, Co2+, Fe2+, and Zn2+ bind to the protein and inhibit the Mn2+-activated enzyme. The 56,000-dalton phosphoprotein phosphatase was found to interact with regulin, a spectrin-associated protein also isolated from reticulocytes, and with skeletal muscle phosphatase inhibitor 2. The interaction was followed by changes in the enzymatic activity and by quenching of fluorescence from the coumarin derivative of the phosphatase. Homogeneous regulin (Mr approximately 230,000) increases the activity of the enzyme severalfold; this stimulation is Mn2+-dependent. Inhibitor 2 decreases enzyme activity but only if the two proteins are preincubated in the absence of Mn2+. Comparable differences in the effect of Mn2+ were also observed in parallel experiments in which changes in fluorescence from the coumarin-labeled 56,000-dalton phosphatase were measured. In these experiments, it was shown that Mn2+ enhances the interaction between regulin and the 56,000-dalton phosphatase, but inhibits the interaction between the phosphatase and inhibitor 2.
D000818:Animals; D003035:Cobalt / Q000494:pharmacology; D003374:Coumarins; D003598:Cytoskeletal Proteins / Q000494:pharmacology*; D005453:Fluorescence; D007501:Iron / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000037:antagonists & inhibitors; D011506:Proteins / Q000494:pharmacology*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D014357:Trypsin / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology; D000097847:Protein Phosphatase Inhibitory Proteins
D003035:Cobalt; D003374:Coumarins; D003598:Cytoskeletal Proteins; D007501:Iron; D008345:Manganese; D010749:Phosphoprotein Phosphatases; D011506:Proteins; D014357:Trypsin; D015032:Zinc; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins; C030123:coumarin