Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols (benzophenone-4- Communicated by Russell F. Doolittle, May 23, 1986 ABSTRACT The thiol-specific photoactivatable reagent benzophenone-4-iodoacetamide can be incorporated into myosin subfragment 1 (Si), accompanied by an increase of Ca2+-ATPase and the loss of K+-ATPase activities, a characteristic property of S1 when reactive sulfhydryl 1 (SH-1) Is modified. After trypsin cleavage, 25-kDa, 50-kDa, and 20-kDa fragments were found upon NaDodSO4/polyacrylamide gel electrophoresis of the unphotolyzed sample, whereas only the 50-kDa fragment and a 45-kDa fragment appeared In the photolyzed sample, indicating that the NH2-terminal 25-kDa fragment was crosslinked to the COOH-terminal 20-kDa fragment via SH-1. When photolysis 'was carried out in the presence of Mg2+ and ATP or Mg2e and adenosine 5-[,B, imidoltriphosphate (AdoPP[NHJP), a 70-kDa band, attribu-

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The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin S1, and upon photolysis, an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.

An acute presentation of an optic disc hemorrhage can indicate true optic nerve head damage or can be a result of tractional forces on the vitreopapillary interface. An isolated optic disc hemorrhage secondary to vitreopapillary traction (VPT) can mimic the clinical presentation of a glaucomatous process or that of an underlying ocular or systemic condition. This article highlights the use of spectral domain optical coherence tomography (SD-OCT) 5 line raster (5LR) in differentiating true optic disc hemorrhages from those as a result of tractional forces. Two examples are given of patients presenting to our clinic with unilateral optic disc hemorrhages and various underlying disease processes as risk factors. With the use of SD-OCT 5LR imaging, VPT was implicated as the cause of the disc hemorrhages in both patients. Although this imaging tool alone is not enough to rule out a disease process such as glaucoma or prevent the need for additional diagnostic testing, SD-OCT 5LR is a noninvasive and valuable clinical tool in distinguishing VPT from other etiologies of an optic nerve hemorrhage.

Regulatory light chain (RLC) mutants, RLC-C18 and RLC-C165, containing a single cysteine at positions 18 and 165 near the N and C terminus, respectively, were each labeled with benzophenone 4-iodoacetamide and exchanged into myosin in their phosphorylated or unphosphorylated forms and then photolyzed. SDS-PAGE showed that, for RLC-C18, the intrachain photo-cross-linking in myosin was inhibited by phosphorylation. For myosin containing RLC-C165, the yield of one intrachain cross-linked band decreased significantly whereas the other was unaffected by phosphorylation. Peptide mapping in conjunction with mass spectrometry showed that Cys165 was cross-linked to site(s) within Ala17-Lys34 independent of the phosphorylation of Ser19. This clearly demonstrates that the proximity between the N- and C-terminal regions of RLC is not affected by phosphorylation. In addition, Cys165 could also be cross-linked to the region of Phe133-Arg143; however, this type of cross-linking was inhibited in the phosphorylated state. For RLC-C18, the cross-linking took place with the region of Glu124-Arg132 or Phe133-Arg143, also only in the unphosphorylated state. Thus, phosphorylation changes the spatial relationship between the region of Glu124-Arg143 and Cys18 and Cys165. In scallop myosin, the region corresponding to Glu124-Arg143 is located at the interfaces between RLC and the essential light chain as well as the heavy chain [Xie, X. , et al. (1994) Nature 368, 306-312]. In light of that work, our results suggest that the region of Glu124-Arg143 is involved in the phosphorylation-dependent signaling and the change in its spatial relationship with respect to the N and C termini of RLC may underlie the activation of the smooth muscle myosin.