Peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, required Ca2+ as an essential co factor and the half-maximal activity was attained at 40~60/-iM Ca2+. Other divalent cations were practically inactive except for Sr2+, which was about 50% as active as Ca2+ when tested at 10mM. However, Sr2+ at less than the concentration of 100/wr had little or no activity. The direct Ca2+-binding for the enzymeshowed a sigmoidal curve with a transition midpoint of about 1 10 /iM, indicating that the binding is cooperative. Analysis of Hill plots of the data revealed that the enzyme binds 3mol of Ca2+/mol of protein with an apparent dissociation constant of 1 10 /iM. A conformational change upon Ca2+-binding was also described for the enzymeusing UV-difference spectra. The alteration could be attributed to an increased exposure of the aromatic residues to a more aqueous environment, as has been described for Ca2 +binding proteins such as calmodulin. Phosphatidylserine enhanced the reaction velocity and concomitantly reduced the Ca2 +-requirement for the enzyme. These effects were stimulated by the addition of diacylglycerol. Diacylglycerol alone had little or no effect. On the other hand, calmodulin had no effect on the enzymatic activity over a wide range of Ca2+ concentrations. These suggest that the activity and Ca2 +-sensitivity of peptidylarginine deiminase is increased at the cell membrane.

Objective: A number of activating mutations of the thyrotropin receptor (TSHR) have been found in autonomously functioning thyroid nodules (AFTNs) in European patients. We aimed to study TSHR mutation in AFTNs in Japanese patients because no TSHR activating mutation has been found by previous incomplete studies. Design: A typical AFTN developed in a 69-year-old Japanese woman was studied. Methods: The entire exon 10 of the TSHR cDNA was sequenced. Functional studies were done by sitedirected mutagenesis and transfection of a mutant construct into COS-7 cells. Results: We identi®ed a novel heterozygous TSHR gene mutation, Leu512!Arg (L512R; CTG!CCG), from the AFTN. The mutation was not detected in the adjacent normal thyroid tissue. COS-7 cells transfected with L512R mutant TSHR expression vector exhibited a 3.3-fold increase in basal cAMP level compared with that of cells transfected with wild-type TSHR DNA, con®rming that the mutation was the direct cause of the AFTN.TSHR activating mutations involving the third transmembrane helix reported to date are S505R/N and V509A as well as L512R. An in vitro site-directed mutagenesis study encompassing residues 505± 513 revealed that mutations involving residues other than these three did not show constitutive activation. Conclusion: This is the ®rst TSHR activating mutation found in a Japanese patient, although true prevalence of TSHR activating mutations in AFTNs developed in Japanese patients remains to be elucidated. In addition, functional studies suggested that amino acid residues in the third transmembrane helix maintaining inactive conformation of the TSHR seem to be located on the same surface of the a-helix, possibly making interhelical bonds with another helix.

Previously, we reported two Spanish siblings with congenital hypothyroidism due to total failure of iodide transport. These were the only cases reported to date who received long-term iodide treatment over 10 yr. We examined the sodium/iodide symporter (NIS) gene of these patients. A large deletion was observed by long and accurate PCR using primers derived from introns 2 and 7 of the NIS gene. PCR-direct sequencing revealed a deletion of 6192 bases spanning from exon 3 to intron 7 and an inverted insertion of a 431-base fragment spanning from exon 5 to intron 5 of the NIS gene. The patients were homozygous for the mutation, and their mother was heterozygous. In the mutant, deletion of exons 3-7 was suggested by analysis using programs to predict exon/intron organization, resulting in an in-frame 182-amino acid deletion from Met(142) in the fourth transmembrane domain to Gln(323) in the fourth exoplasmic loop. The mutant showed no iodide uptake activity when transfected into COS-7 cells, confirming that the mutation was the direct cause of the iodide transport defect in these patients. Further, the mutant NIS protein was synthesized, but not properly expressed, on the cell surface, but was mostly accumulated in the cytoplasm, suggesting impaired targeting to the plasma membrane.

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We designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI), STI-Sepharose was found to be an effective affinity adsorbent for purification of the enzyme. The specific binding of peptidylarginine deiminase to STI-Sepharose was observed in the presence of calcium ion, and the enzyme could be selectively eluted from the affinity adsorbent by washing with chelator. A 1,800-fold purification with a 50% yield was achieved in the three-step procedure, which involved DEAE-Sephacel ion-exchange and STI-Sepharose affinity chromatography. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and the recovery were considerably higher than have been obtained by any procedures previously reported. The specific interaction of peptidylarginine deiminase with STI immobilized on Sepharose was also investigated quantitatively by frontal affinity chromatography. In this method, a peptidylarginine deiminase solution was applied continuously to an STI-Sepharose column and the retardation of the elution front was measured as a parameter of the strength of the interaction. The dissociation constant for the enzyme with STI was found to be 2.3 X 10(-7)M. This value was in good agreement with that obtained by kinetic analysis in our previous studies. Peptidylarginine deiminase required millimolar Ca2+ for the binding to STI-Sepharose. The Ca2+ dependence of the enzyme binding was quite similar to that of the enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)