778.1-Pos Board # B363.1 – Effects of Ca2+ and Temperature on the Force-generating Transition in Cardiac Muscle Studied by Photolysis of Caged-phosphate

Abstract

The genomes of a wide variety of organisms have now been sequenced; a major challenge ahead is to understand the function, regulation and modification of the many encoded gene products. We have been carrying out proteomics approaches to the identification and analysis of signalling pathways in yeast. 121 of 122 protein kinases were cloned and purifed from yeast as GST fusions and analyzed for their ability to phosphorylate 60 different yeast substrates. More than 93% of the kinases exhibited activities that are 5 fold or higher, relative to controls, including 18 of 24 previously uncharacterized kinases. Many protein kinases had novel activities; for example 27 yeast kinases were found to phosphorylate Tyr. In addition, we have now cloned 6000 open reading frames and overexpressed their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with a variety of different proteins, nucleic acids and phospholipids. As examples, we have probed yeast proteome chips with calmodulin and six different phospholipids. These studies revealed many new calmodulin and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. They can also be used to screen protein-drug interactions and to detect posttranslational modifications.

Activation Kinetics of Skinned Cardiac Muscle by Laser Photolysis of Nitrophenyl-EGTA

“…Exponentials were fit to the simulated transients, and the resultant values for k Pi are listed in Table 2. Over the range of [Ca 21 ] simulated, k Pi at 2 or 4 mM final [Pi] was insensitive to calcium similar to previous studies of skeletal Homsher, 1990, 1992) and cardiac muscle (Martin et al, 2003) but slightly different from cardiac studies that reported a twofold increase in k Pi over this range of [Ca 21 ] (Araujo and Walker, 1996).…”

“…This is followed by P i release, which stabilizes a second strongly bound force bearing state, AM.ADP. Studies of both skeletal (Millar and Homsher, 1990;Walker et al, 1992) and cardiac muscle (Araujo and Walker, 1996;Martin et al, 2003) have reported k Pi to be either slightly or not affected by [Ca 21 ], and when compared to k tr in the same preparation, two times faster (Millar and Homsher, 1990;Regnier et al, 1995;Tesi et al, 2000). These results indicate that the tension-generating step is not directly Ca-regulated but suggest a step before force generation is able to sense the steady [Ca 21 ] via the activation level of the thin filament.…”