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T E Garabedian
Publications and source records attributed to T E Garabedian.
Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate.
The active-site topology of smooth muscle myosin has been investigated by direct photoaffinity-labeling studies with [3H]ADP. Addition of vanadate (Vi) and Co2+ enabled [3H]ADP to be stably trapped at the active site (t1/2 greater than 5 days at 0 degrees C). The extraordinary stability of the myosin.Co2+.[3H]ADP.Vi complex allowed it to be purified free of excess [3H]ADP before irradiation began and ensured that only active-site residues became labeled. Following UV irradiation, approximately 10% of the trapped [3H]ADP became covalently attached at the active site. All of the [3H]ADP incorporated into the 200-kDa heavy chain, confirming earlier results using untrapped [alpha-32P]ATP [Maruta, H., & Korn, E. (1981) J. Biol. Chem. 256, 499-502]. After extensive trypsin digestion of labeled subfragment 1, HPLC separation methods combined with alkaline phosphatase treatment allowed two labeled peptides to be isolated. Sequence analysis of both labeled peptides indicated that Glu-185 was the labeled residue. Since Glu-185 has been previously identified as a residue at the active site of smooth myosin using [3H]UDP as a photolabel [Garabedian, T. E., & Yount, R. G. (1990) J. Biol. Chem. 265, 22547-22553], these results provide further evidence that Glu-185, located immediately adjacent to the glycine-rich loop, is located in the purine binding pocket of the active site of smooth muscle myosin.
Adenosine Diphosphate↗
Direct photoaffinity labeling of gizzard myosin with [3H]uridine diphosphate places Glu185 of the heavy chain at the active site.
The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with [3H]UDP. [3H] UDP was stably trapped at the active site by addition of vanadate (Vi) and Co2+. The extraordinary stability of the myosin.Co2+.[3H]UDP.Vi complex (t1/2 greater than 5 days at 0 degrees C) allowed it to be purified free of extraneous [3H]UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped [3H]UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results (Maruta, H., and Korn, E. (1981) J. Biol. Chem. 256, 499-502) using [3H]UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a "zero-length" cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by [3H]UTP photolabeling in Acanthamoeba myosin II (Atkinson, M. A., Robinson, E. A., Appella, E., and Korn, E. D. (1986) J. Biol. Chem. 261, 1844-1848).
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