This proposal requests funds for the purchase of a high sensitivity amino acid analyzer as part of a new and highly sophisticated instrumentation core. The instrument will be placed in the newly renovated Molecular Biology Core Laboratory which will be completed in July, 1990. The amino acid analyzer is a necessary component to complete the trio which also includes two instruments on hand already, an Applied Biosystems pulsed- liquid phase protein sequencer and a Milligen peptide synthesizer. With the addition of an amino acid analyzer to the Core Laboratory, it will be: 1, possible to more efficiently order samples for sequencing knowing the general amount of material available; 2, possible to characterize samples that fail to sequence due to a blocked amino terminus or insufficient sample; 3, possible to provide a characterization of peptides synthesized in the Core Laboratory routinely rather than await for a time convenient for Dr. Rosenberry and his staff (note that there, high sensitivity is not necessary); 4, provide amino acid composition data as part of the characterization of proteins, especially those isolated from SDS gels. The five major users display a wide variety of interests, but a central theme is the characterization of proteins. Dr. Merrick wishes to determine the primary amino acid sequence of eukaryotic translation factors by a combination of protein and cDNA sequencing. Subsequent studies would focus on protein active sites and site-directed mutagenesis. Dr. Miller is investigating the pathway of protein degradation in Salmonella by characterization of a number endo- and exopeptidases which recognize small peptides as opposed to complete proteins. In addition to characterization of the proteases at the level of primary sequence, active sites and substrate specificity will be examined. Dr. Wood is studying the enzyme transcarboxylase which is made up of three different subunits in a ratio of 12:12:6. Protein chemistry and cloned genes are being examined for active sites and portions of the molecules necessary for subunit-subunit interactions. In a similar manner, enzymes involved with autotrophic growth with CO or CO2 and H2 and the enzymes involved in polyphosphate metabolism will be characterized. Dr. Rosenberry is trying to determine the unique biochemical features of different acetyl-cholinesterase forms in synapses. By cDNA and protein sequencing, unique subunits will be characterized as will be their glycoinositol phospholipid anchors. Dr. Ikebe is studying the regulation of smooth muscle contraction. His focus is on determination of the phosphorylation sites in myocin light chain kinase and caldesmon and the role of phosphorylation in effecting of these two proteins.
