An investigation of the SH1-SH2 and SH1-ATPase distances in myosin subfragment-1 by resonance energy transfer using nanosecond fluorimetry

The separation between the two reactive thiols SH1 (Cys-704) and SH2 (Cys-694) and that between SH1 and the active site of myosin subfragment-1 were further investigated by Forster energy transfer techniques. The SH1-SH2 distance was determined with the probe 5[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (AEDANS) attached to SH1 as the energy donor and 5-(iodoacetamido)fluorescein (IAF) attached to SH2 as energy acceptor. The results derived from measurements of donor lifetimes yielded a donor-acceptor separation in the range 26-52A, with the distance R(2/3) based on rapid and isotropic probe motions being 40 A. These parameters were not sensitive to added MgADP, in agreement with previous results obtained by using the steady-state method. The SH1-SH2 distance was also determined with AEDANS attached to SH1 and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) attached to SH2. The range in R for the AEDANS/DDPM pair was 12-36 A, with R(2/3) equal to 27 A. The transfer efficiency between these two probes increased by an average of 38% upon addition of MgADP. These results are in agreement with those previously reported (Dalbey, R.E.; Weiel, J.; Yount, R.G. (1983) Biochemistry 22, 4696-4706), but the uncertainty in choosing an appropriate value of the orientation factor to describe the AEDANS-DDPM separation does not allow a unique interpretation of the observed increase in energy transfer because it could reflect either an increase in the average orientation factor or a decrease in the donor-acceptor separation. Nevertheless, the results are consistent with the notion that nucleotide binding induces structural perturbations that can be sensed by SH1 and SH2. The distance between SH1 and the ATPase site was determined with AEDANS linked to SH1 and the nucleotide analogue 2'(3')-O-(2,4,6,-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) noncovalently bound to the active site as energy acceptor. The bound TNP-ADP was highly immobilized, with a depolarization factor approaching unity. The sepration between AEDANS at SH1 and TNP-ADP at the active site was in the range 15-44 A. The actual minimal separation between SH1 and the active site is probably less than 15 A, which suggests that direct interaction between the two sites cannot be ruled out from energy transfer results.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25503/1/0000044.pd

Causes of Sudden Death Related to Sexual Activity : Results of a Medicolegal Postmortem Study from 2001 to 2005

Sexual activity (SA), combined with organic heart disease, may cause sudden death (SD). However, the causes of SD related to SA are not known well. The aim of this study was to assess the causes of SD related to SA. From August 2001 to November 2005, all autopsies (n=1,379) performed at Kyungpook National University were prospectively searched for SD cases related to SA. Fourteen cases (46±11 yr old, 9 males) of SD related to SA were found. All were heterosexual. The toxicologic study was negative in all. Ten cases were witnessed; during SA in 4 cases, just after SA in another 4 cases, 2 and 5 hr after in 1 each case. In 4 unwitnessed cases the victims were found dead less than 12 hr from the end of their SA. The partners were steady extramarital partners (n=8), prostitutes (n=2), marital partner (n=1) and unknown (n=3). The causes of the SD were as follows; coronary artery disease in 6, subarachnoid hemorrhage with ruptured berry aneurysm in 4, fibromuscular dysplasia of the atrioventricular nodal artery in 2, and unknown in 2. Coronary artery disease and subarachnoid hemorrhage with ruptured berry aneurysm were important as causes of SD related to SA